Ioanna Marinou

Rheumatoid arthritis association at 6q23

The Wellcome Trust Case Control Consortium (WTCCC) identified nine single SNPs putatively associated with rheumatoid arthritis at P = 1 × 10−5 − 5 × 10−7 in a genome-wide association screen. One, rs6920220, was unequivocally replicated (trend P = 1.1 × 10−8) in a validation study, as described here. This SNP maps to 6q23, between the genes oligodendrocyte lineage transcription factor 3 (OLIG3) and tumor necrosis factor-α–induced protein 3 (TNFAIP3).

Re-evaluation of putative rheumatoid arthritis susceptibility genes in the post-genome wide association study era and hypothesis of a key pathway underlying susceptibility

Rheumatoid arthritis (RA) is an archetypal, common, complex autoimmune disease with both genetic and environmental contributions to disease aetiology. Two novel RA susceptibility loci have been reported from recent genome-wide and candidate gene association studies. We, therefore, investigated the evidence for association of the STAT4 and TRAF1/C5 loci with RA using imputed data from the Wellcome Trust Case Control Consortium (WTCCC). No evidence for association of variants mapping to the TRAF1/C5 gene was detected in the 1860 RA cases and 2930 control samples tested in that study. Variants mapping to the STAT4 gene did show evidence for association (rs7574865, P = 0.04). Given the association of the TRAF1/C5 locus in two previous large case–control series from populations of European descent and the evidence for association of the STAT4 locus in the WTCCC study, single nucleotide polymorphisms mapping to these loci were tested for association with RA in an independent UK series comprising DNA from >3000 cases with disease and >3000 controls and a combined analysis including the WTCCC data was undertaken. We confirm association of the STAT4 and the TRAF1/C5 loci with RA bringing to 5 the number of confirmed susceptibility loci. The effect sizes are less than those reported previously but are likely to be a more accurate reflection of the true effect size given the larger size of the cohort investigated in the current study.

Independent associations of anti-cyclic citrullinated peptide antibodies and rheumatoid factor with radiographic severity of rheumatoid arthritis

Several recent publications have established a strong association between anti-cyclic citrullinated peptide antibody (anti-CCP)-positive rheumatoid arthritis (RA) and carriage of shared epitope (SE) alleles. Although anti-CCP have also been associated with more severe RA, the issue of whether this is independent of rheumatoid factor (RF) has not been addressed. To identify associations between RF, anti-CCP, SE status and radiological damage, we studied a large cross-sectional cohort with longstanding RA. Individuals (n = 872) enrolled in the study all fulfilled the American College of Rheumatology criteria for RA, had a minimum disease duration of 3 years, and at least one definite radiographic erosion was present in hands or feet. Radiographs were scored blind at study entry by a single musculoskeletal radiologist using a modified Larsens score. Anti-CCP and RF levels were determined using enzyme-linked immunosorbent assay, and DRB1 typing was performed using polymerase chain reaction based methodology. Both anti-CCP and RF levels were strongly associated with radiographic severity (P < 0.0001). In subgroups stratified for both anti-CCP and RF status, evidence of independent associations of both antibodies with radiographic outcome was found (P < 0.0001). An association of SE alleles with radiographic severity was present only in RF-negative individuals. Anti-CCP positivity was associated with SE status with evidence of a gene-dose effect, most markedly in RF-negative individuals (P < 0.01). Anti-CCP and RF status are independent severity factors for RA, with SE alleles playing at most a secondary role. Our data support the view that previously described associations between SE and radiological severity, especially in RF-negative patients, may be indirect and due to an association with anti-CCP.

The Mal/TIRAP S180L and TLR4 G299D polymorphisms are not associated with susceptibility to, or severity of, rheumatoid arthritis

