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Background No reliable biomarkers to predict response to TNF inhibitors (TNFi) in RA patients currently exist. The aims of this study were to replicate changes in gene co-expression modules that were previously reported in response to TNFi therapy in RA; to test if changes in module expression are specific to TNFi therapy; and to determine whether module expression transitions towards a disease-free state in responding patients. Method Published transcriptomic data from the whole blood of disease-free controls (n = 10) and RA patients, treated with the TNFi adalimumab (n = 70) or methotrexate (n = 85), were studied. Treatment response was assessed using the EULAR response criteria following 3 or 6 months of treatment. Change in transcript expression between pre- and post-treatment was recorded for previously defined modules. Linear mixed models tested whether modular expression after treatment transitioned towards a disease-free state. Results For 25 of the 27 modules, change in expression between pre- and post-treatment in the adalimumab cohort replicated published findings. Of these 25 modules, six transitioned towards a disease-free state by 3 months (P Conclusion This study provides independent validation of changes in module expression in response to therapy in RA. However, these effects are not specific to TNFi. Further studies are required to determine whether specific modules could assist molecular classification of therapeutic response.

Despite the success of TNF-inhibitor therapy in rheumatoid arthritis treatment, up to 40% of patients fail to respond adequately. This study aimed to identify transcriptome-based biomarkers of adalimumab response in rheumatoid arthritis (RA) to aid timely switching in non-responder patients and provide a better mechanistic understanding of the pathways involved in response/non-response.The Affymetrix Human Transcriptome Array 2.0 (HTA) was used to measure the transcriptome in whole blood at pre-treatment and at 3 months in EULAR good- and non-responders to adalimumab therapy. Differential expression of transcripts was analysed at the transcript level using multiple linear regression. Differentially expressed genes were validated in independent samples using OpenArray™ RT-qPCR.In total, 813 transcripts were differentially expressed between pre-treatment and 3 months in adalimumab good-responders. No significant differential expression was observed between good- and non-responders at either time-point and no significant changes were observed in non-responders between time-points. OpenArray™ RT-qPCR was performed for 104 differentially expressed transcripts in good-responders, selected based on magnitude of effect or p value or based on prior association with RA or the immune system, validating differential expression for 17 transcripts.An early transcriptome signature of DAS28 response to adalimumab has been identified and replicated in independent datasets. Whilst treat-to-target approaches encourage early switching in non-responsive patients, registry evidence suggests that this does not always occur. The results herein could guide the development of a blood test to distinguish responders from non-responders at 3 months and support clinical decisions to switch non-responsive patients to an alternative therapy.

ObjectiveIn these studies, we examined the association of single nucleotide polymorphisms (SNPs) of the IL1RN gene with radiographic severity of symptomatic knee osteoarthritis (SKOA) and the risk of incident OA. We also explored these genetic polymorphisms in patients with new onset rheumatoid arthritis (RA).MethodsOver 1000 subjects who met American College of Rheumatology criteria for tibiofemoral OA were selected from three independent, National Institute of Health (NIH)-funded cohorts. CTA and TTG haplotypes formed from three SNPs of the IL1RN gene (rs419598, rs315952, rs9005) were assessed for association with radiographic severity, and risk for incident radiographic OA (rOA) in a nested case–control cohort. These IL1RN haplotypes were also assessed for association with disease activity (DAS28) and plasma inflammatory markers in patients with RA.ResultsCarriage of the IL1RN TTG haplotype was associated with increased odds of more severe rOA compared with age-matched, sex-matched and body mass index-matched individuals. Examination of the osteoarthritis initiative Incidence Subcohort demonstrated that carriage of the TTG haplotype was associated with 4.1-fold (p=0.001) increased odds of incident rOA. Plasma IL-1Ra levels were lower in TTG carriers, while chondrocytes from TTG carriers exhibited decreased secretion of IL-1Ra. In patients with RA, the TTG haplotype was associated with increased DAS28, decreased plasma IL-1Ra and elevations of plasma inflammatory markers (hsCRP, interleukin 6 (IL-6)).ConclusionCarriage of the IL1RN TTG haplotype is associated with more severe rOA, increased risk for incident OA, and increased evidence of inflammation in RA. These data suggest that the IL1RN TTG risk haplotype, associated with decreased IL-1Ra plasma levels, impairs endogenous ‘anti-inflammatory’ mechanisms.

There is much discussion concerning ‘digital transformation’ in healthcare and the potential of artificial intelligence (AI) in healthcare systems. Yet it remains rare to find AI solutions deployed in routine healthcare settings. This is in part due to the numerous challenges inherent in delivering an AI project in a clinical environment. In this article, several UK healthcare professionals and academics reflect on the challenges they have faced in building AI solutions using routinely collected healthcare data.These personal reflections are summarised as 10 practical tips. In our experience, these are essential considerations for an AI healthcare project to succeed. They are organised into four phases: conceptualisation, data management, AI application and clinical deployment. There is a focus on conceptualisation, reflecting our view that initial set-up is vital to success. We hope that our personal experiences will provide useful insights to others looking to improve patient care through optimal data use.

