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TGF-beta is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-beta-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-beta, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-beta-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-beta-induced ECM remodeling and fibrosis.

Objective The aim of this study was to examine whether serum urate-associated genetic variants are associated with early-onset gout. Methods Participants with gout in the Genetics of Gout in Aotearoa study with available genotyping were included (n = 1648). Early-onset gout was defined as the first presentation of gout Results In the Genetics of Gout in Aotearoa study, there were 638 (42.4%) participants with early-onset gout. The ABCG2 rs2231142 gout risk T-allele was present more frequently in participants with early-onset gout compared with the later-onset group. For the other SNPs tested, no differences in risk allele number were observed. In the allelic association analysis, the ABCG2 rs2231142 T-allele was associated with early-onset gout in unadjusted and adjusted models. Analysis of the replication cohorts confirmed the association of early-onset gout with the ABCG2 rs2231142 T-allele, but not with other serum urate-associated SNPs. In the meta-analysis, the odds ratio (95% CI) for early-onset gout for the ABCG2 rs2231142 T-allele was 1.60 (1.41, 1.83). Conclusion In contrast to other serum urate-raising variants, the ABCG2 rs2231142 T-allele is strongly associated with early-onset gout.

Item does not contain fulltext OBJECTIVE: In many rheumatoid arthritis (RA) patients, disease is controlled with anti-tumor necrosis factor (anti-TNF) biologic therapies. However, in a significant number of patients, the disease fails to respond to anti-TNF therapy. We undertook the present study to examine the hypothesis that rare and low-frequency genetic variants might influence response to anti-TNF treatment. METHODS: We sequenced the coding region of 750 genes in 1,094 RA patients of European ancestry who were treated with anti-TNF. After quality control, 690 genes were included in the analysis. We applied single-variant association and gene-based association tests to identify variants associated with anti-TNF treatment response. In addition, given the key mechanistic role of TNF, we performed gene set analyses of 27 TNF pathway genes. RESULTS: We identified 14,420 functional variants, of which 6,934 were predicted as nonsynonymous 2,136 of which were further predicted to be "damaging." Despite the fact that the study was well powered, no single variant or gene showed study-wide significant association with change in the outcome measures disease activity or European League Against Rheumatism response. Intriguingly, we observed 3 genes, of 27 with nominal signals of association (P < 0.05), that were involved in the TNF signaling pathway. However, when we performed a rigorous gene set enrichment analysis based on association P value ranking, we observed no evidence of enrichment of association at genes involved in the TNF pathway (Penrichment = 0.15, based on phenotype permutations). CONCLUSION: Our findings suggest that rare and low-frequency protein-coding variants in TNF signaling pathway genes or other genes do not contribute substantially to anti-TNF treatment response in patients with RA.

ObjectivesMast 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.MethodsSynovial 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).ResultsHigh 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.ConclusionsSynovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

Background Activation of mast cells through FceRI plays an important role in acute allergic reactions. However, little is known about the function of mast cells in patients with chronic allergic inflammation or the effect of repeated FceRI triggering occurring in such responses. Objective We aimed to identify changes in mast cell function after repeated FceRI triggering and to correlate these changes to chronic allergic responses in tissue. Methods Human cord blood–derived mast cells were treated for 2 weeks with anti-IgE. The function of naive or treated mast cells was analyzed by means of RNA sequencing, quantitative RT-PCR, flow cytometry, and functional assays. Protein secretion was measured with ELISAs and multiplex assays. Results We observed several changes in mast cell function after repeated anti-IgE triggering. Although the acute response was dampened, we identified 289 genes significantly upregulated after repeated anti-IgE. Most of these genes (84%) were not upregulated after a single anti-IgE stimulus, indicating a significantly different response mode characterized by increased antigen presentation, response to bacteria, and chemotaxis. Changes in mast cell function were related to changes in expression of the transcription factors RXRA and BATF and others. Importantly, we found a substantial overlap between genes upregulated after repeated anti-IgE triggering and genes upregulated in tissue from patients with chronic allergy, in particular those of patients with chronic rhinosinusitis. Conclusion Our study provides evidence for intrinsic modulation of mast cell function on repeated FceRI-mediated activation. The overlap with gene expression in tissues is suggestive of a direct link between repeated IgE-mediated activation of mast cells and chronic allergy.

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-gamma or via Fc epsilon RI 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. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

Genetics in rheumatoid arthritis (RA) has moved from the finding of HLA-shared epitope decades ago toward the understanding of the role of HLA in RA and the findings of ∼100 additional genetic risk variants for disease susceptibility as well as several risk variants for severe disease. These findings increased our understanding of RA abnormality. Still, the mechanisms by which many of the variants exhibit their effect are not yet understood.

