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Aim: TNF inhibitor drugs are a treatment option for rheumatoid arthritis, but response is not universal. Response is typically measured using the composite 4-component (4C) disease activity score 28 (DAS28) which contains more subjective measures. This study used a validated 2-component (2C) DAS28 score to determine whether SNPs associated with response were replicated in the UK population. Materials & methods: A literature review identified TNF inhibitor response SNPs. Linear regression was conducted to replicate associations with 4C or 2C-DAS28 response. Results: Eighteen independent SNPs were analyzed in 1828 patients. One and four associations with 4C and 2C-DAS28 response respectively were identified (p ≤ 0.05). Conclusion: Further genetic associations were replicated using the 2C-DAS28 which may reflect the objective nature of 2C-AS28.

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.

BACKGROUND: Studies in critically ill patients suggest a relationship between mechanical power (an index of the energy delivered by the ventilator, which includes driving pressure, respiratory rate, tidal volume and inspiratory pressure) and complications. OBJECTIVE: We aimed to assess the association between intra-operative mechanical power and postoperative pulmonary complications (PPCs). DESIGN: Post hoc analysis of a large randomised clinical trial. SETTING: University-affiliated academic tertiary hospital in Melbourne, Australia, from February 2015 to February 2019. PATIENTS: Adult patients undergoing major noncardiothoracic, nonintracranial surgery. INTERVENTION: Dynamic mechanical power was calculated using the power equation adjusted by the respiratory system compliance (CRS). Multivariable models were used to assess the independent association between mechanical power and outcomes. MAIN OUTCOME MEASURES: The primary outcome was the incidence of PPCs within the first seven postoperative days. The secondary outcome was the incidence of acute respiratory failure. RESULTS: We studied 1156 patients (median age [IQR]: 64 [55 to 72] years, 59.5% men). Median mechanical power adjusted by CRS was 0.32 [0.22 to 0.51] (J min-1)/(ml cmH2O-1). A higher mechanical power was also independently associated with increased risk of PPCs [odds ratio (OR 1.34, 95% CI, 1.17 to 1.52); P

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.

Rheumatoid arthritis is a complex, inflammatory autoimmune disease, which is characterized by pain, swelling and joint damage driven by the altered behavior of a number of different cell types such as synovial fibroblasts macrophages and lymphocytes. The mechanism underlying pathogenesis is unclear but increasing evidence points to altered epigenetic regulation within these cell types which promotes the activated destructive behavior that underlies disease pathogenesis. This review summarizes the key epigenetic modifications in the most important cells types in rheumatoid arthritis, which are associated with disease activity. We also discuss emerging avenues of research focusing on readers of epigenetic markers which may serve to be potential therapeutic targets.

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.

Background 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. Methods 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. Results 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. Conclusions 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.

Introduction Amongst critically ill trauma patients admitted to ICU and still alive and in ICU after 24 hours, it is unclear which trauma scoring system offers the best performance in predicting in-hospital mortality. Methods The Australia and New Zealand Intensive Care Society Adult Patient Database and Victorian State Trauma Registry were linked using a unique patient identification number. Six scoring systems were evaluated: the Australian and New Zealand Risk of Death (ANZROD), Acute Physiology and Chronic Health Evaluation III (APACHE III) score and associated APACHE III Risk of Death (ROD), Trauma and Injury Severity Score (TRISS), Injury Severity Score (ISS), New Injury Severity Score (NISS) and the Revised Trauma Score (RTS). Patients who were admitted to ICU for longer than 24 hours were analysed. Performance of each scoring system was assessed primarily by examining the area under the receiver operating characteristic curve (AUROC) and in addition using standardised mortality ratios, Brier score and Hosmer-Lemeshow C statistics where appropriate. Subgroup assessments were made for patients aged 65 years and older, patients between 18 and 40 years of age, major trauma centre and head injury. Results Overall, 5,237 major trauma patients who were still alive and in ICU after 24 hours were studied from 25 ICUs in Victoria, Australia between July 2008 and January 2018. Hospital mortality was 10.7%. ANZROD (AUROC 0.91; 95% CI 0.90-0.92), APACHE III ROD (AUROC 0.88; 95% CI 0.87-0.90), and APACHE III (AUROC 0.88; 95% CI 0.87-0.89) were the best performing tools for predicting hospital mortality. TRISS had acceptable overall performance (AUROC 0.78; 95% CI 0.76-0.80) while ISS (AUROC 0.61; 95% CI 0.59-0.64), NISS (AUROC 0.68; 95% CI 0.65-0.70) and RTS (AUROC 0.69; 95% CI 0.67-0.72) performed poorly. The performance of each scoring system was highest in younger adults and poorest in older adults. Conclusion In ICU patients admitted with a trauma diagnosis and still alive and in ICU after 24 hours, ANZROD and APACHE III had a superior performance when compared with traditional trauma-specific scoring systems in predicting hospital mortality. This was observed both overall and in each of the subgroup analyses. The anatomical scoring systems all performed poorly in the ICU population of Victoria, Australia.