Background:Tumour necrosis factor inhibitors (TNFi) although effective in the treatment of rheumatoid arthritis (RA), show a variable response rate. Therefore, there is a need to identify treatment response predictors to inform therapy selection in order to practise precision medicine. MicroRNAs (miRNAs) are endogenous, single-stranded, non-coding RNAs that can alter gene expression by regulating messenger RNA translation. There is evidence for miRNA involvement in RA pathogenesis and they may serve as a useful biomarker of treatment response.Objectives:To identify miRNAs associated with response to TNFi in RA.Methods:Biologic naïve patients were selected from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS), a prospective multi-center UK study investigating treatment response biomarkers to TNFi with a primary outcome measure of change in DAS28 scores. Patients were stratified into European League Against Rheumatism (EULAR) good or non-responders based on their 3 or 6-month DAS28-CRP score.Pre-treatment and 3-month post-treatment serum samples were substrates for miRNA profiling, which was conducted by FIRALIS using the HTG EdgeSeq miRNA whole transcriptome V2 targeted sequencing assay. Linear modelling using R package limma compared miRNA expression at (i) pre-treatment and at three-months, in EULAR good-responders and non-responders (ii) longitudinal change in expression from pre-treatment to three-months in EULAR good and non-responders.A literature search was conducted to identify miRNAs associated with RA as a diagnostic and/or treatment response predictor. Data on these miRNAs were extracted from the miRNAs identified in the serum samples. A correction for multiple testing was applied to statistical tests.Results:A total of 54 patients were analysed; of these, 35 (65%) were female, median disease duration [inter-quartile range] was 6 years [2 – 14] (n=51), and 44/51 (86%) patients were on a concomitant disease modifying anti-rheumatic drug. Of the 54 patients, 39 (72%) were classified as EULAR good-responders and 15 (28%) as non-responders. 1880 miRNAs were detected in the serum samples. 64 miRNAs were identified to be associated with RA from the literature, of which, 26 were identified in the serum samples tested.No difference in pre-treatment or three-month miRNA levels was seen comparing EULAR good-responders and non-responders (FDR p). There was a significant differential expression of four miRNAs at 3-months in good-responders compared with pre-treatment levels; miR-125a-3p (downregulated, p-value 0.002), miR-149-3p (upregulated, p-value 0.004), miR-766-3p (downregulated, p-value 0.008), miR-146b-5p (upregulated, p-value 0.006). No significant differences were observed between 3-months and baseline in non-responders.Conclusion:Although no pre-treatment miRNAs were associated with TNFi response, changes in the levels of four miRNAs were detected at 3-months compared to baseline in EULAR good-responders. Future work involves validation of these samples in a larger patient cohort and analysing miRNA levels at 6 and 12 months. Replication and validation of these results in larger studies are required to analyse the role of miRNAs in stratifying EULAR good-responders from non-responders at three-months, and as treatment response predictors to TNFi in RA.Acknowledgements:Joint last-author: Dr. Darren PlantDisclosure of Interests:Trixy David: None declared, Nisha Nair: None declared, James Oliver: None declared, Eric Schordan: None declared, Hüseyin Firat: None declared, Kimme Hyrich Consultant of: consultancy/honoraria from AbbVie, Grant/research support from: Pfizer, UCB, BMS, Ann Morgan: None declared, Anthony G Wilson: None declared, John D Isaacs Speakers bureau: consultancy/speaker fees from AbbVie, Gilead, Roche, UCB, Consultant of: consultancy/speaker fees from AbbVie, Gilead, Roche, UCB, Grant/research support from: Pfizer, Darren Plant: None declared, Anne Barton: None declared

Background Chronic illnesses, such as Rheumatoid Arthritis (RA), are a growing burden on health care systems worldwide. Self-management emphasises the patient’s central role in managing their illness. This is pertinent given the majority of care is provided by the individual themselves; yet how individuals make sense of self-management in everyday life is largely unseen. Objective The purpose of this study was to capture the strengths and concerns of individuals with RA in self-managing their illness, raise awareness of their lived experience and spark a dialogue among stakeholders. Methods A community-based participatory approach, Photovoice, was adopted. A purposive sample of participants were tasked with taking photographs to represent the challenges and solutions to living with RA. Group workshops and semi-structured interviews were conducted to facilitate reflection, dialogue and analysis. Data analysis followed Braun and Clarke’s thematic analysis. Public exhibitions were held throughout the Autumn of 2019. Results Eight women and three men (n = 11) across suburban and urban regions of Ireland were recruited (mean age 57 years, disease duration 4–21 years). Participants identified four main themes which reflected the lived experience of self-managing RA: (i) I’m Here but I’m Not, (ii) Visible Illness, (iii) Medicine in All its Forms, (iv) Mind Yourself. These themes captured the challenge of reduced agency, limited contribution and participation, and a complex relationship between visible and invisible illness. Solutions focused on improving psychological and emotional resilience, particularly through personal reflection and increased agency. Conclusions Our findings suggest that RA is experienced as a fluid relationship between states of masking and surfacing of illness shaped by contextual and situational factors. Photovoice was a highly effective tool to capture and communicate this complexity. Supporting increased agency among individuals with RA to control the (in)visibility of illness and disability can inform the development of future self-management support.

