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Objective Juvenile idiopathic arthritis (JIA) is the most common chronic immune-mediated joint disease among children and encompasses a heterogeneous group of immune-mediated joint disorders classified into 7 subtypes according to clinical presentation. However, phenotype overlap and biologic evidence suggest a shared mechanistic basis between subtypes. This study was undertaken to systematically investigate shared genetic underpinnings of JIA subtypes. Methods We performed a heterogeneity-sensitive genome-wide association study encompassing a total of 1,245 JIA cases (classified into 7 subtypes) and 9,250 controls, followed by fine-mapping of candidate causal variants at each genome-wide significant locus, functional annotation, and pathway and network analysis. We further identified candidate drug targets and drug repurposing opportunities by in silico analyses. Results In addition to the major histocompatibility complex locus, we identified 15 genome-wide significant loci shared between at least 2 JIA subtypes, including 10 novel loci. Functional annotation indicated that candidate genes at these loci were expressed in diverse immune cell types. Conclusion This study identified novel genetic loci shared by JIA subtypes. Our findings identified candidate mechanisms underlying JIA subtypes and candidate targets with drug repurposing opportunities for JIA treatment.

ObjectiveTo ascertain the role of platelet glycoprotein Ib α‐chain (GPIbα) plasma protein levels in cardiovascular, autoimmune, and autoinflammatory diseases and whether its effects are mediated by platelet count.MethodsWe performed a two‐sample Mendelian randomization (MR) study, using both a cis‐acting protein quantitative trait locus (cis‐pQTL) and trans‐pQTL near the GP1BA and BRAP genes as instruments. To assess if platelet count mediated the effect, we then performed a two‐step MR study. Putative associations (GPIbα/platelet count/disease) detected by MR analyses were subsequently assessed using multiple‐trait colocalization analyses.ResultsAfter correction for multiple testing (Bonferroni‐corrected threshold P ≤ 2 × 10−3), GPIbα, instrumented by either cis‐pQTL or trans‐pQTL, was causally implicated with an increased risk of oligoarticular and rheumatoid factor (RF)–negative polyarticular juvenile idiopathic arthritis (JIA). These effects of GPIbα appeared to be mediated by platelet count and were supported by strong evidence of colocalization (probability of all 3 traits sharing a common causal variant ≥0.80). GPIbα instrumented by cis‐pQTL did not appear to affect cardiovascular risk, although the GPIbα trans‐pQTL was associated with an increased risk of cardiovascular diseases and autoimmune diseases but a decreased risk of autoinflammatory diseases, suggesting that this trans‐acting instrument operates through other pathways.ConclusionThe role of platelets in thrombosis is well‐established; however, our findings provide some novel genetic evidence that platelets may be causally implicated in the development of oligoarticular and RF‐negative polyarticular JIA, and indicate that GPIbα may serve as a putative therapeutic target for these JIA subtypes.

ObjectivesJuvenile idiopathic arthritis (JIA) is the most prevalent form of juvenile rheumatic disease. Our understanding of the genetic risk factors for this disease is limited due to low disease prevalence and extensive clinical heterogeneity. The objective of this research is to identify novel JIA susceptibility variants and link these variants to target genes, which is essential to facilitate the translation of genetic discoveries to clinical benefit.MethodsWe performed a genome-wide association study (GWAS) in 3305 patients and 9196 healthy controls, and used a Bayesian model selection approach to systematically investigate specificity and sharing of associated loci across JIA clinical subtypes. Suggestive signals were followed-up for meta-analysis with a previous GWAS (2751 cases/15 886 controls). We tested for enrichment of association signals in a broad range of functional annotations, and integrated statistical fine-mapping and experimental data to identify target genes.ResultsOur analysis provides evidence to support joint analysis of all JIA subtypes with the identification of five novel significant loci. Fine-mapping nominated causal single nucleotide polymorphisms with posterior inclusion probabilities ≥50% in five JIA loci. Enrichment analysis identified RELA and EBF1 as key transcription factors contributing to disease risk. Our integrative approach provided compelling evidence to prioritise target genes at six loci, highlighting mechanistic insights for the disease biology andIL6STas a potential drug target.ConclusionsIn a large JIA GWAS, we identify five novel risk loci and describe potential function of JIA association signals that will be informative for future experimental works and therapeutic strategies.

Current classification of primary inflammatory arthritis begins from the assumption that adults and children are different. No form of juvenile idiopathic arthritis bears the same name as an adult arthritis, a nomenclature gap with implications for both clinical care and research. Recent genetic data have raised questions regarding this adult/pediatric divide, revealing instead broad patterns that span the age spectrum. Combining these genetic patterns with demographic and clinical data, we propose that inflammatory arthritis can be segregated into 4 main clusters, largely irrespective of pediatric or adult onset: seropositive, seronegative (likely including a distinct group that usually begins in early childhood), spondyloarthritis, and systemic. Each of these broad clusters is internally heterogeneous, highlighting the need for further study to resolve etiologically discrete entities. Eliminating divisions based on arbitrary age cutoffs will enhance opportunities for collaboration between adult and pediatric rheumatologists, thereby helping to promote the understanding and treatment of arthritis.

Objective Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease and has a strong genomic component. To date, JIA genetic association studies have had limited sample sizes, used heterogeneous patient populations, or included only candidate regions. The aim of this study was to identify new associations between JIA patients with oligoarticular disease and those with IgM rheumatoid factor (RF)−negative polyarticular disease, which are clinically similar and the most prevalent JIA disease subtypes. Methods Three cohorts comprising 2,751 patients with oligoarticular or RF-negative polyarticular JIA were genotyped using the Affymetrix Genome-Wide SNP Array 6.0 or the Illumina HumanCoreExome-12+ Array. Overall, 15,886 local and out-of-study controls, typed on these platforms or the Illumina HumanOmni2.5, were used for association analyses. High-quality single-nucleotide polymorphisms (SNPs) were used for imputation to 1000 Genomes prior to SNP association analysis. Results Meta-analysis showed evidence of association (P < 1 × 10−6) at 9 regions: PRR9_LOR (P = 5.12 × 10−8), ILDR1_CD86 (P = 6.73 × 10−8), WDFY4 (P = 1.79 × 10−7), PTH1R (P = 1.87 × 10−7), RNF215 (P = 3.09 × 10−7), AHI1_LINC00271 (P = 3.48 × 10−7), JAK1 (P = 4.18 × 10−7), LINC00951 (P = 5.80 × 10−7), and HBP1 (P = 7.29 × 10−7). Of these, PRR9_LOR, ILDR1_CD86, RNF215, LINC00951, and HBP1 were shown, for the first time, to be autoimmune disease susceptibility loci. Furthermore, associated SNPs included cis expression quantitative trait loci for WDFY4, CCDC12, MTP18, SF3A1, AHI1, COG5, HBP1, and GPR22. Conclusion This study provides evidence of both unique JIA risk loci and risk loci overlapping between JIA and other autoimmune diseases. These newly associated SNPs are shown to influence gene expression, and their bounding regions tie into molecular pathways of immunologic relevance. Thus, they likely represent regions that contribute to the pathology of oligoarticular JIA and RF-negative polyarticular JIA.