Background Gout progresses through three stages: hyperuricemia, deposition of monosodium urate (MSU) crystals, and innate immune system response to MSU crystals. Genome wide association studies (GWAS) have provided insight into the molecular control of progression to hyperuricemia. However, less is known about the progression from hyperuricemia to gout. Objectives To conduct a GWAS for gout (where an immune response to MSU crystals has occurred) using 5,835 cases - the largest GWAS of gout to date. Methods The GWAS comprised 3 data sets: NZ/Eurogout (2,365 clinically-ascertained cases; 1,485 controls), the Health Professionals Follow-Up (HPFS) and Nurses9 Health Studies (NHS) (1,038 cases, self-ascertained using ACR criteria; 1,095 controls), and UK Biobank (2,432 cases, ascertained by self-report of gout, hospital records, and/or use of urate-lowering therapy; 102,989 controls). The NZ/Eurogout samples were genotyped using the Illumina CoreExome v24 bead chip array (547,644 markers), the HPFS/NHS samples using the Illumina OmniExpress v12 bead chip array (730,525 markers), and the UK Biobank samples using an Affymetrix Axiom array (820,967 markers). UK Biobank genotypes had been imputed to ∼73.3M SNPs. Neither the NZ/Eurogout nor NHS/HPFS genotype sets were imputed. Markers common to all three data sets (279,939) were associated with gout (adjusted for sex, age) within each data set separately using PLINK 1.9. An inverse-variance weighted meta-analysis was done with meta v4.4 in R. Results There were seven loci with genome-wide significant (P Conclusions All seven loci have been previously associated with serum urate levels in GWAS. Our data emphasise the relative importance of genetic control of serum urate, compared to the genetic control of MSU crystal formation or the innate immune response, in determining gout. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and multiple organs of which pathogenesis is poorly understood. Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Objectives Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Methods Skin biopsy-derived RNAs from fourteen early SSc patients and six healthy individuals were sequenced with ion-torrent and analysed using DEseq2. Protein-coding and non-coding genes annotated in GENCODEV7 were analysed. Significant long non-coding RNAs were independently replicated in a Northern American dataset. Results 4901 genes with a fold change >1.5 and a false discovery rate of less than 5% were detected in patients versus controls. Upregulated coding genes clustered in immunological, cell adhesion and keratin-related processes as previously found by microarray studies. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. 42% of these antisense genes had a concurrent deregulated sense gene. The majority of the sense-antisense genes had a similar effect sizes in an independent North American dataset with three genes (OTUD6B-AS1, CTBP1-AS2 and HMGN3-AS1) exceeding the study-wide Bonferroni-corrected ρ-value (PBonf Conclusions For the first time we highlight that together with coding genes, (antisense) long noncoding RNAs are deregulated in skin tissue of SSc patients suggesting a novel class of genes involved in pathogenesis of SSc. Disclosure of Interest None declared

Innate immune cells, such as monocytes, can adopt a long-lasting pro-inflammatory phenotype, a phenomenon called 'trained immunity'. In trained immunity, increased cytokine levels of genes, like interleukin (IL)-6 and tumor necrosis factor (TNF)-α, are observed, which are associated with increased histone 3 lysine 4 trimethylation (H3K4me3) in the promoter region. As systemic IL6 and TNFα levels are increased in rheumatoid arthritis (RA) patients and monocytes are known to be the primary producers of TNFα and IL6, we hypothesized that 'trained immunity' signals may be observed at these genes in monocytes from RA patients. CD14+ monocytes were isolated from untreated RA patients and paired age-matched healthy controls. H3K4me3, mRNA, protein and serum levels of IL6 and TNFα were evaluated by chromatin immunoprecipitation, reverse-transcription quantitative PCR and enzyme-linked immunosorbent assays. Despite elevated serum levels of TNFα and IL6 in the tested RA patients (P

Background Traditionally, immunological responses have been studied in-vitro whereby immune cells are subject to certain stimuli following which alterations in RNA and/or protein expression are measured. Recently, we have challenged this model by showing that mast cells (cells specialised in allergic reactions) triggered repeatedly through their Ig Fc epsilon receptor undergo a reprogramming of their responses (Suurmond et al. JACI, 2016). The differential expression of genes upon a single trigger were dampened and de-novo transcribed genes were observed. The latter belonged to the antigen presentation and pathogen defence response pathways implying a dramatic change in the cellular response mode. We have now extended this work by challenging mast cells with plate-bound IgG in a repeated mode, a model relevant for arthritic diseases where mast cells and IgG immune complexes are likely to interact. Materials and methods Human cord blood-derived mast cells were treated for 2 weeks with plate-bound IgG. The expression profile of naive or treated mast cells was measured through RNA sequencing, quantitative RT-PCR, flow cytometry. Protein secretion was measured with ELISA and Luminex assays. Metabolic changes were measured using HPLC mass-spectrometry. Results Similar to our previous work on Fc Epsilon receptor, we observe a dampening of the normal IgG responses with a set of novel genes upregulated. Interestingly, de-novo expressed genes consisted of DHCR7 and DHCR24, key enzymes in the cholesterol pathway. Pathway analysis confirms an enrichment of genes in this pathway following repeated IgG triggering. Preliminary data on metabolic profiling reveals a decrease in phospholipid levels in repeatedly activated mast cells. Conclusions Our study provides evidence that mast cells are also reprogrammed upon repeated IgG triggering. In contrast to repeated Fc Epsilon Receptor triggering, different pathways are affected, implying stimulus-specific effects. Our work has important implications for revisiting the traditional immunological models in the (in-vitro) modelling of chronic (arthritic) diseases.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and multiple organs of which the pathogenesis is poorly understood. Here we studied differentially expressed coding and non-coding genes in relation to SSc pathogenesis with a specific focus on antisense non-coding RNAs. Skin biopsy-derived RNAs from fourteen early SSc patients and six healthy individuals were sequenced with ion-torrent and analysed using DEseq2. Overall, 4901 genes with a fold change >1.5 and a false discovery rate < 5% were detected in patients versus controls. Upregulated genes clustered in immunological, cell adhesion and keratin-related processes. Interestingly, 676 deregulated non-coding genes were detected, 257 of which were classified as antisense genes. Sense genes expressed opposite of these antisense genes were also deregulated in 42% of the observed sense-antisense gene pairs. The majority of the antisense genes had a similar effect sizes in an independent North American dataset with three genes (CTBP1-AS2, OTUD6B-AS1 and AGAP2-AS1) exceeding the study-wide Bonferroni-corrected ρ-value (PBonfcombined = 1.1x10-9, 1.4x10-8, 1.7x10-6, respectively). In this study, we highlight that together with coding genes, (antisense) long non-coding RNAs are deregulated in skin tissue of SSc patients suggesting a novel class of genes involved in pathogenesis of SSc.

