Inflammatory genes TNFα and IL6 display no signs of increased H3K4me3 in circulating monocytes from untreated rheumatoid arthritis patients.

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<0.05), ex vivo isolated monocytes displayed similar H3K4me3 levels to healthy controls in the promoter region of TNFα and IL6. Concordantly, mRNA and protein levels of IL6 and TNFα were similar before and after lipopolysaccharide stimulation between patients and controls. Together, with the current number of individuals tested we have not detected enhanced trained immunity signals in circulating monocytes from untreated RA patients, despite increased IL6 and TNFα serum levels.

A novel long non-coding RNA in the rheumatoid arthritis risk locus TRAF1-C5 influences C5 mRNA levels.

Long non-coding RNAs (lncRNAs) can regulate the transcript levels of genes in the same genomic region. These locally acting lncRNAs have been found deregulated in human disease and some have been shown to harbour quantitative trait loci (eQTLs) in autoimmune diseases. However, lncRNAs linked to the transcription of candidate risk genes in loci associated to rheumatoid arthritis (RA) have not yet been identified. The TRAF1 and C5 risk locus shows evidence of multiple eQTLs and transcription of intergenic non-coding sequences. Here, we identified a non-coding transcript (C5T1lncRNA) starting in the 3' untranslated region (UTR) of C5. RA-relevant cell types express C5T1lncRNA and RNA levels are further enhanced by specific immune stimuli. C5T1lncRNA is expressed predominantly in the nucleus and its expression correlates positively with C5 mRNA in various tissues (P=0.001) and in peripheral blood mononuclear cells (P=0.02) indicating transcriptional co-regulation. Knockdown results in a concurrent decrease in C5 mRNA levels but not of other neighbouring genes. Overall, our data show the identification of a novel lncRNA C5T1lncRNA that is fully located in the associated region and influences transcript levels of C5, a gene previously linked to RA pathogenesis.

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

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

Fine mapping the TAGAP risk locus in rheumatoid arthritis.

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 5,500 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 × 10(-8), odds ratio=0.87). This SNP remained highly significant upon conditioning on the previous RA risk variant (rs394581, P=2.2 × 10(-5)) or on a SNP previously associated with celiac disease and type I diabetes (rs1738074, P=1.7 × 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.

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

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

Contribution of Fcgamma receptor IIIA gene 158V/F polymorphism and copy number variation to the risk of ACPA-positive rheumatoid arthritis.

BACKGROUND: Fcgamma receptors (FcgammaRs) are potent immune modulators. FcgammaR 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 FcgammaR genes may be the cause of inconsistent findings in previous RA studies on FcgammaR 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 FcgammaRIIIA 158V/F SNP associates differently with ACPA-positive and ACPA-negative RA and to assess if the FcgammaRIIIA gene CNV affects the association of the FcgammaRIIIA 158V/F SNP with RA and whether the FcgammaRIIIA gene CNV confers risk for RA. METHODS: 945 patients with RA and 388 healthy controls, all Dutch-Caucasians, were included in the study. FcgammaRIIIA 158V/F SNP was genotyped using Sequenom. CNV of the FcgammaRIIIA 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 FcgammaRIIIA 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 FcgammaRIIIA 158 VV genotype confers risk for ACPA-positive RA; this association increased slightly after correction for CNV of the FcgammaRIIIA gene. CNV itself is not associated with RA susceptibility.

IL-10 promotor haplotypes associated with susceptibility to and severity of bacterial corneal ulcers.

Interleukin-10 plays an important role in modulating inflammation and antimicrobial defences. In animal models for bacterial corneal ulcers, high IL-10 levels were associated with a better clinical outcome. We investigated whether IL-10 promotor haplotypes, known to determine IL-10 expression in vitro, are associated with susceptibility to and/or clinical outcome of bacterial corneal ulcers in patients. IL-10 promotor polymorphisms C-819T, G-1082A, A-2763C, and A-2849G for 83 patients with bacterial corneal ulcers and 115 healthy controls were determined by restriction fragment length PCR analysis. For 63 patients and all healthy controls the most frequently occurring IL-10 promotor haplotypes were inferred from these data using the program SNPHAP. A significant underrepresentation of the A-2849A genotype was observed in patients as compared to healthy controls. Both the -2763A allele and the IL-10.1 promotor haplotype were associated with a poor clinical outcome, whereas a favourable clinical outcome was seen in patients carrying the IL-10.2 promotor haplotype. Together, IL-10 promotor haplotypes associated with low IL-10 levels seem to protect against the onset of bacterial corneal ulcers. Once a corneal ulcer has developed, patients carrying IL-10 haplotypes associated with a high IL-10 expression may have a favourable outcome.

Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study.

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.

The TRAF1/C5 region is a risk factor for polyarthritis in juvenile idiopathic arthritis.

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 x 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.

Association of the autoimmunity locus 4q27 with juvenile idiopathic arthritis.

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

Interleukin 10: a new risk marker for the development of restenosis after percutaneous coronary intervention.

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.