Annals of the rheumatic diseases 76(5), 906-913 (2017). doi:10.1136/annrheumdis-2016-210324, Published by BMJ Publ. Group, London

Objective Children with childhood-onset rheumatoid arthritis (RA) include those with rheumatoid factor or anti–citrullinated protein antibody–positive juvenile idiopathic arthritis. To test the hypothesis that adult-onset RA–associated variants are also associated with childhood-onset RA, we investigated RA-associated variants at 5 loci in a cohort of patients with childhood-onset RA. We also assessed the cumulative association of these variants in susceptibility to childhood-onset RA using a weighted genetic risk score (wGRS). Methods A total of 155 children with childhood-onset RA and 684 healthy controls were genotyped for 5 variants in the PTPN22, TRAF1/C5, STAT4, and TNFAIP3 loci. High-resolution HLA–DRB1 genotypes were available for 149 cases and 373 controls. We tested each locus for association with childhood-onset RA via logistic regression. We also computed a wGRS for each subject, with weights based on the natural log of the published odds ratios (ORs) for the alleles investigated, and used logistic regression to test the wGRS for association with childhood-onset RA. Results Childhood-onset RA was associated with TNFAIP3 rs10499194 (OR 0.60 [95% confidence interval 0.44–0.83]), PTPN22 rs2476601 (OR 1.61 [95% confidence interval 1.11–2.31]), and STAT4 rs7574865 (OR 1.41 [95% confidence interval 1.06–1.87]) variants. The wGRS was significantly different between cases and controls (P < 2 × 10−16). Individuals in the third to fifth quintiles of wGRS had a significantly increased disease risk compared to baseline (individuals in the first quintile). Higher wGRS was associated with increased risk of childhood-onset RA, especially among males. Conclusion The magnitude and direction of the association between TNFAIP3, STAT4, and PTPN22 variants and childhood-onset RA are similar to those observed in RA, suggesting that adult-onset RA and childhood-onset RA share common genetic risk factors. Using a wGRS, we have demonstrated the cumulative association of RA-associated variants with susceptibility to childhood-onset RA.

Objective.Follistatin-like protein 1 (FSTL-1) is a secreted glycoprotein overexpressed in certain inflammatory diseases. Our objective was to correlate FSTL-1 levels with gene expression, known biomarkers, and measures of disease activity in systemic juvenile idiopathic arthritis (sJIA), including macrophage activation syndrome (MAS).Methods.FSTL-1 serum levels were measured by ELISA in 28 patients with sJIA, including 7 patients who developed MAS, and 30 healthy controls. Levels were correlated with erythrocyte sedimentation rate (ESR), ferritin, and soluble interleukin-2 receptor-α (sIL-2Rα). Gene expression based on FSTL-1 levels was analyzed in peripheral blood mononuclear cells (PBMC).Results.Serum levels of FSTL-1 were elevated at time of presentation of sJIA (mean 200.7 ng/ml) and decreased to normal (mean 133.7 ng/ml) over 24 months (p < 0.01). FSTL-1 levels were markedly elevated during acute MAS (mean 279.8 ng/ml) and decreased to normal following treatment (p < 0.001). FSTL-1 levels correlated with serum markers of inflammation, including sIL-2Rα and ferritin. Ferritin/ESR ratio was superior to ferritin, sIL-2Rα, and FSTL-1 in discriminating MAS from new-onset sJIA. PBMC from patients with FSTL-1 levels > 200 ng/ml showed altered expression of genes related to innate immunity, erythropoiesis, and natural killer cell dysfunction. Two patients with the highest FSTL-1 levels at disease onset (> 300 ng/ml) ultimately developed MAS.Conclusion.Elevated pretreatment serum FSTL-1 levels in sJIA are associated with dysregulated gene expression suggestive of occult MAS, and may have utility in predicting progression to overt MAS. Ferritin/ESR ratio may be superior to ferritin alone in discriminating overt MAS from new-onset sJIA.

ObjectiveParticipants in the Atherosclerosis Prevention in Paediatric Lupus Erythematosus (APPLE) trial were randomised to placebo or atorvastatin for 36 months. The primary endpoint, reduced carotid intima medial thickness (CIMT) progression, was not met but atorvastatin-treated participants showed a trend of slower CIMT progression. Post-hoc analyses were performed to assess subgroup benefit from atorvastatin therapy.MethodsSubgroups were prespecified and defined by age (> or ≤15.5 years), systemic lupus erythematosus (SLE) duration (> or ≤24 months), pubertal status (Tanner score ≥4 as post-pubertal or ResultsSignificant interaction effects with trends of less CIMT progression in atorvastatin-treated participants were observed in pubertal (3 CIMT segments), high hsCRP (2 CIMT segments), and the combined high hsCRP and pubertal group (5 CIMT segments). No significant treatment effect trends were observed across subgroups defined by age, SLE duration, LDL for CIMT or other outcome measures.ConclusionsPubertal status and higher hsCRP were linked to lower CIMT progression in atorvastatin-treated subjects, with most consistent decreases in CIMT progression in the combined pubertal and high hsCRP group. While secondary analyses must be interpreted cautiously, results suggest further research is needed to determine whether pubertal lupus patients with high CRP benefit from statin therapy.ClinicalTrials.gov identifier:NCT00065806.

Author(s): Fotis, Lampros; Shaikh, Nur; Baszis, Kevin; French, Anthony; Tarr, Phillip; Grevich, Sriharsha; Lee, Peggy; Ringold, Sarah; Leroux, Brian; Leahey, Hannah; Yuasa, Megan; Foster, Jessica; Sokolove, Jeremy; Lahey, Lauren; Robinson, William; Newsom, Joshua; Stevens, Anne; Karasawa, Rie; Tamaki, Mayumi; Tanaka, Megumi; Sato, Toshiko; Yudoh, Kazuo; Jarvis, James N; Moncrieffe, Halima; Bennett, Mark F; Tsoras, Monica; Luyrink, Lorie; Xu, Huan; Prahalad, Sampath; Morris, Paula; Dare, Jason; Nigrovic, Peter A; Rosenkranz, Margalit; Becker, Mara; O’Neil, Kathleen M; Griffin, Thomas; Lovell, Daniel J; Grom, Alexei A; Medvedovic, Mario; Thompson, Susan D; Zhu, Lisha; Jiang, Kaiyu; Wong, Laiping; Buck, Michael J; Chen, Yanmin; Moncrieffe, Halima; Brungs, Laura; Liu, Tao; Wang, Ting; Jarvis, James N; Alsaeid, Khaled; Alfailakawi, Jasim; Alenezi, Hamid; Alsaeed, Hazim; Beukelman, Tim; Natter, Marc; Ilowite, Norm; Mieszkalski, Kelly; Burrell, Grendel; Best, Brian; Bristow, Helen; Carr, Shannon; Dennos, Anne; Kaufmann, Rachel; Kimura, Yukiko; Schanberg, Laura; Blier, Peter R; Boneparth, Alexis; Wenderfer, Scott E; Moorthy, L Nandini; Radhakrishna, Suhas M; Sagcal-Gironella, Anna Carmela P; von Scheven, Emily; Gedik, Kader Cetin; Siddique, Salma; Aguiar, Cassyanne L; Erkan, Doruk; Cohen, Ezra; Lee, Yvonne; Dossett, Michelle; Mehta, Darshan; Davis, Roger; Gilbert, Mileka; Goilav, Beatrice; Meidan, Esra

Rheumatoid arthritis (RA), which is estimated to affect ~1% of the adult population, is the most common form of inflammatory arthritis in the world. A substantial majority of individuals with RA have rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPA), and the presence of these antibodies has been associated with a worse prognosis. Some children with juvenile idiopathic arthritis (JIA) have a disease that phenotypically resembles RA, characterized by chronic inflammatory arthritis and the presence of the characteristic biomarkers seen in RA including RF and ACPA Such JIA patients presumably represent a subset of RA patients with childhood onset of RA (CORA), but it is unclear if CORA shares the same risk factors associated with adult-onset RA, which has its peak age of onset in the fifth decade, or if patients with CORA have additional risk factors that, once identified, will offer important insight into the etiopathogenesis of RA. Both genetic and environmental factors have been associated with the risk of developing RA in adults. A recent meta-analysis of genome-wide associations studies of RA brought the total number of loci with confirmed genetic association to 31(1). The strongest association observed to date has been with a subset of alleles encoding the class II Human leukocyte antigens (HLA). RA associated HLA-DRB1 alleles encode DRβ1 chains with conserved amino acid sequence in positions 70-74. The molecular structure encoding the susceptibility residues is termed the shared epitope (SE)(2). While associations between SE and risk of RA are well established, some alleles (e.g. HLA-DRB1*0401) appear to confer a greater degree of risk than others (HLA-DRB1*0101)(3). Tezenas Du Montcel et al proposed a classification based on the amino acid sequence at positions 70-74, and suggested that some classes of SE alleles conferred greater susceptibility than others (4, 5). While SE alleles have been evaluated in childhood cases of RA, often referred to in the literature as “seropositive juvenile rheumatoid arthritis” or “polyarticular RF+ juvenile idiopathic arthritis”, most prior cohorts were small, used low resolution HLA typing, and thus have been limited to evaluation of the presence or absence of SE (6-11). Our objectives were to investigate the association between SE-encoding HLA-DRB1 alleles and CORA, and to explore a possible hierarchy of risk conferred by SE alleles based on the amino-acid sequence in position 70-74, in a large cohort of CORA patients. We also sought to compare the frequencies of SE genotypes in children with CORA versus published frequencies in adults with RA.

