Darren Plant

Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment

Background The NLRP3-inflammasome, implicated in the pathogenesis of several inflammatory disorders, has been analysed in rheumatoid arthritis (RA). Methods Relative gene expression of NLRP3-inflammasome components was characterised in PBMCs of 29 patients receiving infliximab. A total of 1278 Caucasian patients with RA from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort receiving tumour necrosis factor (TNF) antagonists (infliximab, adalimumab and etanercept) were genotyped for 34 single nucleotide polymorphisms (SNPs), spanning the genes NLRP3, MEFV and CARD8. Regression analyses were performed to test for association between genotype and susceptibility and treatment response (disease activity score across 28 joints (DAS28) and EULAR improvement criteria) at 6u2005months, with secondary expression quantitative trait loci (eQTL) analyses. Results At baseline, gene expression of ASC, MEFV, NLRP3-FL, NLRP3-SL and CASP1 were significantly higher compared with controls whereas CARD8 was lower in the patients. Caspase-1 and interleukin-18 levels were significantly raised in patients with RA. SNPs in NLRP3 showed association with RA susceptibility and EULAR response to anti-TNF in the BRAGGSS cohort, and in monocytes but not B cells, in eQTL analysis of 283 healthy controls. CARD8 SNPs were associated with RA susceptibility and DAS28 improvement in response to anti-TNF and eQTL effects in monocytes and B cells. Conclusions This study found evidence of modulation of the NLRP3-inflammasome in patients with RA prior to receiving infliximab and some evidence of association for SNPs at NLRP3 and CARD8 loci with RA susceptibility and response to anti-TNF. The SNPs associated with susceptibility/response are not the main eQTL variants for either locus, and the associations with treatment response require replication in an independent cohort.

Genetic markers of rheumatoid arthritis susceptibility in anti-citrullinated peptide antibody negative patients

Introduction There are now over 30 confirmed loci predisposing to rheumatoid arthritis (RA). Studies have been largely undertaken in patients with anticyclic citrullinated peptide (anti-CCP) positive RA, and some genetic associations appear stronger in this subgroup than in anti-CCP negative disease, although few studies have had adequate power to address the question. The authors therefore investigated confirmed RA susceptibility loci in a large cohort of anti-CCP negative RA subjects. Methods RA patients and controls, with serological and genetic data, were available from UK Caucasian patients (n=4068 anti-CCP positive, 2040 anti-CCP negative RA) and 13,009 healthy controls. HLA-DRB1 genotypes and 36 single nucleotide polymorphisms were tested for association between controls and anti-CCP positive or negative RA. Results The shared epitope (SE) showed a strong association with anti-CCP positive and negative RA, although the effect size was significantly lower in the latter (effect size ratio=3.18, p<1.0E-96). A non-intronic marker at TNFAIP3, GIN1/C5orf30, STAT4, ANKRD55/IL6ST, BLK and PTPN22 showed association with RA susceptibility, irrespective of the serological status, the latter three markers remaining significantly associated with anti-CCP negative RA, after correction for multiple testing. No significant association with anti-CCP negative RA was detected for other markers (eg, AFF3, CD28, intronic marker at TNFAIP3), though the study power for those markers was over 80%. Discussion In the largest sample size studied to date, the authors have shown that the strength of association, the effect size and the number of known RA susceptibility loci associated with disease is different in the two disease serotypes, confirming the hypothesis that they might be two genetically different subsets.

Association of HLA-DRB1 Haplotypes With Rheumatoid Arthritis Severity, Mortality, and Treatment Response