BACKGROUND Toll-like receptors (TLRs), including TLR4, have been implicated in the pathogenesis of rheumatoid arthritis (RA). Signalling by these receptors involves interactions with intracellular proteins, including the MyD88 adapter-like (Mal) protein. Recently, a polymorphism (Mal S180L) has been described which contributes to susceptibility to common infectious diseases and inhibits proinflammatory cytokine production. A non-synonymous variant in the extracellular domain of TLR4 (G299D) has been shown to interrupt TLR4-mediated signalling, resulting in endotoxin hyporesponsiveness. OBJECTIVE To investigate the role of TLR4 G299D and Mal S180L variants in RA. METHODS A total of 964 Caucasians with RA and 965 controls were genotyped. Deviation from Hardy-Weinberg equilibrium was tested for each single nucleotide polymorphism in cases and controls separately using a chi(2) test with a threshold of p<0.05. The odd ratios were calculated with asymptotic 95% confidence intervals, and p values <0.05 were considered significant. Epistasis was assessed using both stratified analysis and the linkage disequilibrium-based statistic. RESULTS Mal S180L genotypes were similar in cases and controls (OR = 0.9, 95% CI 0.7 to 1.0, p = 0.2). Similarly, no difference for TLR4 G299D genotypes was seen (OR = 1.7, 95% CI 0.3 to 11.1, p = 0.5). No association with either rheumatoid factor or anti-cyclic citrullinated peptide status or with radiological damage was detected. Finally, no evidence of epistasis was detected between Mal S180L and TLR4 G299D and RA susceptibility. CONCLUSIONS The Mal S180L and TLR4 G299D polymorphisms do not contribute to RA susceptibility or severity either individually or in combination.

The interferon induced with helicase domain 1 A946T polymorphism is not associated with rheumatoid arthritis

An important feature of autoimmune diseases is the overlap of pathophysiological characteristics. Clustering of autoimmune diseases in families suggests that genetic variants may contribute to autoimmunity. The aim of the present study was to investigate the role of the interferon induced with helicase domain 1 (IFIH1) A946T (rs1990760 A>G) variant in rheumatoid arthritis (RA), as this was recently associated with susceptibility to type 1 diabetes. A total of 965 Caucasians with RA and 988 healthy controls were genotyped for IFIH1 A946T. Gene expression of IFIH1 was measured in peripheral blood leukocytes using real-time PCR. Genotypes were equally distributed in both RA cases and healthy controls (odds ratio for allele C = 0.9, 95% confidence interval = 0.8–1.0, P = 0.3). No association was detected after stratification by sex, age at onset, rheumatoid factor status, anti-cyclic citrullinated peptide status or radiological joint damage. Levels of IFIH1 mRNA were approximately twofold higher in blood leucocytes of RA cases compared with healthy controls (P < 0.0001). These results indicate that the IFIH1 is upregulated in RA but that the A946T variant does not contribute significantly to the genetic background of RA.

Association between radiographic severity of rheumatoid arthritis and shared epitope alleles: differing mechanisms of susceptibility and protection

Objective: To investigate the association of a recently described classification of Human leukocyte antigen (HLA)-DRB1 shared epitope alleles with rheumatoid factors (RF) and anti-cyclic citrullinated peptide (CCP) production and radiological severity in rheumatoid arthritis (RA). Methods: Patients with RA (n = 962) were studied. Genotyping of DRB1 alleles and assays for RF and anti-CCP were performed. Radiological severity was measured using the modified Larsen score. Results: In accordance with previous reports, we found carriage of S2 alleles (K-R-A-A at positions 71–74) to be associated with more severe disease with a gene–dose effect (p = 0.0059), and also associated with the presence of anti-CCP and RF (p<0.001). Carriage of S1 alleles (D-E-R-A-A at positions 70–74) was associated with less severe disease (p = 0.01), however there was no association between S1 and either anti-CCP or RF, suggesting that the basis for this possible protective effect was not related to autoantibody-producing B cells. Conclusions: These data suggest that multiple biological mechanisms underlie the DRB1 association with rheumatoid arthritis severity.

Genetic influences modulating the radiological severity of rheumatoid arthritis

This review focuses on the contribution of genetic markers to the severity of radiological damage in rheumatoid arthritis (RA). Currently available biomarkers of more severe disease include elevated erythrocyte sedimentation rates or C-reactive protein levels and rheumatoid factor (RF) or anticyclic citrullinated protein antibodies positivity; however, these biomarkers explain a relatively modest proportion of the variance in radiological damage. An important role of genetic factors on RA severity has recently emerged but studies to date have generally been of low statistical power and many have not been replicated. Genetic markers have a number of advantages over conventional biomarkers; genotypes are stable, measurable at disease onset, remain unchanged by treatment and are amenable to high-throughput assays. The recent advances in genome-wide genetic analysis should lead to a more comprehensive understanding of RA severity genes. This knowledge could be used, along with existing biomarkers, to therapeutically target subjects at risk of poor radiological outcome.