Objective More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjogren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. Methods All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. Results We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67–86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18–123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. Conclusion The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

Objective To explore biologic correlates to age at onset in patients with juvenile idiopathic arthritis (JIA) using peripheral blood mononuclear cell (PBMC) gene expression analysis. Methods PBMCs were isolated from 56 healthy controls and 104 patients with recent-onset JIA (39 with persistent oligoarticular JIA, 45 with rheumatoid factor–negative polyarticular JIA, and 20 with systemic JIA). RNA was amplified and labeled using NuGEN Ovation, and gene expression was assessed with Affymetrix HG-U133 Plus 2.0 GeneChips. Results A total of 832 probe sets revealed gene expression differences (false discovery rate 5%) in PBMCs from children with oligoarticular JIA whose disease began before age 6 years (early-onset disease) compared with those whose disease began at or after age 6 years (late-onset disease). In patients with early-onset disease, there was greater expression of genes related to B cells and less expression of genes related to cells of the myeloid lineage. Support vector machine analyses identified samples from patients with early- or late-onset oligoarticular JIA (with 97% accuracy) or from patients with early- or late-onset polyarticular JIA (with 89% accuracy), but not from patients with systemic JIA or healthy controls. Principal components analysis showed that age at onset was the major classifier of samples from patients with oligoarticular JIA and patients with polyarticular JIA. Conclusion PBMC gene expression analysis reveals biologic differences between patients with early-and late-onset JIA, independent of classification based on the number of joints involved. These data suggest that age at onset may be an important parameter to consider in JIA classification. Furthermore, pathologic mechanisms may vary with age at onset, and understanding these processes may lead to improved treatment of JIA.

Objective We previously observed the association of the co-occurrence of the HLA–DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)–positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs. Methods SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription–PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay. Results A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti–cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5. Conclusion The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.

Objective Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA. Methods Synovial fluid samples obtained from children with oligoarticular JIA, polyarticular JIA, or systemic JIA were compared. Two-dimensional gel electrophoresis for protein separation and matrix-assisted laser desorption ionization−time-of-flight mass spectrometry and quadripole time-of-flight mass spectrometry for protein identification were used for this study. Synovial fluid cells were analyzed by polymerase chain reaction (PCR) for the presence of haptoglobin messenger RNA (mRNA). Results The synovial fluid proteome of the samples was delineated. The majority of proteins showed overexpression in JIA synovial fluid as compared with noninflammatory control samples. There were 24 statistically significantly differentially expressed spots (>2-fold change; P < 0.05) between the subtypes of JIA. PCR analysis revealed haptoglobin mRNA, suggesting that haptoglobin is locally produced in an inflamed joint in JIA. Conclusion Despite the similar histologic appearance of inflamed joints in patients with different subtypes of JIA, there are differences in protein expression according to the subtype of JIA. Haptoglobin is differentially expressed between the subtypes of JIA and is locally produced in an inflamed joint in JIA. Haptoglobin and other differentially expressed proteins may be potential biomarkers in JIA.

Objective The aim of this study was to quantitate risk and to examine heterogeneity for HLA at high resolution in patients with the most common subtypes of juvenile idiopathic arthritis (JIA), IgM rheumatoid factor–negative polyarticular JIA and oligoarticular JIA. Use of 4-digit comprehensive HLA typing enabled great precision, and a large cohort allowed for consideration of both age at disease onset and disease subtype. Methods Polymerase chain reaction–based high-resolution HLA typing for class I and class II loci was accomplished for 820 patients with JIA and 273 control subjects. Specific HLA epitopes, potential interactions of alleles at specific loci and between loci (accounting for linkage disequilibrium and haplotypic associations), and an assessment of the current International League of Associations for Rheumatology classification criteria were considered. Results An HLA–DRB1/DQB1 effect was shown to be exclusively attributable to DRB1 and was similar between patients with oligoarticular JIA and a younger subgroup of patients with polyarticular JIA. Furthermore, patients with polyarticular JIA showed age-specific related effects, with disease susceptibility in the group older than age 6 years limited to an effect of the HLA–DRB1*08 haplotype, which is markedly different from the additional susceptibility haplotypes, HLA–DRB1*1103/1104, found in the group with oligoarticular JIA and the group of younger patients with polyarticular JIA. Also in contrast to findings for oligoarticular JIA, patients with polyarticular arthritis had no evidence of an HLA class I effect. Markers associated with a reduced risk of disease included DRB1*1501, DRB1*0401, and DRB1*0701. DRB1*1501 was shown to reduce risk across the whole cohort, whereas DRB1*0401 and DRB1*0701 were protective for selected JIA subtypes. Surprisingly, the disease predisposition mediated by DPB1*0201 in individuals without any disease-predisposing DRB1 alleles was great enough to overcome even the very strong protective effect observed for DRB1*1501. Conclusion Inherited HLA factors in JIA show similarities overall as well as differences between JIA subtypes.

Macrophage Activation Syndrome (MAS) is a severe, potentially fatal condition associated with excessive activation of macrophages and T cells leading to an overwhelming inflammatory reaction. The main manifestations of MAS include fever, hepatosplenomegaly, lymphadenopathy, severe cytopenias, serious liver disease, and disseminated intravascular coagulation [1,2]. The pathognomonic feature of MAS is often found in bone marrow: numerous, well-differentiated macrophages phagocytosing hematopoietic elements. Although MAS has been reported in patients with different rheumatic diseases, it is most strongly associated with Systemic Juvenile Idiopathic Arthritis (SJIA). In fact, it accounts for much of the morbidity and mortality seen in this form of JIA. At least 10% of the patients with SJIA develop MAS [3]. The true incidence of MAS might be much higher since there are no validated diagnostic criteria and mild instances of MAS are not always recognized [4,5]. It is now recognized that MAS bears close resemblance to a group of histiocytic disorders collectively known as hemophagocytic lymphohistiocytosis (HLH) [2,6]. HLH is a term that describes a spectrum of disease processes characterized by accumulations of well-differentiated mononuclear cells with a macrophage phenotype [7]. In the contemporary classification of histiocytic disorders, HLH is further subdivided into primary, or familial HLH, and secondary, or reactive HLH (ReHLH) [7]. Clinically, however, they may be difficult to distinguish from each other [9]. Familial hemophagocytic lymphohistiocytosis (FHLH) is a constellation of rare autosomal recessive immune disorders. The clinical symptoms of FHLH usually become evident within the first 2 months of life although initial presentation as late as 22 years of age has been reported [8]. Secondary HLH tends to occur in older children and more often is associated with an identifiable infectious episode, most notably Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infection. The exact pathophysiological relationship between MAS and HLH is not understood. Some pediatric rheumatologists view MAS as ReHLH occurring in a setting of a rheumatologic disease [6]. The pathological mechanisms of HLH/MAS are not fully understood. In HLH, there is uncontrolled proliferation of T cells and macrophages that has been linked to decreased NK-cell and cytotoxic T-cell function [10] often due to mutations in the gene encoding perforin [11]. Perforin is a protein which cytolytic cells utilize to induce apoptosis of target cells such as tumor cells or cells infected by viruses. It has been hypothesized by some authors that abnormal cytotoxic cells may fail to provide appropriate apoptotic signals for removal of activated macrophages and T cells during the contraction stage of certain immune responses [12]. Our recent observations suggest that as in HLH, MAS patients have profoundly depressed NK-cell function, often associated with abnormal perforin expression [13]. More recently, mutations in another gene, MUNC13-4, have been implicated in the development of hemophagocytic lymphohistiocytosis in about 10-30% of patients with inherited HLH [14]. The protein encoded by the MUNC13-4 gene is an essential effector of the cytolytic secretory pathway. MUNC13-4 protein is involved in vesicle priming function which follows granule docking and precedes plasma granule membrane fusion [14]. Therefore, it is an important player in the intracellular transport of perforin. Although the cytolytic cells of the patients with FHLH caused by MUNC13-4 mutations produce sufficient amounts of perforin, the poor ability to deliver perforin to the surface of the cells leads to profoundly decreased cytolytic activity against target cells. Here we present new data that suggests an association between MAS in SJIA and specific mutations and/or haplotypes in MUNC13-4 gene.