OBJECTIVE Monogenic Behcet's disease (BD)-like conditions are increasingly recognized and to date have been found to predominantly involve loss-of-function variants in TNFAIP3. This study was undertaken to identify genetic and pathobiologic mechanisms associated with a BD-like mucocutaneous ulcerative syndrome and neuromyelitis optica (NMO) occurring in 3 generations of an Irish family (n = 5 cases and 5 familial controls). METHODS Whole-exome sequencing was used to identify potential pathogenic variants in affected family members and determine segregation between affected and unaffected individuals. Relative v-rel reticuloendotheliosis viral oncogene homolog A (RELA) expression in peripheral blood mononuclear cells was compared by Western blotting. Human epithelial and RelA-/- mouse fibroblast experimental systems were used to determine the molecular impact of the RELA truncation in response to tumor necrosis factor (TNF). NF-κB signaling, transcriptional activation, apoptosis, and cytokine production were compared between wild-type and truncated RELA in experimental systems and patient samples. RESULTS A heterozygous cytosine deletion at position c.1459 in RELA was detected in affected family members. This mutation resulted in a frameshift p.His487ThrfsTer7, producing a truncated protein disrupting 2 transactivation domains. The truncated RELA protein lacks a full transactivation domain. The RELA protein variants were expressed at equal levels in peripheral mononuclear cells. RelA-/- mouse embryonic fibroblasts (MEFs) expressing recombinant human RELAp.His487ThrfsTer7 were compared to those expressing wild-type RELA; however, there was no difference in RELA nuclear translocation. In RelA-/- MEFs, expression of RELAp.His487ThrfsTer7 resulted in a 1.98-fold higher ratio of cleaved caspase 3 to caspase 3 induced by TNF compared to wild-type RELA (P = 0.036). CONCLUSION Our data indicate that RELA loss-of-function mutations cause BD-like autoinflammation and NMO via impaired NF-κB signaling and increased apoptosis.

Background As chronic illnesses, such as rheumatoid arthritis (RA), place an increased burden on health‐care systems, the ability of individuals to self‐manage these diseases is crucial. Objective To identify and synthesize the lived experience of self‐management described by adults living with RA. Design A systematic search of five electronic databases (MEDLINE, CINAHL, EMBASE, PsycINFO and ASSIA) was undertaken to identify relevant studies. Data were extracted and quality‐assessed using CASP guidelines. A meta‐synthesis was conducted based on Thomas and Harden's thematic synthesis approach. Results The search identified 8423 publications. After removing duplicates, 6527 records remained of which 32 studies met the inclusion criteria. Quality of studies was moderate to high, yet a considerable lack of reflection on researcher bias was evident. Our analysis identified 28 dimensions of self‐management RA across six domains: (a) cognitive‐emotional, (b) behavioural, (c) social, (d) environmental, (e) physical and (f) technological. Cognitive‐emotional experiences dominated the analysis. Renegotiating ‘the self’ (self‐concept, self‐esteem, self‐efficacy) was a key focus of self‐management among individuals with RA. Conclusion Our findings highlight the focus of ‘the self’ as a central concern in the self‐management of RA. Standardized self‐management programmes may primarily focus on disease management and daily functioning. However, we suggest that personal biographies and circumstances should move to the fore of self‐management support. Registration PROSPERO International Prospective Register of Systematic Reviews 2018: CRD42018100450. Patient or Public Contribution Patient and public involvement was not explicit in this review. However, three authors provided a patient perspective on the self‐management of arthritis and autoimmune disease.

Background: In view of the growth of published papers, there is an increasing need for studies that summarise scientific research. An increasingly common review is a 'Methodology scoping review', which provides a summary of existing analytical methods, techniques and software, proposed or applied in research articles, which address an analytical problem or further an analytical approach. However, guidelines for their design, implementation and reporting are limited. Methods: Drawing on the experiences of the authors, which were consolidated through a series of face-to-face workshops, we summarise the challenges inherent in conducting a methodology scoping review and offer suggestions of best practice to promote future guideline development. Results: We identified three challenges of conducting a methodology scoping review. First, identification of search terms; one cannot usually define the search terms a priori and the language used for a particular method can vary across the literature. Second, the scope of the review requires careful consideration since new methodology is often not described (in full) within abstracts. Third, many new methods are motivated by a specific clinical question, where the methodology may only be documented in supplementary materials. We formulated several recommendations that build upon existing review guidelines. These recommendations ranged from an iterative approach to defining search terms through to screening and data extraction processes. Conclusion: Although methodology scoping reviews are an important aspect of research, there is currently a lack of guidelines to standardise their design, implementation and reporting. We recommend a wider discussion on this topic., 22 pages, 2 tables