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 23262 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 center dot 72 (0 center dot 58-0 center dot 88), P=0 center dot 0006]. The variants of this SNP were associated with different C1q serum levels in healthy controls (P=0 center dot 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 center dot 83 (0 center dot 69-1 center dot 00), P=0 center dot 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.

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.

A common allele at the TAGAP gene locus demonstrates a suggestive, but not conclusive association with risk of rheumatoid arthritis (RA). To fine map the locus, we conducted comprehensive imputation of CEU HapMap single-nucleotide polymorphisms (SNPs) in a genome-wide association study (GWAS) of 5500 RA cases and 22 621 controls (all of European ancestry). After controlling for population stratification with principal components analysis, the strongest signal of association was to an imputed SNP, rs212389 (P 3.9 x 10(-8), odds ratio 0.87). This SNP remained highly significant upon conditioning on the previous RA risk variant (rs394581, P = 2.2 x 10(-5)) or on a SNP previously associated with celiac disease and type I diabetes (rs1738074, P = 1.7 x 10(-4)). Our study has refined the TAGAP signal of association to a single haplotype in RA, and in doing so provides conclusive statistical evidence that the TAGAP locus is associated with RA risk. Our study also underscores the utility of comprehensive imputation in large GWAS data sets to fine map disease risk alleles. Genes and Immunity (2011) 12, 314-318; doi:10.1038/gene.2011.8; published online 10 March 2011

Rheumatoid arthritis (RA) is characterized by inflammatory arthritis and localized destruction of bone and cartilage. The severity of joint destruction is highly variable between patients and, according to the findings of twin studies, substantially influenced by genetic factors (1). Nevertheless, the precise contribution of genetic factors has yet to be determined. To date, only a small number of genetic risk factors have been identified, and apart from HLA, the association of none of these factors with RA severity has been convincingly replicated. In contrast, the genetics of susceptibility to RA has been considerably boosted, largely due to the findings of genome-wide association studies. In addition to the HLA–DRB1 shared epitope alleles, several new susceptibility factors have been identified, and their association with RA has been independently replicated: PTPN22, TRAF1/C5, OLIG3/TNFAIP3, and STAT4. Intriguingly, for many of these genetic risk factors, the associations are confined to RA patients positive for anti–citrullinated protein antibody (ACPA). It remains unknown whether genetic factors also affect the severity of joint destruction in ACPA-positive and ACPA-negative RA in different ways. Nonetheless, compelling evidence demonstrates that ACPA-positive RA patients have a more destructive disease course than do ACPA-negative RA patients. A recent meta-analysis of 2 genome-wide association studies identified 6 new risk loci (rs4810485 [CD40], rs1678542 [KIF5A/PIP4K2C], rs42041 [CDK6], rs2812378 [CCL21], rs4750316 [PRKCQ], and rs3890745 [MMEL1/TNFRSF14]) as susceptibility factors for autoantibody-positive RA (2). In the present study, we investigated the association between these single-nucleotide polymorphisms (SNPs) and the rate of radiologic joint destruction in RA patients and in ACPA-positive RA in particular using a large longitudinal cohort. A cohort of ACPA-positive RA patients was used for replication analysis. We found that a genetic variant in the CD40 gene is associated with the rate of joint destruction in ACPA-positive RA patients.

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 = 1.66 × 10−11], OR 0.78 [P = 5.6 × 10−5], and OR 0.91 [P = 1.8 × 10−3], 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.

Contains fulltext : 71178.pdf (Publisher’s version ) (Closed access) OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.

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).

In their published article, Giles et al. ([1][1]) explored functional consequences of rs17611 in the TRAF1-C5 region and propose a mechanism for its contribution to rheumatoid arthritis (RA) pathology, as this SNP would associate with RA. To substantiate the claim that the SNP associates with RA

Objective Certain HLA-DRB1 alleles and single-nucleotide polymorphisms (SNPs) are associated with rheumatoid arthritis (RA). Our objective was to examine the combined effect of these associated variants, calculated as a cumulative genetic risk score (GRS) on RA predisposition, as well as the number of autoantibodies (none, one or two present).Method We calculated four GRSs in 4956 patients and 4983 controls from four European countries. All four scores contained data on 22 non-HLA-risk SNPs, and three scores also contained HLA-DRB1 genotypes but had different HLA typing resolution. Most patients had data on both rheumatoid factor (RF) and anti-citrullinated proteins antibodies (ACPA). The GRSs were standardised (std. GRS) to account for population heterogeneity. Discrimination between patients and controls was examined by receiveroperating characteristics curves, and the four std. GRSs were compared across subgroups according to autoantibody status.Results The std. GRS improved its discriminatory ability between patients and controls when HLA-DRB1 data of higher resolution were added to the combined score. Patients had higher mean std. GRS than controls (p=7.9x10(-156)), and this score was significantly higher in patients with autoantibodies (shown for both RF and ACPA). Mean std. GRS was also higher in those with two versus one autoantibody (p=3.7x10(-23)) but was similar in patients without autoantibodies and controls (p=0.12).Conclusions The GRS was associated with the number of autoantibodies and to both RF and ACPA positivity. ACPA play a more important role than RF with regards to the genetic risk profile, but stratification of patients according to both RF and ACPA may optimise future genetic studies.

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.