Juvenile idiopathic arthritis (JIA) is an autoimmune disorder mediated by Th1 immune responses. CTLA-4, expressed on the T cell surface, plays a negative role in regulating T cell activation. Single-nucleotide polymorphisms (SNPs) in CTLA4 have been implicated in susceptibility to several autoimmune disorders, including JIA. This study was undertaken to test 3 functional CTLA4 variants for association with JIA.Families of 531 children with JIA were genotyped for SNPs located in the promoter region (C-318T), exon 1 (A49G), and the 3'-untranslated region (CT60) of CTLA4 by polymerase chain reaction amplification and digestion. Family Based Association Testing (FBAT) was used to test CTLA4 SNPs and haplotypes for association with JIA. A second independent cohort of300 children with JIA and500 controls were genotyped for case-control analyses. Case-control analyses of the combined cohorts, as well as meta-analyses of published association studies of CTLA4 and JIA, were performed.There were no deviations of transmission of any of the CTLA4 variants to children with JIA, or JIA subtypes, determined by FBAT. No significant associations between CTLA4 C-318T or A49G SNPs and JIA were found in 650 JIA cases and 350 controls. Similarly, no significant associations with CT60 variants were found in800 JIA cases and500 controls. The meta-analysis also failed to confirm an association between JIA and CTLA4 variants.These results suggest that C-318T, A49G, CT60, and haplotypes tagged by these CTLA4 SNPs are not associated with JIA or major JIA subtypes.

Objective Systemic juvenile idiopathic arthritis (JIA) is frequently associated with the development of macrophage activation syndrome. This study was undertaken to better understand the relationship between systemic JIA and macrophage activation syndrome. Methods Gene expression profiles were examined in 17 patients with untreated new-onset systemic JIA, 5 of whom showed evidence of subclinical macrophage activation syndrome (of whom 2 eventually developed overt macrophage activation syndrome). Peripheral blood mononuclear cells (PBMCs) were separated on Ficoll gradients, and purified RNA was analyzed using Affymetrix GeneChip expression arrays. A fraction of the PBMCs were used for flow cytometry to define the cellular composition of the samples. Results Two hundred twenty-five differentially expressed genes (P < 0.05) that distinguished patients with systemic JIA from healthy controls (n = 30) were identified. Clustering analysis indicated that expression patterns correlated with serum ferritin levels. Three main clusters distinguished systemic JIA patients with highly elevated ferritin levels (including those with subclinical macrophage activation syndrome) from those with normal or only moderately elevated ferritin levels. The first cluster comprised genes involved in the synthesis of hemoglobins and structural proteins of erythrocytes. This transcriptional profile was consistent with immature nucleated red blood cells, likely reflective of high red blood cell turnover. Also included were transcripts indicating immature granulocytes. The second cluster was enriched for genes involved in cell cycle regulation. The third cluster was enriched for genes involved in innate immune responses, including those involved in the negative regulation of Toll-like receptor/interleukin-1 receptor–triggered inflammatory cascades and markers of the alternative pathway of macrophage differentiation. Additional differentially expressed genes of interest were those involved in the cytolytic pathway, including SH2D1A and Rab27a. Conclusion These data indicate that gene expression profiling can be a useful tool for identifying early macrophage activation syndrome in patients with systemic JIA.

Objective Anti–cyclic citrullinated peptide (anti-CCP) antibodies have been detected in patients with juvenile rheumatoid arthritis (JRA), particularly in those with polyarticular, rheumatoid factor (RF)-positive JRA. Our objectives were to determine whether anti-CCP antibodies are associated with HLA–DR4 in children with polyarticular JRA, whether anti-CCP antibodies are associated with clinical features of disease, and whether affected sibling pairs (ASPs) with JRA are concordant for this antibody. Methods Stored serum samples obtained from 230 HLA-typed patients with JRA (77 with polyarticular-onset disease and 153 with pauciarticular- or systemic-onset disease), 100 JRA ASPs, and 688 healthy children were tested for anti-CCP antibodies and RF. Results Thirteen percent of the patients with polyarticular-onset JRA and 2% of the other JRA patients exhibited anti-CCP antibodies, compared with only 0.6% of the controls. Fifty-seven percent of RF-positive patients with polyarticular-onset JRA had anti-CCP antibodies. HLA–DR4–positive patients with polyarticular-onset JRA were more likely to have anti-CCP antibodies than were those without HLA–DR4 alleles (odds ratio [OR] 5.20, 95% confidence interval [95% CI] 1.30–20.9). Anti-CCP antibodies were associated with polyarticular onset (OR 7.46, 95% CI 1.99–28.0), a polyarticular disease course (OR 9.78, 95% CI 1.25–76.7), and erosive disease (OR 14.3, 95% CI 3.01–67.9). Concordance rates for anti-CCP antibodies among ASPs were statistically significant. Conclusion These data demonstrate increased anti-CCP antibody formation in HLA–DR4–positive patients with polyarticular-onset JRA. The overall prevalence of anti-CCP antibodies in JRA is low, but a substantial proportion of RF-positive patients with polyarticular-onset JRA have these antibodies. Anti-CCP antibodies in JRA are associated with polyarticular onset, a polyarticular course, and erosive disease.

Objective Juvenile rheumatoid arthritis (JRA) represents a heterogeneous group of disorders with a complex genetic component. A genome scan was performed to detect linkage to JRA in 121 families containing 247 affected children in North America (the JRA Affected Sibpair [ASP] Registry). Methods Genotype data collected for HLA–DR and 386 microsatellite markers were subjected to multipoint nonparametric linkage analysis. Following analysis of the entire set of families, additional analyses were performed after a priori stratification by disease onset type, age at onset, disease course, and selected HLA–DRB1 alleles. Results Linkage of JRA to the HLA region was confirmed (logarithm of odds [LOD] score 2.26). Additional evidence supporting linkage of JRA was observed at 1p36 (D1S214; LOD 1.65), 19p13 (D19S216; LOD 1.72), and 20q13 (D20S100; LOD 1.75). For early-onset polyarticular disease, evidence of linkage was found at chromosome 7q11 (D7S502; LOD 3.47). For pauciarticular disease, evidence supporting linkage was observed on chromosome 19p13 (D19S216; LOD 2.98), the same marker that supported linkage to the “JRA” phenotype. Other regions supporting linkage with JRA disease subtype included 20q13, 4q24, 12q24, and Xp11. Stratification of families based on the presence of the HLA–DR8 allele in affected siblings resulted in significant linkage observed at 2p25 (D2S162/D2S305; LOD 6.0). Conclusion These data support the hypothesis that multiple genes, including at least 1 in the HLA region, influence susceptibility to JRA. These findings for JRA are consistent with findings for other autoimmune diseases and support the notion that common genetic regions contribute to an autoimmune phenotype.