Introduction: Rheumatoid Arthritis (RA) varies from a mild to a severe, unremitting illness characterized by uncontrolled inflammation with consequent damage to cartilage and bone of joints. Individualized therapeutic approaches based on likely outcome would facilitate a personalized therapeutic approach.Areas covered: Genetics is known to contribute a significant component of the variability in RA outcome, estimated at 45–60%. A number of candidate gene studies have been associated with variability in radiologically assessed joint damage; however a more comprehensive genome wide analysis is required to more fully characterize the genetic basis of RA severity.Expert commentary: Genetic profiling of patient presenting with RA has the potential to aid stratification based on predicted prognosis, this would inform the clinical development of a personalized therapeutic approach. It will also result in the identification of novel mediators of tissue damage in RA.

OBJECTIVE: To determine whether using a reweighted disease activity score that better reflects joint synovitis, i.e., the 2-component Disease Activity Score in 28 joints (DAS28) (based on swollen joint count and C-reactive protein level), produces more clinically relevant treatment outcome trajectories compared to the standard 4-component DAS28.METHODS: Latent class mixed modeling of response to biologic treatment was applied to 2,991 rheumatoid arthritis (RA) patients in whom treatment with a biologic disease-modifying antirheumatic drug was being initiated within the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort, using both 4-component and 2-component DAS28 scores as outcome measures. Patient groups with similar trajectories were compared in terms of pretreatment baseline characteristics (including disability and comorbidities) and follow-up characteristics (including antidrug antibody events, adherence to treatments, and blood drug levels). We compared the trajectories obtained using the 4- and 2-component scores to determine which characteristics were better captured by each.RESULTS: Using the 4-component DAS28, we identified 3 trajectory groups, which is consistent with previous findings. We showed that the 4-component DAS28 captures information relating to depression. Using the 2-component DAS28, 7 trajectory groups were identified; among them, distinct groups of nonresponders had a higher incidence of respiratory comorbidities and a higher proportion of antidrug antibody events. We also identified a group of patients for whom the 2-component DAS28 scores remained relatively low; this group included a high percentage of patients who were nonadherent to treatment. This highlights the utility of both the 4- and 2-component DAS28 for monitoring different components of disease activity.CONCLUSION: Here we show that the 2-component modified DAS28 defines important biologic and clinical phenotypes associated with treatment outcome in RA and characterizes important underlying response mechanisms to biologic drugs.

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.

A. Palotie on työryhmän Int IBD Genetics Consortium jäsen. One goal of human genetics is to understand the genetic basis of disease, a challenge for diseases of complex inheritance because risk alleles are few relative to the vast set of benign variants. Risk variants are often sought by association studies in which allele frequencies in case subjects are contrasted with those from population-based samples used as control subjects. In an ideal world we would know population-level allele frequencies, releasing researchers to focus on case subjects. We argue this ideal is possible, at least theoretically, and we outline a path to achieving it in reality. If such a resource were to exist, it would yield ample savings and would facilitate the effective use of data repositories by removing administrative and technical barriers. We call this concept the Universal Control Repository Network (UNICORN), a means to perform association analyses without necessitating direct access to individual-level control data. Our approach to UNICORN uses existing genetic resources and various statistical tools to analyze these data, including hierarchical clustering with spectral analysis of ancestry; and empirical Bayesian analysis along with Gaussian spatial processes to estimate ancestry-specific allele frequencies. We demonstrate our approach using tens of thousands of control subjects from studies of Crohn disease, showing how it controls false positives, provides power similar to that achieved when all control data are directly accessible, and enhances power when control data are limiting or even imperfectly matched ancestrally. These results highlight how UNICORN can enable reliable, powerful, and convenient genetic association analyses without access to the individual-level data.

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

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.