ObjectivesIdentifying blood-based biomarkers that predict treatment response in RA is a clinical priority. We investigated differential DNA methylation as a candidate biomarker of response for the first-line drug used in RA, MTX.MethodsDNA methylation was measured in DNA samples from individuals recruited to the Rheumatoid Arthritis Medication Study. Differentially methylated positions were compared between whole blood samples collected at baseline and at 4 weeks from patients who, by 6 months, had a good (n = 34) or poor response (n = 34) to MTX using linear modelling, adjusting for gender, age, cell composition, baseline 28-joint disease activity score (DAS28) and smoking status. Analyses also compared methylation with changes in DAS28 and changes in swollen joint count and tender joint count, and changes in CRP over the initial 6 months after MTX commencement. Differentially methylated positions showing significant differences with any response parameter were tested using pyrosequencing in an independent group of 100 patients from the Rheumatoid Arthritis Medication Study.ResultsIn the discovery group, two CpG sites showed methylation changes at 4 weeks associated with clinical EULAR response by 6 months. Significant changes in methylation for three differentially methylated positions associated with change in tender joint counts, three with change in swollen joint count and a further four with change in CRP. Of the 12 CpGs, four showed replicated association in an independent dataset of samples from the Rheumatoid Arthritis Medication Study.ConclusionThese data represent an advance on current practice by contributing to a personalized medicine strategy allowing an escalation or change in therapy as early as 4 weeks.

rs26232, located in intron one of C5orf30, is associated with the susceptibility to and severity of rheumatoid arthritis (RA). Here, we investigate the relationship between this variant and the biological activities of rheumatoid arthritis synovial fibroblasts (RASFs). RASFs were isolated from the knee joints of 33 RA patients. The rs26232 genotype was determined and cellular migration, invasion, and apoptosis were compared using in vitro techniques. The production of adhesion molecules, chemokines, and proteases was measured by ELISA or flow cytometry. Cohort genotypes were CC n = 16, CT n = 14, TT n = 3. In comparison with the RASFs of the CT genotype, the CC genotype showed a 1.48-fold greater invasiveness in vitro (p = 0.02), 1.6-fold higher expression intracellular adhesion molecule (ICAM)-1 (p = 0.001), and 5-fold IFN-&gamma, inducible protein-10 (IP-10) (p = 0.01). There was no association of the rs26232 genotype with the expression levels of either total C5orf30 mRNA or any of the three transcript variants. The rs26232 C allele, which has previously been associated with both the risk and severity of RA, is associated with greater invasive activity of RASFs in vitro, and with higher expression of ICAM-1 and IP-10. In resting RASFs, rs26232 is not a quantitative trait locus for C5orf30 mRNA, indicating a more complex mechanism underlying the genotype‒phenotype relationship.

ObjectivesWe sought to investigate whether genetic effects on response to TNF inhibitors (TNFi) in rheumatoid arthritis (RA) could be localised by considering known genetic susceptibility loci for relevant traits and to evaluate the usefulness of these genetic loci for stratifying drug response.MethodsWe studied the relation of TNFi response, quantified by change in swollen joint counts ( Δ SJC) and erythrocyte sedimentation rate ( Δ ESR) with locus-specific scores constructed from genome-wide assocation study summary statistics in 2938 genotyped individuals: 37 scores for RA; scores for 19 immune cell traits; scores for expression or methylation of 93 genes with previously reported associations between transcript level and drug response. Multivariate associations were evaluated in penalised regression models by cross-validation.ResultsWe detected a statistically significant association between Δ SJC and the RA score at the CD40 locus (p=0.0004) and an inverse association between Δ SJC and the score for expression of CD39 on CD4 T cells (p=0.00005). A previously reported association between CD39 expression on regulatory T cells and response to methotrexate was in the opposite direction. In stratified analysis by concomitant methotrexate treatment, the inverse association was stronger in the combination therapy group and dissipated in the TNFi monotherapy group. Overall, ability to predict TNFi response from genotypic scores was limited, with models explaining less than 1% of phenotypic variance.ConclusionsThe association with the CD39 trait is difficult to interpret because patients with RA are often prescribed TNFi after failing to respond to methotrexate. The CD39 and CD40 pathways could be relevant for targeting drug therapy.

Involving patients in research broadens a researcher’s field of influence and may generate novel ideas. Preclinical research is integral to the progression of innovative healthcare. These are not patient-facing disciplines and implementing meaningful public and patient involvement (PPI) can be a challenge. A discussion forum and thematic analysis identified key challenges of implementing public and patient involvement for preclinical researchers. In response we developed a “PPI Ready” planning canvas. For contemporaneous evaluation of public and patient involvement, a psychometric questionnaire and an open source tool for its evaluation were developed. The questionnaire measures information, procedural and quality assessment. Combined with the open source evaluation tool, researchers are notified if public and patient involvement is unsatisfactory in any of these areas. The tool is easy to use and adapts a psychometric test into a format familiar to preclinical scientists. Designed to be used iteratively across a research project, it provides a simple reporting grade to document satisfaction trend over the research lifecycle.