The last decade has seen a dramatic technological revolution. The characterisation of the majority of the common variations in our genetic code in 2003 precipitated the discovery of the genetic risk factors predisposing to Rheumatoid Arthritis development and progression. Prior to 2007, only a handful of genetic risk factors had been identified, HLA, PTPN22 and CTLA4. Since then, over 100 genetic risk loci have been described, with the prediction that an ever-increasing number of risk alleles with consistently decreasing effect sizes will be discovered in the years to come. Each risk locus harbours multiple candidate genes and the proof of causality of each of these candidates is as yet unknown. An enrichment of these RA-associated genes is found in three pathways: T-cell receptor signalling, JAK-STAT signalling and the NF-κB signalling cascade, and currently drugs targeting these pathways are available for the treatment of RA. However, the role that RA-associated genes have in these pathways and how they contribute to disease is not always clear. Major efforts in understanding the contribution of genetic risk factors are currently under way with studies querying the role of genetic variation in gene expression of coding and non-coding genes, epigenetic marks and other regulatory mechanisms yielding ever more valuable insights into mechanisms of disease. Recent work has suggested a possible enrichment of non-coding RNAs as well as super-enhancers in RA genetic loci indicating possible new insights into disease mechanism. This review brings together these emerging genetic data with an emphasis on the immunogenetic links these findings have provided and what we expect the future will bring.

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.

Objective: Juvenile idiopathic arthritis (JIA) is a chronic disorder in which both genetic and environmental factors are involved. Recently, we identified the TRAF1/C5 region (located on chromosome 9q33–34) as a risk factor for rheumatoid arthritis (RA) (p combined = 1.4×10 −8 ). In the present study the association of the TRAF1/C5 region with the susceptibility to JIA was investigated. Methods: A case–control association study was performed in 338 Caucasian patients with JIA and 511 healthy individuals. We genotyped the single nucleotide polymorphism rs10818488 as a marker for the TRAF1/C5 region. Results: The A allele was associated with the susceptibility to rheumatoid factor-negative polyarthritis with an 11% increase in allele frequency (OR 1.54, 95% CI 1.09 to 2.18; p = 0.012). This association was stronger when combining subtypes with a polyarticular phenotype (OR 1.46, 95% CI 1.12 to 1.90; p = 0.004). In addition, we observed a trend towards an increase in A allele frequency in patients with extended oligoarthritis versus persistent oligoarthritis (49%, 38% respectively); p = 0.055. Conclusions: Apart from being a well replicated risk factor for RA, TRAF1/C5 also appears to be a risk factor for the rheumatoid factor-negative polyarthritis subtype of JIA and, more generally, seems to be associated with subtypes of JIA characterised by a polyarticular course.

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10−9). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10−9), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA–approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA., Author Summary A current challenge in human genetics is to follow-up “hits” from genome-wide association studies (GWAS) to guide drug discovery for complex traits. Previously, we identified a common variant in the CD40 locus as associated with risk of rheumatoid arthritis (RA). Here, we fine-map the CD40 signal of association through a combination of dense genotyping and exonic sequencing in large patient collections. Further, we demonstrate that the RA risk allele is a gain-of-function allele that increases the amount of CD40 on the surface of primary human B lymphocyte cells from healthy control individuals. Based on these observations, we develop a high-throughput assay to recapitulate the biology of the RA risk allele in a system suitable for a small molecule drug screen. After a series of primary screens and counter screens, we identify small molecules that inhibit CD40-mediated NF-kB signaling in human B cells. While this is only the first step towards a more comprehensive effort to identify CD40-specific inhibitors that may be used to treat RA, our study demonstrates a successful strategy to progress from a GWAS to a drug screen for complex traits such as RA.

Objective The significance of non–rheumatoid arthritis (RA) autoantibodies in patients with RA is unclear. The aim of this study was to assess associations of autoantibodies with autoimmune risk alleles and with clinical diagnoses from the electronic medical records (EMRs) among RA cases and non-RA controls. Methods Data on 1,290 RA cases and 1,236 non-RA controls of European genetic ancestry were obtained from the EMRs of 2 large academic centers. The levels of anti–citrullinated protein antibodies (ACPAs), antinuclear antibodies (ANAs), anti–tissue transglutaminase antibodies (AGTAs), and anti–thyroid peroxidase (anti-TPO) antibodies were measured. All subjects were genotyped for autoimmune risk alleles, and the association between number of autoimmune risk alleles present and number of types of autoantibodies present was studied. A phenome-wide association study (PheWAS) was conducted to study potential associations between autoantibodies and clinical diagnoses among RA cases and non-RA controls. Results The mean ages were 60.7 years in RA cases and 64.6 years in non-RA controls. The proportion of female subjects was 79% in each group. The prevalence of ACPAs and ANAs was higher in RA cases compared to controls (each P < 0.0001); there were no differences in the prevalence of anti-TPO antibodies and AGTAs. Carriage of higher numbers of autoimmune risk alleles was associated with increasing numbers of autoantibody types in RA cases (P = 2.1 × 10−5) and non-RA controls (P = 5.0 × 10−3). From the PheWAS, the presence of ANAs was significantly associated with a diagnosis of Sjogren's/sicca syndrome in RA cases. Conclusion The increased frequency of autoantibodies in RA cases and non-RA controls was associated with the number of autoimmune risk alleles carried by an individual. PheWAS of EMR data, with linkage to laboratory data obtained from blood samples, provide a novel method to test for the clinical significance of biomarkers in disease.

The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome-wide association studies (GWASs). First, we assessed the contribution of rare coding variants in the 25 genes to the risk of RA in a pooled sequencing study of 500 RA cases and 650 controls of European ancestry. We observed an accumulation of rare nonsynonymous variants exclusive to RA cases in IL2RA and IL2RB (burden test: p = 0.007 and p = 0.018, respectively). Next, we assessed the aggregate contribution of low-frequency and common coding variants to the risk of RA by dense genotyping of the 25 gene loci in 10,609 RA cases and 35,605 controls. We observed a strong enrichment of coding variants with a nominal signal of association with RA (p A [p.His266Gln]), and a noncoding variant, rs624988, reside on distinct haplotypes and independently contribute to the risk of RA (p = 4.6 x 10(-6)). Overall, our results indicate that variants (distributed across the allele-frequency spectrum) within the protein-coding portion of a subset of biological candidate genes identified by GWASs contribute to the risk of RA. Further, we have demonstrated that very large sample sizes will be required for comprehensively identifying the independent alleles contributing to the missing heritability of RA.