Genetic variants in

OBJECTIVES: To investigate the admission characteristics and hospital outcomes for Indigenous and non-Indigenous patients admitted to intensive units (ICUs) after major trauma. DESIGN, SETTING: Retrospective analysis of Australian and New Zealand Intensive Care Society (ANZICS) Adult Patient Database data from 92 Australian ICUs for the 6-year period, 2010-2015. PARTICIPANTS: Patients older than 17 years of age admitted to public hospital ICUs with a primary diagnosis of trauma. MAIN OUTCOME MEASURES: ICU and overall hospital lengths of stay, hospital discharge destination, and ICU and overall hospital mortality rates for Indigenous and non-Indigenous patients. RESULTS: 23 804 people were admitted to Australian public hospital ICUs after major trauma; 1754 (7.4%) were Indigenous Australians. The population-standardised incidence of admissions was consistently higher for Indigenous Australians than for non-Indigenous Australians (847 per million v 251 per million population; incidence ratio, 3.37; 95% CI, 3.19-3.57). Overall hospital mortality rates were similar for Indigenous and non-Indigenous patients (adjusted odds ratio [aOR], 1.04; 95% CI, 0.82-1.31). Indigenous patients were more likely than non-Indigenous patients to be discharged to another hospital (non-Indigenous v Indigenous: aOR, 0.84; 95% CI, 0.72-0.96) less likely to be discharged home (non-Indigenous v Indigenous: aOR, 1.17; 95% CI, 1.04-1.31). CONCLUSION: The population rate of trauma-related ICU admissions was substantially higher for Indigenous than non-Indigenous patients, but hospital mortality rates after ICU admission were similar. Indigenous patients were more likely to be discharged to a another hospital and less likely to be discharged home than non-Indigenous patients.

Public and patient involvement (PPI) is increasingly an expectation of funders and policy-makers. Not all areas of health research are obvious or accessible to patients. Involving the public in preclinical and laboratory-based research in a mutually beneficial way can be challenging. Fundamentally, there is confusion among preclinical researchers as to what PPI is and how it is applied.1 PPI is not about unleashing the public into your laboratory; rather it is about increasing research relevance and cooperating with people living with rheumatic disease to enable careful and deliberate study design. In practice, how can you involve patients meaningfully and in line with European League Against Rheumatism recommendations2 if you have not already established mutual respect, a supportive environment and a relationship built on equal status? Too often, the literature aimed at guiding researchers …

Objective Hallux valgus (HV) affects ∼36% of Caucasian adults. Although considered highly heritable, the underlying genetic determinants are unclear. We conducted the first genome-wide association study (GWAS) aimed to identify genetic variants associated with HV. Methods HV was assessed in three Caucasian cohorts (n=2263, n=915 and n=1231 participants, respectively). In each cohort, a GWAS was conducted using 2.5 M imputed SNPs. Mixed-effect regression with the additive genetic model adjusted for age, sex, weight and within-family correlations was used for both sex-specific and combined analyses. To combine GWAS results across cohorts, fixed-effect inverse-variance meta-analyses were used. Following meta-analyses, top-associated findings were also examined in an African American cohort (n=327). Results The proportion of HV variance explained by genome-wide genotyped SNPs was 50% in men and 48% in women. A higher proportion of genetic determinants of HV were sex specific. The most significantly associated SNP in men was rs9675316 located on chr17q23-a24 near the AXIN2 gene (p=0.000000546×10−7); the most significantly associated SNP in women was rs7996797 located on chr13q14.1-q14.2 near the ESD gene (p=0.000000721×10−7). Genome-wide significant SNP-by-sex interaction was found for SNP rs1563374 located on chr11p15.1 near the MRGPRX3 gene (interaction p value =0.0000000041×10−9). The association signals diminished when combining men and women. Conclusions The findings suggest that the potential pathophysiological mechanisms of HV are complex and strongly underlined by sex-specific interactions. The identified genetic variants imply contribution of biological pathways observed in osteoarthritis as well as new pathways, influencing skeletal development and inflammation.

Rheumatoid arthritis (RA) is characterised by chronic synovial joint inflammation. Treatment has been revolutionised by tumour necrosis factor alpha inhibitors (TNFi) but each available drug shows a significant non-response rate. We conducted a genome-wide association study of 1752 UK RA TNFi-treated patients to identify predictors of change in the Disease Activity Score 28 (DAS28) and subcomponents over 3-6 months. The rs7195994 variant at the FTO gene locus was associated with infliximab response when looking at a change in the swollen joint count (SJC28) subcomponent (p = 9.74 × 10

Methotrexate and TNF-blocking agents are the DMARDs most commonly prescribed for the treatment of rheumatoid arthritis (RA). However, not all patients treated with these nonbiologic and biologic DMARDs respond satisfactorily and few predictors of treatment efficacy have been identified, despite the fact that these therapies have now been available for many years. Many studies have investigated genetic factors that might predict patient responsiveness to therapies used to treat RA, and epigenetic studies regarding response to treatment are expected to accumulate in the literature in the near future. Herein, we review the advances in identifying genetic and epigenetic predictors of therapeutic responses to methotrexate and/or TNF inhibitors in RA that have been made to date, and highlight important considerations for future studies, such as the need for an improved, preferably biological, outcome measure reflecting response to 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 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.