Objective Levels of interleukin-6 (IL-6) have been shown to correlate with the fever and disease activity of systemic juvenile idiopathic arthritis (JIA). In a previous case–control study, a significant association between the IL-6 −174 nucleotide variant and systemic JIA was noted, and HeLa cell transfection assays show functional differences in levels of transcription of the IL-6 −174 alleles. The present study was undertaken to confirm the previous findings and to assess possible association with variations of the AnTn tract in the promoter. Methods We studied a cohort of JIA families from 3 countries, using transmission disequilibrium testing. Genotyping of the −174 nucleotide variant was done by restriction fragment length polymorphism, heteroduplex analysis, or allelic discrimination. The AnTn tract at −392 to −373 was typed using DNA sequencing. Statistical analysis was performed using the programs Transmit and EHplus. Results There was a significant excess transmission of the −174G allele in the systemic JIA families (P = 0.041). The excess transmission was only to systemic JIA patients with age at onset >5 years (P = 0.007). No significant association with the other subtypes was found. No AnTn alleles or −174/AnTn haplotypes were significantly associated with systemic JIA. Conclusion This study confirms that the IL-6 –174 nucleotide variant is significantly associated with systemic JIA. The significant excess transmission to patients with age at onset >5 years but not to those with age at onset ≤5 years suggests that there may be genetic heterogeneity between the 2 groups.

Objective To determine if the prevalence of autoimmunity among relatives of patients with juvenile rheumatoid arthritis (JRA) is greater than that among relatives of healthy volunteer control subjects. Methods Interviews were used to obtain histories of the following disorders among living first- and second-degree relatives of 110 patients and 45 controls: alopecia areata, ankylosing spondylitis, dermatomyositis, Graves' disease, Hashimoto thyroiditis, insulin-dependent diabetes mellitus, inflammatory bowel disease, iritis, JRA, multiple sclerosis, psoriasis, RA, systemic lupus erythematosus, and vitiligo. Chi-squares, odds ratios (ORs), and 95% confidence intervals (95% CIs) were calculated. Families of 23 JRA affected sibpairs were interviewed subsequently. Results There were no significant differences between patients and controls with regard to age, sex, ethnicity, or family size. Patients had 1,228 relatives and controls had 496 relatives. Of all the relatives of the patients, 155 had at least 1 autoimmune disorder, compared with 20 relatives of the controls (12.6% versus 4.0%; OR 3.4 [95% CI 2.1–5.7], P < 0.000001). The prevalence of autoimmunity was increased in first-degree and in second-degree relatives of patients (16.1% and 10.6%, respectively). The prevalence of Hashimoto thyroiditis was significantly higher in the relatives of patients (OR 3.5 [95% CI 1.6–7.9], P = 0.0008). The prevalences of other disorders were not significantly different. JRA affected sibpair families had an increased prevalence of autoimmunity (15.0%). A history of arthritis was found significantly more frequently in the JRA affected sibpair families, but not in the simplex families. Conclusion These data demonstrate that the prevalence of autoimmunity is significantly higher among first- and second-degree relatives of JRA patients. This suggests that clinically different autoimmune phenotypes may share common susceptibility genes, which may act as risk factors for autoimmunity.

Objective To test for linkage between the HLA region and juvenile rheumatoid arthritis (JRA), with stratification by onset and course types, in a cohort of affected sibling pairs (ASPs). Methods Eighty pairs of siblings with JRA who were registered with the Research Registry for JRA ASPs (sponsored by the National Institute of Arthritis and Musculoskeletal and Skin Diseases) were typed for HLA–DR. The observed ratio of sharing of none, one, or both parental DR alleles was compared against the expected ratio of 1:2:1 by goodness-of-fit chi-square tests. A group of 265 unrelated control subjects served as a comparison population for HLA–DR allele frequencies among patients, by Fisher's exact test. Results Overall, there was excess sharing of 2 DR alleles among ASPs with JRA. The observed ratio of sharing 0, 1, or 2 DR alleles was 8:40:32, instead of the expected ratio of 20:40:20 (P < 0.001). When stratified by JRA onset type, excess allele sharing was demonstrated among ASPs who were concordant for onset type (P = 0.002). This was true for both pauciarticular and polyarticular onset. When stratified by disease course, excess allele sharing was also demonstrated among ASPs who were concordant for disease course (P < 0.001). This was true for both the pauciarticular and the polyarticular course. Among the 32 ASPs who shared two DR alleles, 5 pairs had both DR8 and DR11, which was significantly more frequent (P < 0.0001) than the incidence in the control group (n = 0). Conclusion This study of an independent cohort of multiplex families confirms the previously reported linkage between pauciarticular JRA and the HLA–DR region that was identified using a different analytic method in a cohort of simplex families. Additionally, this study establishes evidence for linkage between polyarticular JRA and the HLA–DR region.

Objective To identify features of the T cell receptors (TCRs) present on clonally expanded T cells in the joints of patients with similar types of childhood rheumatic disease. Vbeta8 and Vbeta20 TCRs were selected as prototypic for polyarticular juvenile rheumatoid arthritis (JRA) and pauciarticular/juvenile spondylarthropathy (SpA), respectively. Methods The portion of the TCR beta chain involved in antigen recognition in the synovial tissue, synovial fluid, and peripheral blood from patients with JRA and juvenile SpA was cloned and sequenced. The frequency of expanded clonotypes, size of expansions, the Jbeta region, and sequence motifs were determined for >2,000 sequences. Results The majority of Vbeta20 and Vbeta8 clonal expansions were found in the joint rather than the peripheral blood. While instances of both Vbeta8 and Vbeta20 clonal expansion were detected in all disease types, the features of these expanded clonotypes were specific for disease type and Vbeta family. For example, Vbeta20 clonal expansion was characterized by many small expanded clonotypes in samples from patients with pauciarticular JRA and juvenile SpA while single large Vbeta8-specific expansions were found only in patients with polyarticular disease. Motifs specific to individual patients were identified, and for Vbeta20 clonotypes, a motif was found in synovial tissue samples. Conclusion Identification of common TCR features in oligoclonal expansions within individual patients and between patients with the same type of JRA suggests the recognition of a common or limited group of antigens in these diseases.

Background To investigate the autoinflammatory hereditary periodic fever syndrome genes MVK and TNFRSF1A, and the NLRP1 and IL1 genes, for association with juvenile idiopathic arthritis (JIA). Methods For MVK, TNFRSF1A and NLRP1 pair-wise tagging SNPs across each gene were selected and for IL1A SNPs from a prior meta-analysis were included. 1054 UK Caucasian JIA patients were genotyped by Sequenom iPlex MassARRAY and allele and genotype frequencies compared with 5380 unrelated healthy UK Caucasian controls. Results Four SNPs were significantly associated with UK JIA: rs2071374 within intron 4 of IL1A (ptrend=0.006), rs2228576 3’ of TNFRSF1A (ptrend=0.009) and 2 SNPs, rs11836136 and rs7957619, within MVK (ptrend=0.006, ptrend=0.005 respectively). In all cases the association appeared to be driven by the systemic-onset JIA (SoJIA) subtype. Genotype data for the two MVK SNPs was available in a validation cohort of 814 JIA (oligoarticular and RF negative polyarticular) cases and 3058 controls from the US. Replication was not confirmed, however, further suggesting that this association is specific to SoJIA. Conclusions These findings extend the observations of the relevance of studying monogenic loci as candidates for complex diseases. We provide novel evidence of association of MVK and TNFRSF1A with UK JIA, specifically driven by association with SoJIA and further confirm that the IL1A SNP association with SoJIA is subtype specific. Replication is required in independent cohorts.