At the population level, rheumatoid arthritis (RA) is generally viewed as autoimmune in nature with a small subgroup of cases having a palindromic form or systemic autoinflammatory disorder (SAID) phenotype. Herein, we describe resistant cases of classical autoantibody associated RA that had clinical, genetic and therapeutic responses indicative of coexistent autoinflammatory disease. Five patients with clinically overlapping features between RA and SAID including polysynovitis and autoantibody/shared epitope positivity, and who had abrupt severe self-limiting attacks including fevers and serositis, are described. Mutations or single nucleotide polymorphisms in recognised autoinflammatory pathways were evident. Generally, these cases responded poorly to conventional Disease-modifying anti-rheumatic drugs (DMARD) treatment with some excellent responses to colchicine or interleukin 1 pathway blockade. A subgroup of RA cases have a mixed autoimmune-autoinflammatory phenotype and genotype with therapeutic implications.

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 6 months, 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.

Rheumatoid arthritis (RA) is a complex disease affecting approximately 0.5–1% of the population. While there are effective biologic therapies, in up to 40% of patients, disease activity remains inadequately controlled. Therefore, identifying factors that predict, prior to the initiation of therapy, which patients are likely to respond best to which treatment is a research priority and DNA methylation is increasingly being explored as a potential theranostic biomarker. DNA methylation is thought to play a role in RA disease pathogenesis and in mediating the relationship between genetic variants and patient outcomes. The role of DNA methylation has been most extensively explored in cancer medicine, where it has been shown to be predictive of treatment response. Studies in RA, however, are in their infancy and, while showing promise, further investigation in well-powered studies is warranted.

Background Polymorphisms of the peptidylarginine deiminase type 4 ( PADI4 ) gene confer susceptibility to rheumatoid arthritis (RA) in East Asian people. However, studies in European populations have produced conflicting results. This study explored the association of the PADI4 genotype with RA in a large UK Caucasian population. Methods The PADI4_94 (rs2240340) single nucleotide polymorphism (SNP) was directly genotyped in a cohort of unrelated UK Caucasian patients with RA (n=3732) and population controls (n=3039). Imputed data from the Wellcome Trust Case Control Consortium (WTCCC) was used to investigate the association of PADI4_94 with RA in an independent group of RA cases (n=1859) and controls (n=10 599). A further 56 SNPs spanning the PADI4 gene were investigated for association with RA using data from the WTCCC study. Results The PADI4_94 genotype was not associated with RA in either the present cohort or the WTCCC cohort. Combined analysis of all the cases of RA (n=5591) and controls (n=13 638) gave an overall OR of 1.01 (95% CI 0.96 to 1.05, p=0.72). No association with anti-CCP antibodies and no interaction with either shared epitope or PTPN22 was detected. No evidence for association with RA was identified for any of the PADI4 SNPs investigated. Meta-analysis of previously published studies and our data confirmed no significant association between the PADI4_94 genotype and RA in people of European descent (OR 1.06, 95% CI 0.99 to 1.13, p=0.12). Conclusion In the largest study performed to date, the PADI4 genotype was not a significant risk factor for RA in people of European ancestry, in contrast to Asian populations.

OBJECTIVE: To define interactions between the HLA-DRB1 shared epitope (SE), PTPN22, and smoking in cyclic citrullinated peptide (CCP) antibody- and rheumatoid factor (RF)-positive and -negative rheumatoid arthritis (RA). METHODS: Data on approximately 5,000 RA patients and approximately 3,700 healthy controls recruited from 6 centers in the UK were analyzed; not all centers had both genotype data and smoking data available for study. The magnitude of association was assessed in autoantibody-positive and -negative subgroups. The effect of smoking on antibody status among cases was assessed following adjustment for year of birth and center, using Mantel-Haenszel analysis. Analyses of the combined effects of PTPN22, HLA-DRB1 SE, and smoking were performed using additive and multiplicative models of interaction within a logistic regression framework. RESULTS: The combined effects of PTPN22, HLA-DRB1 SE, and smoking were defined, with no evidence of departure from a multiplicative model. Within the case population, all 3 factors were independently associated with the generation of CCP antibodies (odds ratio [OR] 11.1, P < 0.0001), whereas only HLA-DRB1 SE and smoking were independently associated with RF production (OR 4.4, P < 0.0001). There was some evidence of increasing likelihood of antibody positivity with heavier smoking. Finally, we demonstrated that smoking was associated with the generation of both CCP and RF antibodies (OR 1.7, P = 0.0001). CONCLUSION: PTPN22 appears to be primarily associated with anticitrulline autoimmunity, whereas HLA-DRB1 SE is independently associated with RF. This study has confirmed associations of specific gene-environment combinations with a substantially increased risk of developing RA. Further work is needed to determine how these data can be used to inform clinical practice.