Background Long noncoding RNAs (lncRNAs) are an emerging class of noncoding transcripts involved in the regulation of gene expression in health and disease. We have recently identified a novel lncRNA, H19X, which is strictly regulated by TGFβ in a dose and time dependent manner. Objectives To detail the differential expression of H19X in SSc and other fibrotic diseases and to characterize its function in fibrotic diseases. Methods Skin and lung biopsies from patients with SSc, idiopathic lung fibrosis (IPF), and healthy controls (HC) were obtained from cohorts at four different expert centers. Expression of H19X was analyzed by RNA Sequencing Ilumina HiSeq2000 and real-time PCR respectively. H19X was silenced in skin fibroblasts using locked nucleic acid antisense oligonucleotides (LNA GapmeRs), followed by qPCR analyses, immunofluorescence staining, SIRCOL assay, contraction assay and Western blot. In situ hybridization of H19X on SSc dermal fibroblast was performed using Stellaris FISH probes. Results H19X expression was consistently upregulated across all four cohorts (SSc n=34, HC n=26). Notably, the upregulation was also consistent across different subsets of patients. H19X showed a similar expression profile in clinically non-involved skin compared to established fibrotic skin. Moreover, expression of H19X was also significantly increased in SSc interstitial lung disease patients versus HC (n=11 each, p Conclusions H19X is a novel lncRNA which is a key factor in mediating the pro-fibrotic effects of TGFβ in systemic sclerosis and other fibrotic diseases. Disclosure of Interest E. Pachera: None declared, S. Assassi: None declared, G. Salazar: None declared, M. Frank-Bertoncelj: None declared, R. Dobrota: None declared, M. Brock: None declared, F. Kurreeman: None declared, J. de Vries-Bouwstra: None declared, T. Messemaker: None declared, C. Feghali-Bostwick: None declared, J. Distler: None declared, G. Kania: None declared, O. Distler Grant/research support from: from Bayer, Sanofi, Ergonex, Boehringer Ingelheim, Actelion, Pfizer, Consultant for: from 4 D Science, Actelion, Active Biotec, Bayer, BiogenIdec, BMS, Boehringer Ingelheim, EpiPharm, Ergonex, espeRare foundation, Genentech/Roche, GSK, Inventiva, Lilly, medac, MedImmune, Pharmacyclics, Pfizer, Serodapharm, Sinoxa

Background and objective Rheumatoid Arthritis (RA) is a heterogeneous disease affecting approximately 1–2% of the European population. Development of RA is affected by both genetic and environmental triggers. In the present study we hypothesised that these environmental triggers might induce changes in the epigenome of immune cells. A concept that describes such a mechanism is known as ‘trained immunity’, which shows that isolated monocytes previously exposed to micro-organisms display elevated H3K4me3 on the promotor regions of immune genes resulting in enhanced RNA- and protein levels of TNFa and IL6 upon re-stimulation with inflammatory triggers. The phenomenon that innate cells triggered by past insults more readily react to new non-specific stimuli could contribute to the onset of auto-immunity. We wished to test whether the innate immune system of RA patients displays signs of “trained immunity” by analysing immune responsiveness and epigenetic changes of CD14 + monocytes isolated from both untreated RA patients and age-matched healthy controls. Methods CD14 + monocytes were isolated from both untreated RA patients and age-matched healthy controls. Secreted TNFa and IL6 levels were measured using ELISA in the supernatant of naive and LPS-stimulated monocytes from both RA patients and non-RA controls. RNA levels of TNFa and IL6 in naive and LPS-stimulated monocytes from both groups were examined using RT-qPCR. Results Both RNA and protein levels of TNFa and IL6 were low in unstimulated monocytes isolated from RA patients and healthy controls but no large differences were observed. After LPS-stimulation, the relative RNA levels (expressed in fold change relative to household gene) increased but were similar between patients and controls for TNFa (RA: 1.7 ± 0,4, non-RA: 1.8 ± 0.9) and IL6 (RA: 2.9 ± 1,3, non-RA: 3.3 ± 2.6). Concordantly, no large differences were detected for secreted cytokine levels, although secreted TNFa (RA: 4.3ng/ml ± 2.9, non-RA: 2.8ng/ml ± 2.6) and IL6 (RA: 67.2mg/ml ± 14.6, non-RA: 50.8mg/ml ± 17.3) levels seem slightly elevated in RA monocytes. Conclusions In general, we could not detect large differences on RNA level or protein level of TNFa and IL6 between RA and non-RA patients. Therefore, we did not find evidence that the concept of trained immunity may contribute to the development of RA.

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.

Summary 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.

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.

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, Author Summary Celiac disease (CD) and rheumatoid arthritis (RA) are two autoimmune diseases characterized by distinct clinical features but increased co-occurrence in families and individuals. Genome-wide association studies (GWAS) performed in CD and RA have identified the HLA region and 26 non-HLA genetic risk loci in each disease. Of the 26 CD and 26 RA risk loci, previous studies have shown that six are shared between the two diseases. In this study we aimed to identify additional shared risk alleles and, in doing so, gain more insight into shared disease pathogenesis. We first empirically investigated the distribution of putative risk alleles from GWAS across both diseases (after removing known risk loci for both diseases). We found that CD risk alleles are non-randomly distributed in the RA GWAS (and vice versa), indicating that CD risk alleles have an increased prior probability of being associated with RA (and vice versa). Next, we performed a GWAS meta-analysis to search for shared risk alleles by combing the RA and CD GWAS, performing both directional and opposite allelic effect analyses, followed by replication testing in independent case-control datasets in both diseases. In addition to the already established six non-HLA shared risk loci, we observed statistically robust associations at eight SNPs, thereby increasing the number of shared non-HLA risk loci to fourteen. Finally, we used gene expression studies and pathway analysis tools to identify the plausible candidate genes in the fourteen associated loci. We observed remarkable overrepresentation of T-cell signaling molecules among the shared genes.