The initial T cell receptor (TCR) alpha gene rearrangements were analyzed in fetal and neonatal thymocyte hybridomas by Southern blotting. Interestingly, in 30% of all thymocyte hybridomas and in all fetal day 16 thymocyte hybridomas the most proximal J alpha 50 (psi J alpha) gene was rearranged. This rearrangement was found on one chromosome only and mostly in association with a delta rearrangement on the homologous chromosome. J alpha 50 was rearranged to multiple target genes based on the variable size of the restriction fragments. In addition, delta rearrangement was found with a concomitant alpha rearrangement in the majority of hybridomas and it was not only associated with J alpha 50 but with several other rearranged J alpha genes as well. Our results clearly demonstrate that T cell precursors are not pre-committed to either delta or alpha rearrangement but a flexible progenitor responds to multiple regulatory signals during T cell differentiation and they do not support the notion that delta rec-psi J alpha rearrangement is required for cell commitment to TCR alpha gene rearrangement.

Juvenile idiopathic arthritis (JIA) contains a heterogeneous mixture of arthritis phenotypes of unknown etiology. Human leukocyte antigen (HLA) associations have been observed for multiple subtypes, suggesting a role for adaptive immune mistargeting (1, 2). The participation of antibodies in disease pathogenesis is supported by several observations, including the prevalence of autoantibodies (such as ANA and anti-DEK), circulating immune complexes, and complement consumption in blood and joint fluid in some patients (3-6). One remarkable feature of the epidemiology of JIA is age of onset. The peak of JIA incidence occurs around 2-3 years of age (1, 7, 8). This peak is composed primarily of patients in the oligoarticular subgroup, but is evident also in seronegative polyarticular JIA, psoriatic JIA, and potentially systemic JIA (7, 9-11). By contrast, onset before age 1 year is unusual. The basis for this epidemiological pattern is unknown. One possibility is that it reflects the point of first contact between a genetically susceptible host and a specific environmental trigger. Supporting this concept, younger and older JIA patients exhibit different HLA associations, even within the same ILAR subtype (1, 2). A complementary possibility is that some feature of early childhood immunity may favor initiation of arthritis. Children and adults differ immunologically in multiple respects (12). For example, the proportion of circulating lymphocytes that are naive is initially high and falls gradually. Circulating IgG nadirs at approximately 3-4 months of age with the decay of maternal antibodies, rising to 60% of adult levels by 1 year and to adult levels by 10 years of age (13). Humoral immunity is also functionally immature in young children, evident most strikingly in poor responses to polysaccharide antigens in the first 18-24 months of life (14). Another potentially important difference between pediatric and adult immunity concerns IgG glycosylation. Approximately 3% of the mass of IgG is carbohydrate, representing principally 2 branched glycans that attach to a canonical asparagine (Asn297) in each heavy chain (Figure 1A) (15). These oligosaccharides reside within the Fc region, where they help to maintain its three-dimensional conformation (16). Polymorphisms in IgG glycans modulate its ability to bind Fc receptors and fix complement, and thus are highly determinative of antibody effector function (15, 17). In particular, IgG glycans lacking galactose (G0) bind mannose-binding lectin, thereby facilitating activation of complement (Figure 1B) (18-20). Interestingly, one analysis of IgG glycans in healthy individuals found that children, like older adults, exhibited an excess of pro-inflammatory G0 IgG glycoforms (21), though a smaller survey did not identify this pattern (22). Figure 1 Structure of IgG-associated N-glycans Hypogalactosylation of IgG may be particularly relevant for arthritis. Studies over the last 30 years have demonstrated that adult rheumatoid arthritis (RA) is associated with an increased prevalence of circulating G0 IgG (23-29). Further, elevation in G0 IgG can predate the diagnosis of RA by more than 3 years, varies directly with disease activity, and is particularly pronounced for antibodies directed against citrullinated peptides (28, 30). Finally, animal studies have suggested that antibodies rendered G0 by enzymatic techniques are especially arthritogenic (31). Together, these studies support a role for aberrant IgG glycosylation in the pathogenesis of RA. A similar mechanism may be operative in JIA. Several groups have studied IgG glycans in patients with arthritis beginning before the age of 16 years (24-26, 32, 33). Using diverse methodologies, these series have generally (though not invariably (32)) come to the conclusion that IgG hypogalactosylation is a feature of childhood arthritis. Yet the data have important limitations. First, the number of patients studied was relatively small (total n=100), divided among multiple phenotypic classes using diverse nomenclatures. Second, most patients were sampled while receiving treatment, known to alter IgG glycosylation (27, 29). Finally and most critically, patients were not always matched to controls by age. Indeed, only a very few normal samples have been characterized from children below the age of 6 years, the peak of JIA onset, rendering IgG glycan data from young JIA patients exceptionally difficult to interpret (21, 22). In the present study, we set out to characterize IgG hypogalactosylation in JIA, with a particular focus on early childhood. To achieve this goal, we collected samples from healthy children and patients with JIA naive to disease modifying anti-rheumatic drug (DMARD) treatment, enabling age-normalized analysis of IgG glycan variation by disease subtype, laboratory features and age of onset. Our results establish the association of JIA with aberrant IgG glycosylation, and demonstrate marked elevation in hypogalactosylated IgG in healthy young children, suggesting a possible mechanism favoring onset of JIA in early childhood.

Objective In a genome-wide association study of Caucasian patients with juvenile idiopathic arthritis (JIA), we have previously described findings limited to autoimmunity loci shared by JIA and other diseases. The present study was undertaken to identify novel JIA-predisposing loci using genome-wide approaches. Methods The discovery cohort consisted of Caucasian JIA cases (n = 814) and local controls (n = 658) genotyped on the Affymetrix Genome-Wide SNP 6.0 Array, along with 2,400 out-of-study controls. In a replication study, we genotyped 10 single-nucleotide polymorphisms (SNPs) in 1,744 cases and 7,010 controls from the US and Europe. Results Analysis within the discovery cohort provided evidence of associations at 3q13 within C3orf1 and near CD80 (rs4688011) (odds ratio [OR] 1.37, P = 1.88 × 10−6) and at 10q21 near JMJD1C (rs647989 [OR 1.59, P = 6.1 × 10−8], rs12411988 [OR 1.57, P = 1.16 × 10−7], and rs10995450 [OR 1.31, P = 6.74 × 10−5]). Meta-analysis provided further evidence of association for these 4 SNPs (P = 3.6 × 10−7 for rs4688011, P = 4.33 × 10−5 for rs6479891, P = 2.71 × 10−5 for rs12411988, and P = 5.39 × 10−5 for rs10995450). Gene expression data on 68 JIA cases and 23 local controls showed cis expression quantitative trait locus associations for C3orf1 SNP rs4688011 (P = 0.024 or P = 0.034, depending on the probe set) and JMJD1C SNPs rs6479891 and rs12411988 (P = 0.01 or P = 0.04, depending on the probe set and P = 0.008, respectively). Using a variance component liability model, it was estimated that common SNP variation accounts for approximately one-third of JIA susceptibility. Conclusion Genetic association results and correlated gene expression findings provide evidence of JIA association at 3q13 and suggest novel genes as plausible candidates in disease pathology.