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Background Interleukin (IL)-15 levels are increased in serum, synovium and bone marrow of patients with rheumatoid arthritis (RA). IL-15 influences both the innate and the adaptive immune response; its major role is activation and proliferation of T cells. There are also emerging data that IL-15 affects osteoclastogenesis. The authors investigated the association of genetic variants in IL15 with the rate of joint destruction in RA.Method 1418 patients with 4885 x-ray sets of both hands and feet of four independent data sets were studied. First, explorative analyses were performed on 600 patients with early RA enrolled in the Leiden Early Arthritis Clinic. Twenty-five single-nucleotide polymorphisms (SNPs) tagging IL-15 were tested. Second, SNPs with significant associations in the explorative phase were genotyped in data sets from Groningen, Sheffield and Lund. In each data set, the relative increase of the progression rate per year in the presence of a genotype was assessed. Subsequently, data were summarised in an inverse weighting meta-analysis.Results Five SNPs were significantly associated with rate of joint destruction in phase 1 and typed in the other data sets. Patients homozygous for rs7667746, rs7665842, rs2322182, rs6821171 and rs4371699 had respectively 0.94-, 1.04-, 1.09-, 1.09- and 1.09- fold rate of joint destruction compared to other patients (p = 4.0x10(-6), p = 3.8x10(-4), p = 5.0x10(-3), p = 5.0x10(-3) and p = 9.4x10(-3)).Discussion Independent replication was not obtained, possibly due to insufficient power. Meta-analyses of all data sets combined resulted in significant results for four SNPs (rs7667746, p Conclusion Genetic variants in IL-15 are associated with progression of joint destruction in RA.

Background Progression of joint destruction in rheumatoid arthritis (RA) is partly heritable; knowledge of genetic factors may increase our understanding of the mechanisms underlying joint destruction. The activity of the Wnt/beta-catenin pathway influences osteoblast differentiation. Dickkopf-1 (Dkk-1) and sclerostin (Sost) are negative regulators and lipoprotein receptor-related protein-5 (LRP-5) and Kremen-1 are transmembrane receptors involved in this pathway.Objective To study variants in the genes encoding these proteins in relation to progression of joint destruction.Methods 1418 patients with RA of four cohorts with 4885 sets of hands and feet x-rays were studied. Explorative analyses were performed on 600 patients with RA from Leiden on single nucleotide polymorphisms (SNPs) tagging Dkk-1, Sost, Kremen-1 and LRP-5. SNPs significantly associating with joint damage progression were subsequently genotyped in cohorts from Groningen (NL), Sheffield (UK) and Lund (Sweden). Data were summarised in meta-analyses. Serum levels of functional Dkk-1 and sclerostin were measured and studied in relation to genotypes.Results In the first cohort, six Dkk-1, three Sost, one Kremen-1 and 10 LRP-5 SNPs were significantly associated with radiological progression of joint destruction. Three Dkk-1 SNPs were associated significantly with progression of joint damage in the meta-analysis, also after correction for multiple testing (rs1896368, rs1896367 and rs1528873). Two Sost SNPs tended to significance (rs4792909 and rs6503475, p=0.07 after false discovery rate correction). Gene-gene interactions between SNPs on Dkk-1 and Sost were seen. Serum levels of Dkk-1 were significantly correlated with the genotypes in rs1896368 (p=0.02).Conclusions Patients with RA carrying risk alleles of genetic variants in Dkk-1 have higher serum levels of functional Dkk-1 and more progressive joint destruction over time.

Objective. Non-adherence to DMARDs is common, but little is known about adherence to biologic therapies and its relationship to treatment response. The purpose of this study was to investigate the association between self-reported non-adherence to s.c. anti-TNF therapy and response in individuals with RA. Methods. Participants about to start s.c. anti-TNF therapy were recruited to a large UK multicentre prospective observational cohort study. Demographic information and disease characteristics were assessed at baseline. Self-reported non-adherence, defined as whether the previous due dose of biologic therapy was reported as not taken on the day agreed with the health care professional, was recorded at 3 and 6 months following the start of therapy. The 28-joint DAS (DAS28) was recorded at baseline and following 3 and 6 months of therapy. Multivariate linear regression was used to examine these relationships. Results. Three hundred and ninety-two patients with a median disease duration of 7 years [interquartile range (IQR) 3–15] were recruited. Adherence data were available in 286 patients. Of these, 27% reported non-adherence to biologic therapy according to the defined criteria at least once within the first 6-month period. In multivariate linear regression analysis, older age, lower baseline DAS28 and ever non-adherence at either 3 or 6 months from baseline were significantly associated with a poorer DAS28 response at 6 months to anti-TNF therapy. Conclusion. Patients with RA who reported not taking their biologic on the day agreed with their health care professional showed poorer clinical outcomes than their counterparts, emphasizing the need to investigate causes of non-adherence to biologics.