IMPORTANCEnAdvances have been made in identifying genetic susceptibility loci for autoimmune diseases, but evidence is needed regarding their association with prognosis and treatment response.nnnOBJECTIVEnTo assess whether specific HLA-DRB1 haplotypes associated with rheumatoid arthritis (RA) susceptibility are also associated with radiological severity, mortality, and response to tumor necrosis factor (TNF) inhibitor drugs.nnnDESIGN, SETTING, AND PARTICIPANTSnThe Norfolk Arthritis Register (NOAR; 1691 patients and 2811 radiographs; recruitment: 1989-2008; 2008 as final follow-up) was used as a discovery cohort and the Early Rheumatoid Arthritis Study (421 patients and 3758 radiographs; recruitment: 1986-1999; 2005 as final follow-up) as an independent replication cohort for studies of radiographic outcome. Mortality studies were performed in the NOAR cohort (2432 patients; recruitment: 1990-2007; 2011 as final follow-up) and studies of treatment response in the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort (1846 patients enrolled at initiation of TNF inhibitor; recruitment: 2006-2010; 2011 as final follow-up). Longitudinal statistical modeling was performed to integrate multiple radiograph records per patient over time. All patients were from the United Kingdom and had self-reported white ancestry.nnnEXPOSURESnSixteen HLA-DRB1 haplotypes defined by amino acids at positions 11, 71, and 74.nnnMAIN OUTCOMES AND MEASURESnRadiological outcome using the Larsen score (range: 0 [none] to 200 [severe joint damage]) and erosions of the hands and feet on radiographs, all-cause mortality, and treatment response measured by change in Disease Activity Score based on 28 joint counts and European League Against Rheumatism (EULAR) response.nnnRESULTSnPatients with RA and valine at position 11 of HLA-DRB1 had the strongest association with radiological damage (OR, 1.75 [95% CI, 1.51-2.05], Pu2009=u20094.6E-13). By year 5, the percentages of patients with erosions of the hands and feet were 48% of noncarriers (150/314) of valine at position 11, 61% of heterozygote carriers (130/213), and 74% of homozygote carriers (43/58). Valine at position 11 also was associated with higher all-cause mortality in patients with inflammatory polyarthritis (hazard ratio, 1.16 [95% CI, 1.03-1.31], Pu2009=u2009.01) (noncarriers: 319 deaths in 1398 patients over 17,196 person-years, mortality rate of 1.9% per year; carriers: 324 deaths in 1116 patients in 13,208 person-years, mortality rate of 2.5% per year) and with better EULAR response to TNF inhibitor therapy (OR, 1.14 [95% CI, 1.01-1.30], Pu2009=u2009.04) (noncarriers: 78% [439/561 patients] with moderate or good EULAR response; heterozygote carriers: 81% [698/866]; and homozygote carriers: 86% [277/322]). The risk hierarchy defined by HLA-DRB1 haplotypes was correlated between disease susceptibility, severity, and mortality, but inversely correlated with TNF inhibitor treatment response.nnnCONCLUSIONS AND RELEVANCEnAmong patients with RA, the HLA-DRB1 locus, which is associated with disease susceptibility, was also associated with radiological severity, mortality, and treatment response. Replication of these findings in other cohorts is needed as a next step in evaluating the role of HLA-DRB1 haplotype analysis for management of RA.

The role of rheumatoid arthritis genetic susceptibility markers in the prediction of erosive disease in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register

Objectives. Recent whole-genome and candidate gene association studies in RA have identified a number of single nucleotide polymorphisms (SNPs) that predispose to disease with moderate risk. It remains poorly understood how recently identified genetic factors may contribute to RA severity. We therefore sought to investigate the role of recently identified RA susceptibility SNP markers in predicting erosive outcome in patients with recent-onset inflammatory polyarthritis (IP). Methods. DNA and X-ray data were available for 1049 patients who were registered between 1990 and 2003 with the Norfolk Arthritis Register (NOAR); a primary care-based inception cohort of patients with recent-onset IP. Demographic and clinical data were recorded at inclusion, and at yearly assessments thereafter. Patients were genotyped for 18 SNP markers. The presence of serum anti citrullinated peptide antibodies (ACPAs) was assessed in samples collected at inclusion to the NOAR. The association of serological and genetic markers with poor radiological (Larsen) score at Years 1 and 5, and erosions at Years 1 and 5 was investigated. Results. Baseline ACPA positivity was associated with erosive disease and higher radiological damage. SNP markers within the TRAF1/C5 locus were associated with erosive disease at Year 1 [rs2900180: odds ratio (OR) 1.53 (95% CI 1.14, 2.05)] and Year 5 [rs2900180: OR 1.47 (95% CI 1.07, 2.02)]. None of the SNP markers tested was associated with Larsen score. Conclusion. Our results are in keeping with a previous report and suggest that the TRAF1/C5 region is associated with risk of development of radiological erosions in IP/RA patients. The finding requires replication in other large data sets.