Susceptibility to childhood onset rheumatoid arthritis: investigation of a weighted genetic risk score that integrates cumulative effects of five genetic risk variants Sampath Prahalad, Milton Brown, Susan D Thompson, Michael Zwick, David Cutler, Lori A Ponder, Jennifer Prozonic, Sheila Angeles-Han, Larry B Vogler, Christine W Kennedy, Carol A Wallace, Carol Wise, Marilynn G Punaro, Ann M Reed, Jane L Park, Elizabeth D Mellins, John F Bohnsack, David N Glass

Background/Purpose: Monocytes phenotype changes in different microenvironments: the proinflammatory M1, regulatory M2, and M2-like phenotypes are each regulated by specific transcriptional factors (TFs). We have observed altered phenotypes in blood monocytes in systemic onset juvenile idiopathic arthritis (sJIA), including a decreased M1 cells, an increased mixed M1/M2 cells, and reduced secretion of IL-1, despite an increased IL-1b response to LPS (PMID 22281427). Here, we investigate whether these monocyte phenotypes are affected by IL-1 blockade. We analyzed monocytes from RAPPORT (RAndomized Placebo Phase study Of Rilonacept in the Treatment of sJIA) patients to determine levels of TFs involved in monocyte polarization and expression of genes related to IL-1 secretion, before and after treatment with Rilonacept, an IL-1 trap. Methods: Subjects on the Rilonacept arm received active drug from week 0 for a total of 24 weeks; subjects on the placebo arm received placebo for 4 weeks, then Rilonacept for 20 weeks. Blood samples were obtained at week 0, 2, 4, 14 and 24. We used real time PCR to measure M1 associated genes: Interferon Regulatory factor (IRF) family IRF5, STAT1; M2 associated genes IRF4, STAT6, Kruppel-Like Factor 4 (KLF4) and peroxisome proliferator-activated receptor-γ (PPAR-γ); IL-1 secretion related genes: RAB39, RAB27A, RAB27B, P2RX7, and IL-1β. All TFs levels were normalized by the average levels of 3 housekeeping genes. Results: 30 non-paired RNA samples from 15 subjects were tested. Samples from subjects treated with Rilonacept for ≥10 weeks (Late RAPPORT) showed decreased expression of M2 genes, especially KLF4, compared to those untreated or treated for

Background/Purpose: Genome-wide association studies have identified susceptibility loci for many autoimmune diseases, including rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). About 5% of children with JIA have rheumatoid factor positive arthritis that is phenotypically similar to adult seropositive RA, thus representing childhood onset RA (CORA). To understand the genetic architecture of CORA risk relative to other autoimmune diseases, we genotyped CORA cases and controls on the Immunochip, a custom array designed by the Immunochip Consortium to fine map autoimmune disease-associated loci shared across 11 autoimmune phenotypes. Methods: Genotyping was completed on 340 CORA cases (mean onset age: 10.2 ± 4.2 yrs) and 11624 controls. Standard SNP and sample QC was performed (e.g., removing samples with call rate

Objective—To examine both the source of follistatin-like protein 1 (FSTL-1) and factors that induce its expression in arthritis and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by over-expression of FSTL-1. Methods—FSTL-1 expression patterns were analyzed by immunohistochemistry on joints derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to TGF-β, IL-1β, TNF-α, and IL-6. Finally, sera and synovial fluids from children with oligoarticular, polyarticular, and systemic-onset JRA were assayed for FSTL-1 using a custom ELISA. FSTL-1 concentrations were correlated with erythrocyte sedimentation rate (ESR) and platelet count. Results—Immunohistochemical staining of joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGF-β, IL-1β, TNF-α, and IL-6. The IL-1β response was significantly greater than the TNF-α response (p < 0.05). Only the systemic-onset JRA subtype had elevated serum and synovial fluid FSTL-1 concentrations. Synovial fluid concentrations were 2–3-fold higher than serum concentrations. The elevation in serum FSTL-1 concentrations seen in systemic-onset JRA correlated closely with elevated ESR and platelet count. Conclusion—These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1β is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA. Juvenile rheumatoid arthritis (JRA) encompasses a heterogeneous group of diseases that are important causes of morbidity in children. JRA affects an estimated 250,000 children in the United States. The American College of Rheumatology (ACR) has classified JRA into a number of subtypes, including systemic-onset, polyarthritis, and oligoarthritis (1). Each of these subtypes has a different clinical presentation, prognosis, and response to specific therapies, suggesting that they differ in their pathogenesis and pathophysiology. For

Introduction Previous observations suggest that active systemic juvenile idiopathic arthritis (sJIA) is associated with a prominent erythropoiesis gene-expression signature. The aim of this study was to determine the association of this signature with peripheral blood mononuclear cell (PBMC) subpopulations and its specificity for sJIA as compared with related conditions. Methods The 199 patients with JIA (23 sJIA and 176 non-sJIA) and 38 controls were studied. PBMCs were isolated and analyzed for multiple surface antigens with flow cytometry and for gene-expression profiles. The proportions of different PBMC subpopulations were compared among sJIA, non-sJIA patients, and controls and subsequently correlated with the strength of the erythropoiesis signature. Additional gene-expression data from patients with familial hemophagocytic lymphohistiocytosis (FHLH) and from a published sJIA cohort were analyzed to determine whether the erythropoiesis signature was present. Results Patients with sJIA had significantly increased proportions of immature cell populations, including CD34+ cells, correlating highly with the strength of the erythropoiesis signature. The erythropoiesis signature strongly overlapped with the gene-expression pattern in purified immature erythroid precursors. The expansion of immature cells was most prominently seen in patients with sJIA and anemia, even in the absence of reticulocytosis. Patients with non-sJIA and anemia did not exhibit the erythropoiesis signature. The erythropoiesis signature was found to be prominent in patients with FHLH and in a published cohort of patients with active sJIA, but not in patients with inactive sJIA. Conclusions An erythropoiesis signature in active sJIA is associated with the expansion of CD34+ cells, also is seen in some patients with FHLH and infection, and may be an indicator of ineffective erythropoiesis and hemophagocytosis due to hypercytokinemia.

To identify differences in peripheral blood gene expression between patients with different subclasses of juvenile idiopathic arthritis (JIA) and healthy controls in a multicenter study of patients with recent-onset JIA prior to treatment with disease-modifying antirheumatic drugs (DMARDs) or biologic agents.Peripheral blood mononuclear cells (PBMCs) from 59 healthy children and 136 patients with JIA (28 with enthesitis-related arthritis [ERA], 42 with persistent oligoarthritis, 45 with rheumatoid factor [RF]-negative polyarthritis, and 21 with systemic disease) were isolated from whole blood. Poly(A) RNA was labeled using a commercial RNA amplification and labeling system (NuGEN Ovation), and gene expression profiles were obtained using commercial expression microarrays (Affymetrix HG-U133 Plus 2.0).A total of 9,501 differentially expressed probe sets were identified among the JIA subtypes and controls (by analysis of variance; false discovery rate 5%). Specifically, 193, 1,036, 873, and 7,595 probe sets were different in PBMCs from the controls compared with those from the ERA, persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA patients, respectively. In patients with persistent oligoarthritis, RF-negative polyarthritis, and systemic JIA subtypes, up-regulation of genes associated with interleukin-10 (IL-10) signaling was prominent. A hemoglobin cluster was identified that was underexpressed in ERA patients but overexpressed in systemic JIA patients. The influence of JAK/STAT, ERK/MAPK, IL-2, and B cell receptor signaling pathways was evident in patients with persistent oligoarthritis. In systemic JIA, up-regulation of innate immune pathways, including IL-6, Toll-like receptor/IL-1 receptor, and peroxisome proliferator-activated receptor signaling, were noted, along with down-regulation of gene networks related to natural killer cells and T cells. Complement and coagulation pathways were up-regulated in systemic JIA, with a subset of these genes being differentially expressed in other subtypes as well.Expression analysis identified differentially expressed genes in PBMCs obtained early in the disease from patients with different subtypes of JIA and in healthy controls, providing evidence of immunobiologic differences between these forms of childhood arthritis.