Genetic case-control association studies often include data on clinical covariates, such as body mass index (BMI), smoking status, or age, that may modify the underlying genetic risk of case or control samples. For example, in type 2 diabetes, odds ratios for established variants estimated from low–BMI cases are larger than those estimated from high–BMI cases. An unanswered question is how to use this information to maximize statistical power in case-control studies that ascertain individuals on the basis of phenotype (case-control ascertainment) or phenotype and clinical covariates (case-control-covariate ascertainment). While current approaches improve power in studies with random ascertainment, they often lose power under case-control ascertainment and fail to capture available power increases under case-control-covariate ascertainment. We show that an informed conditioning approach, based on the liability threshold model with parameters informed by external epidemiological information, fully accounts for disease prevalence and non-random ascertainment of phenotype as well as covariates and provides a substantial increase in power while maintaining a properly controlled false-positive rate. Our method outperforms standard case-control association tests with or without covariates, tests of gene x covariate interaction, and previously proposed tests for dealing with covariates in ascertained data, with especially large improvements in the case of case-control-covariate ascertainment. We investigate empirical case-control studies of type 2 diabetes, prostate cancer, lung cancer, breast cancer, rheumatoid arthritis, age-related macular degeneration, and end-stage kidney disease over a total of 89,726 samples. In these datasets, informed conditioning outperforms logistic regression for 115 of the 157 known associated variants investigated (P-value = 1×10−9). The improvement varied across diseases with a 16% median increase in χ2 test statistics and a commensurate increase in power. This suggests that applying our method to existing and future association studies of these diseases may identify novel disease loci., Author Summary This work describes a new methodology for analyzing genome-wide case-control association studies of diseases with strong correlations to clinical covariates, such as age in prostate cancer and body mass index in type 2 diabetes. Currently, researchers either ignore these clinical covariates or apply approaches that ignore the disease's prevalence and the study's ascertainment strategy. We take an alternative approach, leveraging external prevalence information from the epidemiological literature and constructing a statistic based on the classic liability threshold model of disease. Our approach not only improves the power of studies that ascertain individuals randomly or based on the disease phenotype, but also improves the power of studies that ascertain individuals based on both the disease phenotype and clinical covariates. We apply our statistic to seven datasets over six different diseases and a variety of clinical covariates. We found that there was a substantial improvement in test statistics relative to current approaches at known associated variants. This suggests that novel loci may be identified by applying our method to existing and future association studies of these diseases.

The concept of susceptibility genes common to different autoimmune diseases is now firmly established with previous studies demonstrating overlap of loci conferring susceptibility to type 1 diabetes (T1D) with both Coeliac disease and multiple sclerosis. Rheumatoid arthritis (RA) is an archetypal autoimmune disease and we, therefore, targeted putative T1D susceptibility loci for genotyping in UK RA cases and unrelated controls. A novel RA susceptibility locus at AFF3 was identified with convincing evidence for association in a combined sample cohort of 6819 RA cases and 12 650 controls [OR 1.12 95% confidence intervals (CI) 1.07-1.17, P = 2.8 x 10(-7)]. Association of two previously described loci (CTLA-4 and 4q27) with RA was also replicated (OR 0.87, 95% CI 0.82-0.94, P = 1.1 x 10(-4) and OR 0.86, 95% CI 0.79-0.94, P = 5.4 x 10(-4), respectively). These findings take the number of established RA susceptibility loci to 13, only one of which has not been associated with other autoimmune diseases.

Genome-wide association studies (GWAS) have heralded a major breakthrough in the search for genes underlying rheumatoid arthritis (RA). Indeed, GWAS in UK, US and Swedish populations have identified a number of confirmed RA loci.1 Recently, a modestly-sized GWAS was undertaken in a Spanish population comprising 400 patients with RA and 400 controls.2 Association with the KLF12 gene was detected (p=6.5×10−6) and subsequently replicated in an independent cohort of 410 Spanish patients with RA and 394 controls (p=0.01). The most strongly associated single nucleotide polymorphism (SNP) was rs1324913 mapping to intron 1 of the KLF12 gene. The KLF12 protein is a repressor for a transcriptional regulatory factor, AP-2α, which has been implicated in the control of inflammation. Other SNPs at this locus also showed weak evidence for association with RA in UK …

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

Objective A recent meta-analysis of published genome-wide association studies (GWAS) in populations of European descent reported novel associations of markers mapping to the CD40, CCL21 and CDK6 genes with rheumatoid arthritis (RA) susceptibility while a large-scale, case-control association study in a Japanese population identified association with multiple single nucleotide polymorphisms (SNPs) in the CD244 gene. The aim of the current study was to validate these potential RA susceptibility markers in a UK population. Methods A total of 4 SNPs (rs4810485 in CD40, rs2812378 in CCL21, rs42041 in CDK6 and rs6682654 in CD244) were genotyped in a UK cohort comprising 3962 UK patients with RA and 3531 healthy controls using the Sequenom iPlex platform. Genotype counts in patients and controls were analysed with the chi(2) test using Stata. Results Association to the CD40 gene was robustly replicated (p=2 x 10(-4), OR 0.86, 95% CI 0.79 to 0.93) and modest evidence was found for association with the CCL21 locus (p=0.04, OR 1.08, 95% CI 1.01 to 1.16). However, there was no evidence for association of rs42041 (CDK6) and rs6682654 (CD244) with RA susceptibility in this UK population. Following a meta-analysis including the original data, association to CD40 was confirmed (p=7.8 x 10(-8), OR 0.87 (95% CI 0.83 to 0.92). Conclusion In this large UK cohort, strong association of the CD40 gene with susceptibility to RA was found, and weaker evidence for association with RA in the CCL21 locus.