Clinical utility of random anti-tumor necrosis factor drug-level testing and measurement of antidrug antibodies on the long-term treatment response in rheumatoid arthritis.

To investigate whether antidrug antibodies and/or drug non‐trough levels predict the long‐term treatment response in a large cohort of patients with rheumatoid arthritis (RA) treated with adalimumab or etanercept and to identify factors influencing antidrug antibody and drug levels to optimize future treatment decisions.

Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans

Objectives Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. Methods A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. Results Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, Pu200a=u200a4.4×10−9). Conclusions This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.

A re-evaluation of three putative functional single nucleotide polymorphisms in rheumatoid arthritis

In European rheumatoid arthritis (RA) patients, the strongest genetic effects are conferred by the HLA-DRB1 locus1 and the protein tyrosine phosphatase 22 gene ( PTPN22 ).2 A further three susceptibility loci ( OLIG3 / TNFAIP3 , TRAF1 / C5 and STAT4 ) have been confirmed.3 4 All five loci associate with RA in UK patients.5nnFunctional variants of the Fc receptor-like 3 gene ( FCRL3 ), the solute carrier 22 member 4 gene ( SLC22A4 ) and the major histocompatibility complex, class II, transactivator gene ( MHC2TA ) associate with RA in some but not all populations.nnWe have previously investigated polymorphisms in FCRL3 , MHC2TA and SLC22A4 in moderately sized UK studies and not detected evidence of association.6–8 Here we revisit these putative RA …

Correlation of C-reactive protein haplotypes with serum C-reactive protein level and response to anti-tumor necrosis factor therapy in UK rheumatoid arthritis patients: results from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort

IntroductionIn many European countries, restrictions exist around the prescription of anti-tumor necrosis factor (anti-TNF) treatments for rheumatoid arthritis (RA). Eligibility and response to treatment is assessed by using the disease activity score 28 (DAS28) algorithm, which incorporates one of two inflammatory markers, erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). Although DAS28-CRP provides a more reliable measure of disease activity, functional variants exist within the CRP gene that affect basal CRP production.Therefore, we aimed to determine the relation between functional genetic variants at the CRP gene locus and levels of serum CRP in RA patients, and whether these variants, alone or in combination, are correlated with DAS28-CRP and change in DAS28-CRP after anti-TNF treatment.MethodsDNA samples from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) were genotyped for rs1205, rs1800947, and rs3091244 by using either TaqMan or the Sequenom MassARRAY iPLEX system.Estimated haplotypes were constructed for each sample by using the expectation maximization algorithm implemented in the haplo.stats package within the R statistical program.CRP values were log transformed, and the association between single nucleotide polymorphisms (SNPs), haplotypes of SNPs and baseline CRP, baseline DAS28-CRP, and change in DAS28-CRP were evaluated by using linear regression in STATA v.10.ResultsBaseline CRP measurements were available for 599 samples with 442 also having data 6 months after treatment with an anti-TNF. For these 442 samples, the study had > 80% power to detect a clinically meaningful difference of 0.6 DAS28 Units for an allele frequency of 5%. Estimated haplotype frequencies corresponded with previous frequencies reported in the literature. Overall, no significant association was observed between any of the markers investigated and baseline CRP levels. Further, CRP haplotypes did not correlate with baseline CRP (P = 0.593), baseline DAS28-CRP (P = 0.540), or change in DAS28-CRP after treatment with an anti-TNF over a 6-month period (P = 0.302).ConclusionsAlthough CRP genotype may influence baseline CRP levels, in patients with very active disease, no such association was found. This suggests that genetic variation at the CRP locus does not influence DAS28-CRP, which may continue to be used in determining eligibility for and response to anti-TNF treatment, without adjusting for CRP genotype.