Objective A recent high-density fine-mapping (ImmunoChip) study of genetic associations in rheumatoid arthritis (RA) identified 14 risk loci with validated genome-wide significance, as well as a number of loci showing associations suggestive of significance (P = 5 × 10−5 < 5 × 10−8), but these have yet to be replicated. The aim of this study was to determine whether these potentially significant loci are involved in the pathogenesis of RA, and to explore whether any of the loci are associated with a specific RA serotype. Methods A total of 16 single-nucleotide polymorphisms (SNPs) were selected for genotyping and association analyses in 2 independent validation cohorts, comprising 6,106 RA cases and 4,290 controls. A meta-analysis of the data from the original ImmunoChip discovery cohort and from both validation cohorts was carried out, for a combined total of 17,581 RA cases and 20,160 controls. In addition, stratified analysis of patient subsets, defined according to their anti–cyclic citrullinated peptide (anti-CCP) antibody status, was performed. Results A significant association with RA risk (P < 0.05) was replicated for 6 of the SNPs assessed in the validation cohorts. All SNPs in the validation study had odds ratios (ORs) for RA susceptibility in the same direction as those in the ImmunoChip discovery study. One SNP, rs72928038, mapping to an intron of BACH2, achieved genome-wide significance in the meta-analysis (P = 1.2 × 10−8, OR 1.12), and a second SNP, rs911263, mapping to an intron of RAD51B, was significantly associated in the anti-CCP–positive RA subgroup (P = 4 × 10−8, OR 0.89), confirming that both are RA susceptibility loci. Conclusion This study provides robust evidence for an association of RA susceptibility with genes involved in B cell differentiation (BACH2) and DNA repair (RAD51B). The finding that the RAD51B gene exhibited different associations based on serologic subtype adds to the expanding knowledge base in defining subgroups of RA.

Objective Genetic factors account for an estimated 4558% of the variance in joint destruction in rheumatoid arthritis (RA). The serine proteinase granzyme B induces target cell apoptosis, and several in vitro studies suggest that granzyme B is involved in apoptosis of chondrocytes. Serum levels of granzyme B are increased in RA and are also associated with radiographic erosions. The aim of this study was to investigate GZMB as a candidate gene accounting for the severity of joint destruction in RA. Methods A total of 1,418 patients with 4,885 radiograph sets of the hands and feet from 4 independent cohorts were studied. First, explorative analyses were performed in 600 RA patients in the Leiden Early Arthritis Clinic cohort. Fifteen single-nucleotide polymorphisms (SNPs) tagging GZMB were tested. Significantly associated SNPs were genotyped in data sets representing patients from the Groningen, Sheffield, and Lund cohorts. In each data set, the relative increase in the annual rate of progression in the presence of a genotype was assessed. Data were summarized in a meta-analysis. The association of GZMB with the RNA expression level of the GZMB genomic region was tested by mapping expression quantitative trait loci (QTLs) on 1,469 whole blood samples. Results SNP rs8192916 was significantly associated with the rate of joint destruction in the first cohort and in the meta-analysis of all data sets. Patients homozygous for the minor allele of rs8192916 had a higher rate of joint destruction per year compared with other patients (P = 7.8 x 104). Expression QTL of GZMB identified higher expression in the presence of the minor allele of rs8192916 (P = 2.27 x 105). Conclusion SNP rs8192916 located in GZMB is associated with the progression of joint destruction in RA as well as with RNA expression in whole blood.

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.

Aim: A genetic variant has recently reached genome-wide significance for association with TNF-inhibitor response in rheumatoid arthritis patients. Here we undertake a replication study in a UK Caucasian population to test for association with TNF-inhibitor response. Materials & methods: The genetic variant, rs3794271, located within the PDE3A–SLCO1C1 locus was analyzed for correlation with treatment response using both the EULAR classification criteria and absolute change in (Δ)DAS28 scores as outcome measures. Results: Genotype data were available from 1750 TNF-inhibitor treated individuals. However, no evidence for association was observed (EULAR: p = 0.91 and ΔDAS28: p = 0.93). Furthermore, no significant associations were observed upon stratification by the anti-TNF received (p > 0.05). Conclusion: In the largest replication cohort conducted to date, no evidence for association was observed.

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.

Working memory is essential to higher order cognition (e.g. fluid intelligence) and to performance of daily activities. Though working memory capacity was traditionally thought to be inflexible, recent studies report that working memory capacity can be trained and that offline processes occurring during sleep may facilitate improvements in working memory performance. We utilized a 48-h in-laboratory protocol consisting of repeated digit span forward (short-term attention measure) and digit span backward (working memory measure) tests and overnight polysomnography to investigate the specific sleep-dependent processes that may facilitate working memory performance improvements in the synucleinopathies. We found that digit span backward performance improved following a nocturnal sleep interval in patients with Parkinson's disease on dopaminergic medication, but not in those not taking dopaminergic medication and not in patients with dementia with Lewy bodies. Furthermore, the improvements in patients with Parkinson's disease on dopaminergic medication were positively correlated with the amount of slow-wave sleep that patients obtained between training sessions and negatively correlated with severity of nocturnal oxygen desaturation. The translational implication is that working memory capacity is potentially modifiable in patients with Parkinson's disease but that sleep disturbances may first need to be corrected.