Lack of association or interactions between the IL-4, IL-4Rα and IL-13 genes, and rheumatoid arthritis

IntroductionA feature of rheumatoid arthritis (RA) is an imbalance between proinflammatory and anti-inflammatory cytokines. Several recent studies have implicated polymorphism in the IL-4 signalling pathway in the development of erosive RA. The aim of the present study was to investigate the role of polymorphism in the IL-4, IL-4Rα and IL-13 genes in RA, including an examination of epistasis.MethodsA total of 965 Caucasian patients with RA (cases) and 988 healthy control individuals (controls) were genotyped for five variants in the IL-4/IL-13 gene cluster (5q31.1) and two functional variants IL-4Rα (16p12.1). Individual genotype and haplotype frequencies were compared between cases and controls. The odd ratios were calculated with asymptotic 95% confidence intervals, and P values less than 0.05 were considered statistically significant. The potential association with radiological joint damage was also examined. Potential gene interactions were assessed using both stratified analysis and the linkage disequilibrium-based statistic.ResultsGenotype, allele and haplotype frequencies were equally distributed between RA cases and controls. Similarly, no association was detected between these variants and modified Larsen scores. Furthermore, no evidence of epistasis was detected between IL-4 or IL-13 genotypes and IL-4Rα.ConclusionThese results indicate that common variants of the IL-4/IL-13 pathway do not significantly contribute to RA susceptibility and radiological severity.

Evidence of epistasis between interleukin 1 and selenoprotein-S with susceptibility to rheumatoid arthritis

Objective: Selenoprotein-S (SELS) is involved in the stress response within the endoplasmic reticulum (ER) and inflammation. Recently, promoter variants in the SELS gene were shown to be associated with plasma levels of interleukin (IL)6, IL1β and tumour necrosis factor (TNF). It was hypothesised that these variants could influence rheumatoid arthritis (RA) susceptibility and may interact with functional single nucleotide polymorphisms (SNPs) in the genes for IL1, IL6 and TNF. Methods: Genotyping was performed in 988 unrelated healthy controls and 965 patients with RA. Stratified analysis was used to test for interactions. Single gene effects and evidence of epistasis were investigated using the Mantel–Haenszel (M–H) test and the linkage disequilibrium (LD)-based statistic. Results: No association of SELS −105 genotype and RA susceptibility was detected. Stratification of SELS −105 genotypes by IL1 −511 genotypes showed that the disease risk (comparing AA/GA to GG at the SELS −105 locus) in individuals with the GG/AG genotype at the IL1β −511 locus was significantly lower than that in individuals having the AA genotype at the IL1β −511 locus (odds ratio (OR): 0.9 and 2.3, respectively; p = 0.004 by M–H test). Significant epistasis was also detected using the LD-based statistic (p = <0.001). No interaction was observed between SELS −105 and IL6 or TNF variants. Conclusion: Our results reveal evidence of strong epistasis in two genes in the IL1 production pathway and highlight the potential importance of gene–gene interactions in the pathogenesis of RA.

Investigating the viability of genetic screening/testing for RA susceptibility using combinations of five confirmed risk loci

Objective. Five loci—the shared epitope (SE) of HLA-DRB1, the PTPN22 gene, a locus on 6q23, the STAT4 gene and a locus mapping to the TRAF1/C5 genetic region—have now been unequivocally confirmed as conferring susceptibility to RA. The largest single effect is conferred by SE. We hypothesized that combinations of susceptibility alleles may increase risk over and above that of any individual locus alone. Methods. We analysed data from 4238 RA cases and 1811 controls, for which genotypes were available at all five loci. Results. Statistical analysis identified eight high-risk combinations conferring an odds ratio >6 compared with carriage of no susceptibility variants and, interestingly, 10% population controls carried a combination conferring high risk. All high-risk combinations included SE, and all but one contained PTPN22. Statistical modelling showed that a model containing only these two loci could achieve comparable sensitivity and specificity to a model including all five. Furthermore, replacing SE (which requires full subtyping at the HLA-DRB1 gene) with DRB1*1/4/10 carriage resulted in little further loss of information (correlation coefficient between models = 0.93). Conclusions. This represents the first exploration of the viability of population screening for RA and identifies several high-risk genetic combinations. However, given the population incidence of RA, genetic screening based on these loci alone is neither sufficiently sensitive nor specific at the current time.