Clinical utility of random anti-tumour necrosis factor drug testing and measurement of anti-drug antibodies on long-term treatment response in rheumatoid arthritis

BACKGROUNDnUp to 40% of patients with rheumatoid arthritis treated with anti-tumour necrosis factor (TNF) drugs do not respond because of primary inefficacy or loss of response. Although one explanation is that immunogenicity leads to the development of anti-drug antibodies and low drug concentrations, the clinical usefulness of pharmacological monitoring is debated. Our aim was to assess whether the presence of anti-drug antibodies and non-trough drug concentrations could predict treatment response in patients with rheumatoid arthritis treated with anti-TNF drugs.nnnMETHODSn331 patients were selected from a multicentre prospective cohort (160 treated with adalimumab, 171 etanercept). Serum samples were collected at 3, 6, and 12 months after treatment initiation. Anti-drug antibodies were measured with RIA, drug concentrations with ELISAs, and Disease Activity Score in 28 joints (DAS28) at each timepoint. Linear and logistic regression, generalised estimating equation (GEE), and receiver operating characteristic curves were used to test the association and predictive value of anti-drug antibodies and non-trough drug concentrations on treatment response (ΔDAS28).nnnFINDINGSn835 serial samples were tested (414 adalimumab, 421 etanercept). Anti-adalimumab antibodies were detected in 31 (24·8%) of 125 patients who had completed 12 month follow-up and none of the etanercept patients. The presence of anti-drug antibodies was associated with lower adalimumab concentrations (Spearman r=-0·66, p=0·0041). At 3 months, anti-drug antibody formation and low adalimumab concentrations were significant predictors of poor treatment response at 12 months (area under curve [AUC] 0·68, 95% CI 0·54-0·81, and 0·66, 0·55-0·77, respectively; and both combined 0·71, 0·57-0·85). Adalimumab concentration was the most significant independent predictor of ΔDAS28 after adjustment for confounders (regression coefficient 0·12, 95% CI 0·06-0·18; p=0·003). High etanercept concentrations were associated with better treatment response (p=0·01), but low concentrations at 3 months were not a significant predictor of poor treatment response at 12 months (AUC 0·58, 95% CI 0·46-0·70). In the combined GEE model including adalimumab and etanercept, a body-mass index of 30 kg/m(2) or more was associated with low drug concentrations (regression coefficient 0·78, 95% CI 0·37-1·18; p<0·0001).nnnINTERPRETATIONnPharmacological testing in anti-TNF initiated patients is clinically useful even in the absence of trough levels. At 3 months, presence of anti-drug antibodies and low adalimumab concentrations are a significant predictor for poor treatment response at 12 months. Strengths of this study include a large, prospective cohort and use of RIA to measure antibodies (less prone to drug interference). Although non-trough concentrations might have underestimated the frequency of antibodies, their presence still predicted response.nnnFUNDINGnMJ is a MRC Clinical Training Fellow supported by the North West England Medical Research Council Fellowship Scheme in Clinical Pharmacology and Therapeutics, which is funded by the UK Medical Research Council (grant number G1000417/94909), ICON, GlaxoSmithKline, AstraZeneca, and the Medical Evaluation Unit. Arthritis Research UK (grant ref 20385).

A genetic marker at the OLIG3/TNFAIP3 locus associates with methotrexate continuation in early inflammatory polyarthritis: results from the Norfolk Arthritis Register

Whole-genome association studies in rheumatoid arthritis have identified single-nucleotide polymorphisms (SNPs) predisposing to disease with moderate risk. We aimed to investigate the role of these markers in predicting methotrexate (MTX) response, measured by continuation on MTX monotherapy in patients with recent onset inflammatory polyarthritis (IP). In all, 19 SNPs were genotyped in 736 patients treated with MTX following registration, or not more than 3 months before registration, to the Norfolk Arthritis Register. The association of SNPs with MTX continuation by year 1 and by year 2 was investigated using Cox proportional hazard regression models. A SNP within the OLIG3/TNFAIP3 locus (rs6920220) was associated with being less likely to maintain MTX monotherapy at year 1, hazards ratio (HR) 1.73 (1.18, 2.52) and year 2, HR 1.49 (1.11, 2.00); correlating with an increased in adverse events. Weak evidence for an effect at the PTPN22 locus was also observed. These findings require replication in other large datasets.