The FCGR locus encoding the low-affinity Fcγ receptors (FcγR) for immunoglobulin G has largely been missed by genome-wide association studies due to complications with structural variation and segmental duplication. Recently identified copy number variants (CNVs) affecting FCGR3A and FCGR3B have been linked to a number of autoimmune disorders. We have developed and validated a novel quantitative sequence variant assay in combination with an adapted paralogue ratio test to examine independent CNVs carrying FCGR3A and FCGR3B in rheumatoid arthritis (RA) compared with healthy volunteers (n = 1,115 and 654, respectively). Implementation of a robust statistical analysis framework (CNVtools) allowed for systematic batch effects and for the inherent uncertainty of copy number assignment, thus avoiding two major sources of false positive results. Evidence for association with neither duplications nor deletions of FCGR3A was found; however, in line with previous studies, there was evidence of overrepresentation of FCGR3B deletions in RA (odds ratio [OR] 1.50, P = 0.028), which was more apparent in rheumatoid factor positive disease (OR 1.61, P = 0.011). The level of FcγRIIIb, encoded by FCGR3B, expression on neutrophils was shown to correlate with gene copy number. Thus, our results may highlight an important role for neutrophils in the pathogenesis of RA, potentially through reduced FcγRIIIb-mediated immune complex clearance.

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.

Summary Background Persistent inflammation has been proposed to contribute to various stages in the pathogenesis of cardiovascular disease. Interleukin-6 receptor (IL6R) signalling propagates downstream inflammation cascades. To assess whether this pathway is causally relevant to coronary heart disease, we studied a functional genetic variant known to affect IL6R signalling. Methods In a collaborative meta-analysis, we studied Asp358Ala (rs2228145) in IL6R in relation to a panel of conventional risk factors and inflammation biomarkers in 125 222 participants. We also compared the frequency of Asp358Ala in 51 441 patients with coronary heart disease and in 136 226 controls. To gain insight into possible mechanisms, we assessed Asp358Ala in relation to localised gene expression and to postlipopolysaccharide stimulation of interleukin 6. Findings The minor allele frequency of Asp358Ala was 39%. Asp358Ala was not associated with lipid concentrations, blood pressure, adiposity, dysglycaemia, or smoking (p value for association per minor allele ≥0·04 for each). By contrast, for every copy of 358Ala inherited, mean concentration of IL6R increased by 34·3% (95% CI 30·4–38·2) and of interleukin 6 by 14·6% (10·7–18·4), and mean concentration of C-reactive protein was reduced by 7·5% (5·9–9·1) and of fibrinogen by 1·0% (0·7–1·3). For every copy of 358Ala inherited, risk of coronary heart disease was reduced by 3·4% (1·8–5·0). Asp358Ala was not related to IL6R mRNA levels or interleukin-6 production in monocytes. Interpretation Large-scale human genetic and biomarker data are consistent with a causal association between IL6R-related pathways and coronary heart disease. Funding British Heart Foundation; UK Medical Research Council; UK National Institute of Health Research, Cambridge Biomedical Research Centre; BUPA Foundation.

Background: Dysregulated production of TNF has been implicated in the pathogenesis and severity of inflammatory rheumatic diseases, many of which show age-related increased incidence. Ageing is also associated with changes in the immune system including higher systemic levels of pro-inflammatory cytokines. Methylation of DNA is an important regulator of gene expression and changes with age. Objective: In this study we investigated whether the DNA methylation status of the TNF promoter changed with age in peripheral blood leucocytes and macrophages. Methods and results: Using pyrosequencing assays we detected age-related demethylation of CpG motifs (−304, −245 and −239) in the TNF promoter in human peripheral blood cells from 312 healthy controls (0.8% per decade, confidence interval (CI) = 0.44–1.13%, p = 1 × 10−5) and primary monocyte-derived macrophages (MDM) from a separate population of 78 healthy controls (1.4% per decade, CI = 0.79–2.13%, p = 7 × 10−5). Methylation a TNF promoter fragment (−345–+154) resulted in 78% reduction of reporter gene activity compared with the unmethylated promoter construct. Conclusions: These data suggest a potential role of accrued changes in DNA methylation in the development of age-related inflammatory diseases, such as rheumatoid arthritis and polymyalgia rheumatica, in which TNF is a pivotal mediator.