Discovering and following up on genetic associations with complex phenotypes require large patient cohorts. This is particularly true for patient cohorts of diverse ancestry and clinically relevant subsets of disease. The ability to mine the electronic health records (EHRs) of patients followed as part of routine clinical care provides a potential opportunity to efficiently identify affected cases and unaffected controls for appropriate-sized genetic studies. Here, we demonstrate proof-of-concept that it is possible to use EHR data linked with biospecimens to establish a multi-ethnic case-control cohort for genetic research of a complex disease, rheumatoid arthritis (RA). In 1,515 EHR-derived RA cases and 1,480 controls matched for both genetic ancestry and disease-specific autoantibodies (anti-citrullinated protein antibodies [ACPA]), we demonstrate that the odds ratios and aggregate genetic risk score (GRS) of known RA risk alleles measured in individuals of European ancestry within our EHR cohort are nearly identical to those derived from a genome-wide association study (GWAS) of 5,539 autoantibody-positive RA cases and 20,169 controls. We extend this approach to other ethnic groups and identify a large overlap in the GRS among individuals of European, African, East Asian, and Hispanic ancestry. We also demonstrate that the distribution of a GRS based on 28 non-HLA risk alleles in ACPA+ cases partially overlaps with ACPA- subgroup of RA cases. Our study demonstrates that the genetic basis of rheumatoid arthritis risk is similar among cases of diverse ancestry divided into subsets based on ACPA status and emphasizes the utility of linking EHR clinical data with biospecimens for genetic studies.

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. European Journal of Human Genetics (2010) 18, 821-826; doi: 10.1038/ejhg.2010.12; published online 24 February 2010

Contains fulltext : 88498.pdf (Publisher’s version ) (Closed access) 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. 01 juni 2010

BackgroundTwo 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 α-induced protein 3 (TNFAIP3). TNFAIP3 is a negative regulator of NF-κB 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.ObjectiveTo analyse the effect of the 6q23 region on the rate of joint destruction.MethodsFive 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.ResultsTwo 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.ConclusionsThese data associate the 6q23 region with the rate of joint destruction in ACPA+ RA.

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.

Background: Fcγ receptors (FcγRs) are potent immune modulators. FcγR genes encompass a complex region, polymorphic by both single nucleotide polymorphisms (SNPs) and copy number variation (CNV). The heterogeneity of rheumatoid arthritis (RA) combined with the genetic complexity of FcγR genes may be the cause of inconsistent findings in previous RA studies on FcγR SNPs. There is increasing evidence that anti-citrullinated peptide antibody (ACPA)-positive RA and ACPA-negative RA have a different genetic background. Objective: To investigate whether FcγRIIIA 158V/F SNP associates differently with ACPA-positive and ACPA-negative RA and to assess if the FcγRIIIA gene CNV affects the association of the FcγRIIIA 158V/F SNP with RA and whether the FcγRIIIA gene CNV confers risk for RA. Methods: 945 patients with RA and 388 healthy controls, all Dutch-Caucasians, were included in the study. FcγRIIIA 158V/F SNP was genotyped using Sequenom. CNV of the FcγRIIIA gene was determined in 456 patients with RA and 285 controls using multiplex ligation-dependent probe amplification. Associations between genotypes and RA were analysed, stratifying for the presence/absence of ACPA and CNV. Results: In all patients with RA the FcγRIIIA 158V/F SNP was not associated with RA. In ACPA-positive RA (n = 358), the VV genotype was more prevalent in cases than in controls (18.4% vs 13.2%, OR = 1.5, p = 0.05). After stratification for CNV the VV genotype was associated with RA in general (n = 426) (OR = 1.6, 95% CI 0.97 to 2.6, p = 0.05) and with ACPA-positive RA (n = 135) (OR = 2.1, 95% CI 1.2 to 3.8, p = 0.009) but not with ACPA-negative RA. The distribution of CNV was not significantly different between patients with RA and controls. Conclusion: The FcγRIIIA 158 VV genotype confers risk for ACPA-positive RA; this association increased slightly after correction for CNV of the FcγRIIIA gene. CNV itself is not associated with RA susceptibility.

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting both joints and extra-articular tissues. Although some genetic risk factors for RA are well-established, most notably HLA-DRB1 and PTPN22, these markers do not fully account for the observed heritability. To identify additional susceptibility loci, we carried out a multi-tiered, case-control association study, genotyping 25,966 putative functional SNPs in 475 white North American RA patients and 475 matched controls. Significant markers were genotyped in two additional, independent, white case-control sample sets (661 cases/1322 controls from North America and 596 cases/705 controls from The Netherlands) identifying a SNP, rs1953126, on chromosome 9q33.2 that was significantly associated with RA (ORcommon = 1.28, trend Pcomb = 1.45E-06). Through a comprehensive fine-scale-mapping SNP-selection procedure, 137 additional SNPs in a 668 kb region from MEGF9 to STOM on 9q33.2 were chosen for follow-up genotyping in a staged-approach. Significant single marker results (Pcomb, Author Summary Rheumatoid arthritis (RA), a chronic autoimmune disorder affecting ∼1% of the population, is characterized by immune-cell–mediated destruction of the joint architecture. Gene–environment interactions are thought to underlie RA etiology. Variants within HLA-DRB1 and the hematopoietic-specific phosphatase, PTPN22, are well established RA-susceptibility loci, and although other markers have been identified, they do not fully account for the disease heritability. To identify additional susceptibility alleles, we carried out a multi-tiered, case-control association study genotyping >25,000 putative functional SNPs; here we report our finding of RA-associated variants in chromosome 9q33.2. A detailed genetic analysis of this region, incorporating HapMap information, localizes the RA-susceptibility effects to a 70 kb region that includes a portion of PHF19, all of TRAF1, and the majority of the TRAF1-C5 intergenic region, but excludes the C5 coding region. In addition to providing new insights into underlying mechanism(s) of disease and suggesting novel therapeutic targets, these data provide the underpinnings of a genetic signature that may predict individuals at increased risk for developing RA. Indeed, initial analyses of three known genetic risk factors, HLA, PTPN22, and the chromosome 9q33.2 variants described here, suggest a >45-fold difference in RA risk depending on an individual's three-locus genotype.