INTRODUCTION: Genome wide association studies, replicated by numerous well powered validation studies, have revealed a large number of loci likely to play a role in susceptibility to many multifactorial diseases. It is now well established that some of these loci are shared between diseases with similar aetiology. For example, a number of autoimmune diseases have been associated with variants in the PTPN22, TNFAIP3 and CTLA4 genes. Here we have attempted to define overlapping genetic variants between rheumatoid arthritis (RA), type 1 diabetes (T1D) and coeliac disease (CeD). METHODS: We selected eight SNPs previously identified as being associated with CeD and six T1D-associated SNPs for validation in a sample of 3,962 RA patients and 3,531 controls. Genotyping was performed using the Sequenom MassArray platform and comparison of genotype and allele frequencies between cases and controls was undertaken. A trend test P-value < 0.004 was regarded as significant. RESULTS: We found statistically significant evidence for association of the TAGAP locus with RA (P = 5.0 × 10-4). A marker at one other locus, C1QTNF6, previously associated with T1D, showed nominal association with RA in the current study but did not remain statistically significant at the corrected threshold. CONCLUSIONS: In exploring the overlap between T1D, CeD and RA, there is strong evidence that variation within the TAGAP gene is associated with all three autoimmune diseases. Interestingly a number of loci appear to be specific to one of the three diseases currently studied suggesting that they may play a role in determining the particular autoimmune phenotype at presentation.

Genome-wide association studies (GWAS) have contributed to the identification of at least 14 rheumatoid arthritis (RA) susceptibility loci.1 One of the first RA GWAS included 1522 cases and 1850 controls from the USA/Sweden and identified TRAF1/C5 as a novel RA locus.2 This GWAS was recently repeated after including an additional 1550 cases and 3310 controls from the USA and restricting analysis to US subjects.3 In the expanded sample, two novel single nucleotide polymorphisms (SNP) mapping to the REL locus showed association with RA. REL encodes c-Rel, a member of the nuclear factor kappa B family of transcription factors and one of the associated SNP (rs13031237) maps to an intron of this gene. The association was validated in an independent sample of 2604 RA cases and 2882 controls from the USA/Canada, with strong evidence for association in the combined samples (rs13031237, p=3.1×10−14). We aimed to test the …

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.5 Functional 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. We 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 …

Objective:To determine whether rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibodies, or carriage of shared epitope (SE) and PTPN22 genetic susceptibility variants predict response to therapy in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor (TNF) agents.Methods:UK-wide multicentre collaborations were established to recruit a large cohort of patients treated with anti-TNF drugs for RA. Serum RF, anti-CCP antibody and SE status were determined using commercially available kits. PTPN22 R620W genotyping was performed by Sequenom MassArray. Linear regression analyses were performed to investigate the role of these four factors in predicting response to treatment by 6 months, defined as the absolute change in 28-joint Disease Activity Score (DAS28).Results:Of the 642 patients analysed, 46% received infliximab, 43% etanercept and 11% adalimumab. In all, 89% and 82% of patients were RF and anti-CCP positive, respectively. Patients that were RF negative had a 0.48 (95% CI 0.08 to 0.87) greater mean improvement in DAS28 compared to patients that were RF positive. A better response was also seen among patients that were anti-CCP negative. No association was demonstrated between drug response and SE or PTPN22 620W carriage.Conclusion:The presence of RF or anti-CCP antibodies was associated with a reduced response to anti-TNF drugs. However, these antibodies only account for a small proportion of the variance in treatment response. It is likely that genetic factors will contribute to treatment response, but these do not include the well established RA susceptibility loci, SE and PTPN22.

OBJECTIVE--To determine whether a polymorphism within the tumour necrosis factor alpha (TNF alpha) gene is associated with susceptibility to, or severity of, rheumatoid arthritis (RA). METHODS--Consecutive patients with recent onset RA were enrolled in a prospective trial. DNA was collected, disease activity was measured at presentation, and radiographic progression at three years was assessed. Typing of TNF alpha was by polymerase chain reaction and single stranded conformation polymorphism analysis. RESULTS--No association of TNF alpha alleles and susceptibility to, or severity of, RA was demonstrated. CONCLUSIONS--These results indicate that this TNF alpha polymorphism does not play a part in the genetic background of RA.

Tumor necrosis factor alpha (TNF alpha) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNF alpha are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at -308 in the TNF alpha promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNF alpha production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at -308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNF alpha gene regulation and may be responsible for the association of TNF2 with high TNF alpha phenotype and more severe disease in infections such as malaria and leishmaniasis.

The tumor necrosis factor (TNF) alpha gene lies within the class III region of the major histocompatibility complex (MHC), telomeric to the class II and centromeric to the class I region. We have recently described the first polymorphism within the human TNF-alpha locus. This is biallelic and lies within the promoter region. Frequency analysis of the TNF-alpha polymorphism, using the polymerase chain reaction and single-stranded conformational polymorphism, in HLA-typed individuals, reveals a very strong association between the uncommon TNF allele and HLA A1, B8, and DR3 alleles. This is the first association between TNF-alpha and other MHC alleles and raises the possibility that the uncommon TNF-alpha allele may contribute to the many autoimmune associations of the A1,B8,DR3 haplotype.