BackgroundAnti-tumour necrosis factor (anti-TNF) therapy has proved to be highly successful in treating rheumatoid arthritis (RA), although 30–40% of patients have little or no response. The authors hypothesise that this may be genetically determined. In other complex diseases, susceptibility genes have been shown to influence treatment response. The aim of the current study was to investigate the association of markers within confirmed RA susceptibility loci with the response to anti-TNF treatment.MethodsEighteen single nucleotide polymorphisms (SNPs) mapping to 11 genetic loci were genotyped in 1012 patients with RA receiving treatment with etanercept, infliximab or adalimumab. Multivariate linear regression analyses were performed using the absolute change in 28 joint count disease activity score (DAS28) between baseline and 6-month follow-up as the outcome variable, adjusting for confounders. p Values ResultsIn the combined analysis, SNPs mapping to AFF3 and CD226 had a statistically significant association with the response to anti-TNF treatment under an additive model. The G allele at rs10865035, mapping to AFF3, was associated with an improved response to anti-TNF treatment (coefficient −0.14 (95% CI −0.25 to −0.03), p=0.015). At the CD226 SNP rs763361, the C allele conferred reduced response to treatment (coefficient 0.11 (95% CI 0.00 to 0.22), p=0.048).ConclusionThese results suggest that AFF3 and CD226, two confirmed RA susceptibility genes, have an additional role in influencing the response to anti-TNF treatment.

Objectives To investigate the association between genotype at the soluble interleukin 6 receptor (sIL-6R) A358C single nucleotide polymorphism (SNP, rs8192284), previously reported to correlate with soluble receptor levels, and response to anti-TNF therapy in subjects with RA. Methods In a large cohort of Caucasian RA patients treated with anti-TNF medications (total, n = 1050; etanercept, n = 455; infliximab, n = 450; and adalimumab, n = 142), the sIL-6R A358C polymorphism was genotyped using a Taqman 5'-allelic discrimination assay. Linear regression analysis adjusted for baseline 28 joint disease activity score (DAS28), baseline HAQ score, gender and use of concurrent DMARDs was used to assess the association of genotype at this polymorphism with response to anti-TNF therapy, defined by change in DAS28 after 6 months of treatment. Analyses were performed in the entire cohort, and also stratified by an anti-TNF agent. Additional analysis according to the EULAR response criteria was also performed, with the chi-squared test used to compare genotype groups. Results No association between genotype at sIL-6R A358C and response to anti-TNF treatment was detected either in the cohort as a whole or after stratification by anti-TNF agent, in either the linear regression analysis or with response segregated according to EULAR criteria. Conclusions This study shows that genotype at the functional sIL-6R A358C SNP is not associated with response to anti-TNF treatment in patients with RA.

OBJECTIVE: To determine whether the promoter polymorphism tumor necrosis factor (TNF) (-308) is associated with susceptibility to or death from meningococcal sepsis. DESIGN, SETTING, PATIENTS, AND PARTICIPANTS: Association study involving 1321 patients with microbiologically proven invasive meningococcal disease presenting to hospitals throughout United Kingdom during 1998-2001, among whom 134 died. Controls were derived from 1280 northern English blood donors. MEASUREMENTS: DNA from patients and controls was genotyped at TNF (-308). After analysis, DNA was subsequently genotyped at eight other markers in strong linkage disequilibrium with TNF (-308); these markers were IkappaBL (-62), BAT3, LST1, NOTCH4 (+1297), NOTCH4 (+3061), CCHCR1 (+436), CCHCR1 (+2271), and LTalpha. To confirm functional relevance of TNF (-308) in the context of meningococcal disease, TNF secretion by, and TNF messenger RNA expression of macrophages derived from volunteers with known TNF (-308) genotype after exposure to Neisseria meningitidis were measured. MAIN RESULTS: Among cases of meningococcal disease, likelihood of death was shown to be influenced by the age of the affected individual and also with the infecting serogroup, but was not influenced by genotype at TNF (-308) or the other linked markers. However, patients with meningococcal disease, irrespective of whether they died, were more likely to be homozygous for the rare allele at TNF (-308) (odds ratio = 1.93, 95% confidence interval 1.08-3.46), and less likely to be heterozygous for this marker (odds ratio = 0.79, 95% confidence interval 0.64-0.97), compared with the control cohort. There was no association of susceptibility to disease with the other markers studied. Macrophages derived from volunteers homozygous for the rare allele at TNF (-308) expressed higher levels of TNF messenger RNA and secreted higher concentrations of TNF compared with common homozygotes after exposure to N. meningitidis. CONCLUSIONS: Genotype at TNF (-308) modifies cellular TNF secretion in response to N. meningitidis and may influence susceptibility to meningococcal disease, but does not influence the likelihood of death after infection.