Background Rheumatoid arthritis (RA) is a chronic autoimmune disorder affecting ∼1% of the population. The disease results from the interplay between an individual's genetic background and unknown environmental triggers. Although human leukocyte antigens (HLAs) account for ∼30% of the heritable risk, the identities of non-HLA genes explaining the remainder of the genetic component are largely unknown. Based on functional data in mice, we hypothesized that the immune-related genes complement component 5 (C5) and/or TNF receptor-associated factor 1 (TRAF1), located on Chromosome 9q33–34, would represent relevant candidate genes for RA. We therefore aimed to investigate whether this locus would play a role in RA. Methods and Findings We performed a multitiered case-control study using 40 single-nucleotide polymorphisms (SNPs) from the TRAF1 and C5 (TRAF1/C5) region in a set of 290 RA patients and 254 unaffected participants (controls) of Dutch origin. Stepwise replication of significant SNPs was performed in three independent sample sets from the Netherlands (n cases/controls = 454/270), Sweden (n cases/controls = 1,500/1,000) and US (n cases/controls = 475/475). We observed a significant association (p < 0.05) of SNPs located in a haplotype block that encompasses a 65 kb region including the 3′ end of C5 as well as TRAF1. A sliding window analysis revealed an association peak at an intergenic region located ∼10 kb from both C5 and TRAF1. This peak, defined by SNP14/rs10818488, was confirmed in a total of 2,719 RA patients and 1,999 controls (odds ratiocommon = 1.28, 95% confidence interval 1.17–1.39, p combined = 1.40 × 10−8) with a population-attributable risk of 6.1%. The A (minor susceptibility) allele of this SNP also significantly correlates with increased disease progression as determined by radiographic damage over time in RA patients (p = 0.008). Conclusions Using a candidate-gene approach we have identified a novel genetic risk factor for RA. Our findings indicate that a polymorphism in the TRAF1/C5 region increases the susceptibility to and severity of RA, possibly by influencing the structure, function, and/or expression levels of TRAF1 and/or C5., Using a candidate-gene approach, Rene Toes and colleagues identified a novel genetic risk factor for rheumatoid arthritis in theTRAF1/C5 region., Editors' Summary Background. Rheumatoid arthritis is a very common chronic illness that affects around 1% of people in developed countries. It is caused by an abnormal immune reaction to various tissues within the body; as well as affecting joints and causing an inflammatory arthritis, it can also affect many other organs of the body. Severe rheumatoid arthritis can be life-threatening, but even mild forms of the disease cause substantial illness and disability. Current treatments aim to give symptomatic relief with the use of simple analgesics, or anti-inflammatory drugs. In addition, most patients are also treated with what are known as disease-modifying agents, which aim to prevent joint damage. Rheumatoid arthritis is known to have a genetic component. For example, an association has been shown with the part of the genome that contains the human leukocyte antigens (HLAs), which are involved in the immune response. Information on other genes involved would be helpful both for understanding the underlying cause of the disease and possibly for the discovery of new treatments. Why Was This Study Done? Previous work in mice that have a disease similar to human rheumatoid arthritis has identified a number of possible candidate genes. One of these genes, complement component 5 (C5) is involved in the complement system—a primitive system within the body that is involved in the defense against foreign molecules. In humans the gene for C5 is located on Chromosome 9 close to another gene involved in the inflammatory response, TNF receptor-associated factor 1 (TRAF1). A preliminary study in humans of this region had shown some evidence, albeit weak, to suggest that this region might be associated with rheumatoid arthritis. The authors set out to look in more detail, and in a larger group of individuals, to see if they could prove this association. What Did the Researchers Do and Find? The researchers took 40 genetic markers, known as single-nucleotide polymorphisms (SNPs), from across the region that included the C5 and TRAF1 genes. SNPs have each been assigned a unique reference number that specifies a point in the human genome, and each is present in alternate forms so can be differentiated. They compared which of the alternate forms were present in 290 patients with rheumatoid arthritis and 254 unaffected participants of Dutch origin. They then repeated the study in three other groups of patients and controls of Dutch, Swedish, and US origin. They found a consistent association with rheumatoid arthritis of one region of 65 kilobases (a small distance in genetic terms) that included one end of the C5 gene as well as the TRAF1 gene. They could refine the area of interest to a piece marked by one particular SNP that lay between the genes. They went on to show that the genetic region in which these genes are located may be involved in the binding of a protein that modifies the transcription of genes, thus providing a possible explanation for the association. Furthermore, they showed that one of the alternate versions of the marker in this region was associated with more aggressive disease. What Do These Findings Mean? The finding of a genetic association is the first step in identifying a genetic component of a disease. The strength of this study is that a novel genetic susceptibility factor for RA has been identified and that the overall result is consistent in four different populations as well as being associated with disease severity. Further work will need to be done to confirm the association in other populations and then to identify the precise genetic change involved. Hopefully this work will lead to new avenues of investigation for therapy. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040278. • Medline Plus, the health information site for patients from the US National Library of Medicine, has a page of resources on rheumatoid arthritis • The UK's National Health Service online information site has information on rheumatoid arthritis • The Arthritis Research Campaign, a UK charity that funds research on all types of arthritis, has a booklet with information for patients on rheumatoid arthritis • Reumafonds, a Dutch arthritis foundation, gives information on rheumatoid arthritis (in Dutch) • Autocure is an initiative whose objective is to transform knowledge obtained from molecular research into a cure for an increasing number of patients suffering from inflammatory rheumatic diseases • The European league against Rheumatism, an organisation which represents the patient, health professionals, and scientific societies of rheumatology of all European nations