Summary Objective An association between osteoarthritis (OA) and functional polymorphisms in the aspartic acid (d) repeat of the asporin ( ASPN ) gene was reported in Japanese and Han Chinese populations. The aim of this study was to assess the association of variants in the ASPN gene with the presence of radiographic hand and/or knee OA in a US Caucasian population. Methods Ten single nucleotide polymorphisms (SNPs) within the ASPN gene were genotyped in 775 affected siblings with radiographically confirmed hand and/or knee OA, and the allelic, genotypic and haplotypic association results were examined. Results One variant (SNP RS7033979) showed nominal evidence of association with both hand OA ( P =0.042) and knee OA ( P =0.032). Four additional SNPs showed nominal evidence of association with knee OA only. These associations were only observed with genotypic tests; the corresponding allelic and haplotype tests did not corroborate the single-point association results. Conclusion These data suggest that polymorphisms within ASPN are not a major influence in susceptibility to hand or knee OA in US Caucasians.

PURPOSE: The primary goal of the Genetics of Generalized Osteoarthritis (GOGO) study is to identify chromosomal regions associated with increased susceptibility to generalized osteoarthritis (OA). Here we describe the study design and phenotype of the 2728 participants from the 1145 families recruited for this study. METHODS: GOGO is an investigator-initiated collaboration involving seven clinical academic sites and sponsored by GlaxoSmithKline. Family ascertainment was carried out between 1999 and 2002. A qualifying family required self-reported Caucasian ethnicity and at least two affected siblings with clinical hand OA. We hypothesized that this clinical phenotype would facilitate identification of participants with multijoint radiographic OA (rOA) in and beyond the hand. The "gold standard" case definition, however, was based on rOA (Kellgren-Lawrence grade > or =2) involving > or =3 hand joints distributed bilaterally and including at least one distal interphalangeal joint, with two of the three involved joints within a joint group (distal interphalangeal, proximal interphalangeal, or carpometacarpal). Radiographs of hips, knees and spine were also obtained. Additional siblings and living parents from qualifying families, both affected and unaffected, were invited to participate. RESULTS: A total of 2706 participants had complete clinical and radiological examination data. Of these, 2569 participants met clinical examination criteria for affected status; while 1963 (73%) participants met the prespecified radiographic criteria for affected status. This corresponded to a total of 707 families with at least two affected siblings that met the hand rOA criteria. Of those individuals with rOA of the hand, the frequency of rOA at other sites was highest for the knee (51%) and spine (54%), and less common for the hip (25%). Concordance rates among hand affected siblings were greatest for spine (36%) followed by knee (31%) and hip (9%); a total of 53% of the affected sib pairs were concordant for specific patterns of generalized rOA involving the hand and large joints (knees, hips or spine). CONCLUSIONS: GOGO represents a large multicenter collection of families with multiple joint OA that have been characterized both clinically and radiographically. The GOGO study will employ a comprehensive strategy for genetic screening based upon both qualitative and quantitative radiographic trait analyses, circulating biomarkers in a quantitative trait-based analysis, fine mapping, and candidate gene analysis. This sample should provide sufficient power to detect linkage to OA associated genes.

Introduction Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease primarily involving the synovium. Evidence in recent years has suggested that the bone marrow (BM) may be involved, and may even be the initiating site of the disease. Abnormalities in haemopoietic stem cells' (HSC) survival, proliferation and aging have been described in patients affected by RA and ascribed to abnormal support by the BM microenvironment. Mesenchymal stem cells (MSC) and their progeny constitute important components of the BM niche. In this study we test the hypothesis that the onset of inflammatory arthritis is associated with altered self-renewal and differentiation of bone marrow MSC, which alters the composition of the BM microenvironment. Methods We have used Balb/C Interleukin-1 receptor antagonist knock-out mice, which spontaneously develop RA-like disease in 100% of mice by 20 weeks of age to determine the number of mesenchymal progenitors and their differentiated progeny before, at the start and with progression of the disease. Results We showed a decrease in the number of mesenchymal progenitors with adipogenic potential and decreased bone marrow adipogenesis before disease onset. This is associated with a decrease in osteoclastogenesis. Moreover, at the onset of disease a significant increase in all mesenchymal progenitors is observed together with a block in their differentiation to osteoblasts. This is associated with accelerated bone loss. Conclusions Significant changes occur in the BM niche with the establishment and progression of RA-like disease. Those changes may be responsible for aspects of the disease, including the advance of osteoporosis. An understanding of the molecular mechanisms leading to those changes may lead to new strategies for therapeutic intervention.

231 – Table 1. Symmetry of knee OA progression MJS Right Knee MJS Right Knee MJS Left Knee MJS Left Knee Medial Compartment Lateral Compartment Medial Compartment Lateral Compartment Ipsilateral-medial -0.226 (812)* -0.2137 (829)* Ipsilateral-lateral -0.226 (812)* -0.2137 (829)* Contralateral-medial 0.4609 (1117)* -0.0822 (774) 0.4609 (1117)* -0.0257 (788) Contralateral-lateral -0.0257 (788) 0.4462 (721)* -0.0822 (774) 0.4462 (721)* Sum KLG -0.2388 (1174)* -0.00007(819) -0.1596 (1129)* -0.0054 (796) Sum Osteophytes -0.0768 (1172) 0.0241 (815) -0.1483 (1179)* -0.0585 (834) Sum JSN -0.3787 (1164)* -0.0488 (812) -0.4009 (1168)* -0.1094 (832) Values are Pearson R coefficients (N individuals with data available for analysis of a particular set of variables) *p