Objective The HLA–DRB1 locus within the major histocompatibility complex (MHC) at 6p21.3 has been identified as a susceptibility gene for rheumatoid arthritis (RA); however, there is increasing evidence of additional susceptibility genes in the MHC region. The aim of this study was to estimate their number and location. Methods A case–control study was performed involving 977 control subjects and 855 RA patients. The HLA–DRB1 locus was genotyped together with 2,360 single-nucleotide polymorphisms in the MHC region. Logistic regression was used to detect DRB1-independent effects. Results After adjusting for the effect of HLA–DRB1, 18 markers in 14 genes were strongly associated with RA (P < 10−4). Multivariate logistic regression analysis of these markers and DRB1 led to a model containing DRB1 plus the following 3 markers: rs4678, a nonsynonymous change in the VARS2L locus, ∼1.7 Mb telomeric of DRB1; rs2442728, upstream of HLA–B, ∼1.2 Mb telomeric of DRB1; and rs17499655, located in the 5′-untranslated region of DQA2, only 0.1 Mb centromeric of DRB1. In-depth investigation of the DQA2 association, however, suggested that it arose through cryptic linkage disequilibrium with an allele of DRB1. Two non–shared epitope alleles were also strongly associated with RA (P < 10−4): *0301 with anti– cyclic citrullinated peptide–negative RA and *0701 independently of autoantibody status. Conclusion These results confirm the polygenic contribution of the MHC to RA and implicate 2 additional non-DRB1 susceptibility loci. The role of the HLA–DQ locus in RA has been a subject of controversy, but in our data, it appears to be spurious.

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 = 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.

Objective To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients. Methods Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti–tumor necrosis factor α (anti-TNFα). Results The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate ≤ 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor α, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (κ = 0.64). In anti-TNFα–treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score. Conclusion This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.

The therapeutic potential of high dose cytotoxic therapy and stem cell transplantation (SCT) in severe rheumatoid arthritis (RA) was originally supported by animal studies and serendipitous clinical cases where allogeneic and autologous procedures were shown to ameliorate and potentially cure the disease. Phase I and Phase II clinical studies established the feasibility, safety and efficacy of autologous stem cell mobilisation and transplantation. Although it was clear that the effects of high dose chemotherapy and autologous SCT could safely achieve profound responses, sustained control of disease usually required the reintroduction of disease modifying agents. Responses were improved with dose escalation of the conditioning regimen, and also with post-SCT therapy, such as rituximab, but were not observed with graft manipulation. Phase III studies were attempted, but recruitment was compromised by the increasingly widespread use of biological anti-rheumatic agents. Autologous SCT is now only reasonably considered in relatively rare patients whose disease has resisted conventional and biological treatments, and small numbers of cases continue to be registered with the EBMT. Occasional patients treated with allogeneic and syngeneic SCT continue to stimulate academic interest, particularly as some appear to be cured, but significant logistical and toxicity issues mean that routine and widespread application is unrealistic. In summary, SCT continues to have a limited therapeutic potential in rare patients with RA refractory to modern therapy and sufficient fitness for the procedure. From a scientific perspective, ablation of the dysfunctional rheumatoid immune system and its reconstruction with SCT has provided useful insights into the pathophysiology of RA.

Several studies have implicated the NF-kappaB inhibitor-like protein 1 (NFkBIL1) gene located in the class III region of the major histocompatibility complex (MHC) as a possible susceptibility locus for rheumatoid arthritis (RA). Based on limited homology, it has been suggested to be a member of the inhibitor of NF-kappaB (IkappaB) family of proteins, but a role in mRNA processing has also been proposed. We have investigated the expression of NFkBIL1 in RA synovial tissue and characterized its function. Real-time PCR showed the two NFkBIL1 mRNA splice variants are expressed in a tissue-specific manner. Dual immunofluorescent staining of human RA synovium with polyclonal anti-NFkBIL1 antibodies and anti-CD68, anti-CD3 or anti-factor VIII showed that NFkBIL1 was expressed in the rheumatoid synovial lining and sub-lining layers and co-localized in CD68+ and CD3+, but not Factor VIII+ cells. Confocal microscopy of cultured synovial fibroblasts revealed expression in speckled nuclear and homogenous cytoplasmic distributions, suggesting shuttling between the cytoplasmic and nuclear compartments. Functional tests showed that NFkBIL1 isoforms were incapable of associating with NF-kappaB and did not inhibit it, thus disproving the hypothesis that NFkBIL1 functions as an IkappaB. Affinity purification of endogenous NFkBIL1 proteins and co-immunoprecipitation experiments showed that NFkBIL1 can associate with mRNA and with three protein partners, identified by mass spectrometry as leukophysin, translation elongation factor 1 alpha and CTP synthase I. These data support a potential role for NFkBL1 in the pathogenesis of RA and indicates that it may be involved in mRNA processing or the regulation of translation.