Objective. To determine whether genetic variants of the interferon regulatory factor 5 (IRF-5) and Tyk-2 genes are associated with rheumatoid arthritis (RA). Methods. Five single-nucleotide polymorphisms (SNPs) in IRF5 and 3 SNPs in Tyk2 were analyzed in a Swedish cohort of 1,530 patients with RA and 881 controls. A replication study was performed in a Dutch cohort of 387 patients with RA and 181 controls. All patient sera were tested for the presence of autoantibodies against cyclic citrullinated peptides (anti-CCP). Results. Four of the 5 SNPs located in the 5' region of IRF5 were associated with RA, while no association was observed with the Tyk2 SNPs. The minor alleles of 3 of the IRF5 SNPs, which were in linkage disequilibrium and formed a relatively common haplotype with a frequency of ∼0.33, appeared to confer protection against RA. Although these disease associations were seen in the entire patient group, they were mainly found in RA patients who were negative for anti-CCP. A suggestive association of IRF5 SNPs with anti-CCP-negative RA was also observed in the Dutch cohort. Conclusion. Given the fact that anti-CCP-negative RA differs from anti-CCP-positive RA with respect to genetic and environmental risk factor profiles, our results indicate that genetic variants of IRF5 contribute to a unique disease etiology and pathogenesis in anti-CCP-negative RA.

Genetic factors appear to be important in the process of restenosis after percutaneous coronary intervention (PCI), as well as in inflammation, a pivotal factor in restenosis. An important mediator in the inflammatory response is interleukin (IL)-10. Our aim was to study whether genetic variants in IL-10 predispose to the risk of restenosis. The GENetic DEterminants of Restenosis (GENDER) study included 3104 patients treated with successful PCI. Target vessel revascularization (TVR) was chosen as primary end point. Genotyping of the -2849G/ A, -1082G/ A, -592C/A and +4259A/G polymorphisms of the IL-10 gene was performed by MassArray platform. After adjusting for clinical variables, three polymorphisms significantly increased the risk of restenosis (-2849AA: relative risk (RR), 1.7, 95% confidence interval (CI), 1.2-2.5; -1082AA: RR, 1.4, 95% CI, 1.1-1.8 and +4259GG: RR, 2.0, 95% CI, 1.4-2.8). To further exclude possible involvement of neighboring genes due to LD in the IL-10 locus, additional polymorphisms were genotyped. The results reveal that association of the IL-10 gene with restenosis is independent of flanking genes. Our findings demonstrate that IL-10 is associated with restenosis and therefore support the hypothesis that anti-inflammatory genes also may be involved in developing restenosis. Furthermore, they may provide a new targeting gene for drug-eluting stents.

The interleukin (IL)-10 single nucleotide promoter polymorphism (SNP) -2849A is associated with decreased IL-10 production as measured by lipopolysaccharide (LPS) stimulated whole blood cultures. A low innate production of IL-10 using the same assay is associated with an increased risk of familial osteoarthritis (OA). We investigated the association of 7 novel SNP located downstream of the IL-10 transcription start site: -2849,-2763, -1330, -1082, -819, and -592, constituting the 4 ancient haplotypes, with distal interphalangeal (DIP) OA.The study population comprised consecutive patients with and without radiological DIP OA (Kellgren-Lawrence score ofor = 2 in one joint) aged 40-70 years from a cohort of subjects with different types of arthritis in an early stage referred to an Early Arthritis Clinic (EAC). DNA typing for IL-10 SNP as well as radiographs of the hands were performed at clinic enrolment. Patients with rheumatoid arthritis, systemic lupus erythematosus, spondyloarthropathies, and psoriatic arthritis were excluded.The distribution of DIP OA and IL-10 SNP were comparable to representative samples of the Dutch population. In the cohort of 172 subjects, 57 had DIP OA (33%) and 115 (67%) had no DIP OA. No significant association was found between DIP OA and IL-10 SNP and the 4 common haplotypes IL10.1, IL10.2, IL10.3, and IL10.4.Our data suggest that IL-10 SNP, including -2849, which is associated with differential production, do not play a major role in the susceptibility of DIP OA.

Interleukin-10 (IL10) is a cytokine with key regulatory and anti-inflammatory function involved in the pathogenesis of various diseases. Although the large interindividual differences in the production of IL10 have been extensively associated with polymorphisms and haplotypes of the IL10 gene, surprisingly little evidence exists that this variation is actually dictated by IL10 haplotypes. Using the technique of allele-specific transcript quantification, the ratio between two alleles (A and G) of the IL10 gene was characterized in 15 healthy heterozygous individuals. Two groups were identified whereby donors in group 1 exhibited a 1:1 ratio, whereas those in group 2 exhibited a ratio > 1( P < 0.0017). We found that donors heterozygous for haplotype IL10.2 (one of the four ancient IL10 haplotypes) were only prevalent in the group that showed higher allelic expression ratios. In this study we show that IL10 alleles are indeed differentially transcribed in cells from heterozygous individuals and that IL10 haplotypes dictate production of IL10. These findings show that interindividual differences in IL10 protein levels can be explained at the transcriptional level.