Polyarticular juvenile idiopathic arthritis (JIA) is chronic arthritis in more than four joints for more than six weeks with onset before the 16th birthday in a child without other known causes of arthritis (1-3). Polyarticular JIA is divided into rheumatoid factor (RF) positive and negative sub-types, with the RF+ sub-type having positive tests for serum IgM RF on two occasions at least three months apart within the first six months of disease. Ravelli and Martini have recently proposed that RF- polyarticular JIA be divided into 3 subsets: one similar to adult rheumatoid arthritis, another with “dry synovitis” and a third similar to ANA+ early-onset oligoarticular JIA (3). Given this heterogeneity, it is not surprising that children with polyarticular JIA have a wide variety of disease courses and outcomes, ranging from self-limited arthritis with no long-term disability to relentless and destructive arthritis with severe disabilities (4). Unfortunately, our present ability to predict course and outcome is limited, with treatment typically tailored to current disease activity, assessment of which is also imperfect. Global gene expression profiling is a molecular technique that measures in parallel genome-wide expression of thousands of genes in a sample of cells. This technology holds promise for dramatically advancing knowledge of many diseases, including JIA. This approach has already provided important information regarding classification and pathogenesis of several JIA sub-types in studies that generally used small numbers of subjects with varying degrees of clinical diversity (5-9). In the present study, global gene expression profiling of peripheral blood mononuclear cells (PBMC) was used to characterize a relatively large population of children with recent onset polyarticular JIA (both RF- and RF+) who had not been treated with methotrexate, biologics or other disease modifying anti-rheumatic drugs (DMARDs). The goals of applying this technology to JIA are to advance understanding of disease pathogenesis, improve assessment of disease activity, predict response to medications and foresee long-term outcomes. The present work takes a step toward these goals by defining gene expression signatures that appear to be associated with distinct disease processes in subgroups of children with polyarticular JIA.

To examine the relationships between T cell populations at various stages of development, T cell receptor (TcR) gene rearrangements were compared between the four murine populations of (a) early thymocytes, (b) early splenocytes, (c) adult thymocytes and (d) adult splenocytes. TcR alpha gene rearrangements were shown to progress from 5' to 3' regions of the J alpha locus and from 3' to 5' regions of the V alpha locus during the development of T cells in both the thymus and spleen. Thus, the gene rearrangement potentials of proximal genes varied with age, yielding a biased repertoire in the young vs. adult animal. As evidence that gamma/delta and alpha/beta gene rearrangements appeared concomitantly in individual precursors, it was found that: (a) multiple adult thymocytes bore alpha gene rearrangements on one chromosome and delta gene rearrangements on the homologous chromosome, and (b) V gamma 3-J gamma 1 rearrangements, prominent joins in the early gamma/delta T cell population, were also prominent in the early alpha/beta T cell subset. These data illustrate the non-random nature of the developmental TcR gene rearrangement and suggest that alpha/beta and gamma/delta T cell populations derive from related, if not identical, T cell precursor populations.

Background A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this female bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. The JIA genetic risk variant, PTPN22 rs2476601, has been reported to show female-specific association in type 1 diabetes. Objectives We sought to determine whether PTPN22 rs2476601 exhibits a female-specific association with JIA. Methods The discovery sample was drawn from the Australian CLARITY JIA biobank. rs2476601 was genotyped in 413 cases (67% female, mean age 9.1 years) and 690 healthy controls (42% female, mean age 7.1 years). We used logistic regression to assess the association of rs2476601 with JIA in the total sample (males and females). We then explored the role of sex in this association: we adjusted the regression analysis for sex, stratified by sex, and performed a genotype x sex interaction analysis. Sensitivity analyses were performed by restricting to individuals of European descent, subtypes of oligoarticular and polyarticular RF negative disease course, and exclusion of systemic JIA. We sought to replicate the sex-specific associations in an independent JIA case-control sample collected from the USA and Norway and held at the Children9s Hospital of Philadelphia. Analyses were performed using Stata or PLINK. Results The outcomes of analyses are shown in the Table. rs2476601 was associated with all JIA in both the discovery and replication samples, and the association remained when adjusted for sex. When stratified by sex, a strong association was observed for females, but not for males. There was evidence for genotype-by-sex interaction (p=0.0087). Similar patterns were observed in all sensitivity analyses. In the replication sample, we also observed association of rs2476601 with JIA in females, but not in males. Conclusions We have demonstrated, in both a discovery and replication sample, that the association between PTPN22 rs2476601 and JIA appears restricted to females, helping to explain the greater number of females that develop this disease. Disclosure of Interest None declared

Background Juvenile idiopathic arthritis (JIA) is the most common arthritic disease of childhood and is caused by a combination of genes and environment. In the last few years great advances have been made in dissecting the genetic basis of JIA with now 17 confirmed susceptibility loci identified. One of these loci, the MHC region, has been established for many years but the complexity and the broad linkage disequilibrium across the region has rendered fine-mapping associations challenging. Novel imputation strategies can now be utilized to impute HLA classical alleles and amino acids across the region. Objectives The aim of this work was to gain a greater understanding of the associations across the HLA region in all JIA subtypes. Methods Using the dense genotype data obtained from the analysis of the Illumina ImmunoChip in 4574 JIA cases (not including the systemic-onset cases) and 14412 controls, we imputed HLA classical alleles (2-digit and 4-digit resolution) and amino acids across the MHC region (Chr6:29-34Mb) using the SNP2HLA algorithm and a large reference panel. We performed univariate analysis for all markers across the region and tested all amino acids in HLA-DRB1 by performing an omnibus test of amino acid residues for each position. Conditional analysis, including the most significant marker as a covariate, was performed to identify independent effects. Analysis was repeated in the individual subtypes (persistent oligoarthritis (pO) n=1751, extended oligoarthritis (eO) n=658, RF negative polyarthritis (RF-P) n=1525, RF positive polyarthritis (RF+P) n=337, enthesitis related arthritis (ERA) n=183, psoriatic arthritis (jPsA) n=112). Results The omnibus test for all amino acids across HLA-DRB1 showed most significant association at HLA-DRB1 amino acid (AA) 67 and conditioning on all residues at AA 67 found significant association remaining at HLA-DRB1 AA 13 (the glycine residue most associated), suggesting two independent effects in HLA-DRB1. There was evidence for further effects in HLA-DRB1. Conditioning on all alleles of HLA-DRB1 found additional independent effects at HLA-DPB1-0201 and HLA-A AA 95. These associations hold across RF-P and both pO and eO subtypes, HLA-DRB1 AA 13 is most strongly associated in RF+P with the histidine residue driving the association. In the ERA subtype HLA-B27 showed the strongest association. For jPsA no HLA markers reached genome-wide significance. Conclusions Analysis of the MHC region in the largest cohort of JIA cases and controls studied to date has found the strongest association with the HLA-DRB1 region, with additional multiple independent effects. Acknowledgements Juvenile Arthritis Consortium for Immunochip (JACI) Disclosure of Interest None declared

Background Juvenile Idiopathic Arthritis (JIA) patients have arthritis onset of unknown cause that lasts >6 weeks in the inflamed joints of children younger than 16 years. JIA is a complex genetic trait with established increased familial risk and robustly replicated genetic associations (e.g. HLA, PTPN2 ). Multiple single nucleotide polymorphisms (SNPs) in the 22q13.1 locus have reached genome-wide significance in recent genetic association studies using the Immunochip. The 22q13.1 locus contains both IL2RB and RAC2 which are attractive candidates to influence JIA pathology and have respective roles in IL-2 signaling and cell migration. Objectives To identify the statistically, bioinformatically and experimentally most plausible causal SNPs associated with risk of JIA in the 22q13 region. Methods We fine mapped the 22q13.1 locus using data from large scale genotyping projects of a European ancestry cohort of subjects with (3,905) and without JIA (14,388) to predict variants most likely causal at the 22q13.1 locus. Logistic regression conditional analysis was used to define regions of association followed by Bayesian analysis to define variants with large posterior probabilities. Transcription factor binding predictions were used to identify variants for experimental testing by electrophoretic mobility shift assay (EMSA). We limited our investigation to polyarticular RF - and oligoarticular JIA cases to reduce phenotypic heterogeneity. Results Conditional analyses showed independent associations to JIA in both the IL2RB and RAC2 loci. We identified two SNPs that can account for all of the JIA genetic association in this region. Using a Bayesian analysis we identified the SNPs that explained 95% of the posterior probability in the region and are the most likely to be causal. Altered nuclear extract binding was observed with risk and non-risk alleles in the IL2RB locus but not RAC2. These experimental data are consistent with the predicted alteration in transcription factor binding. Conclusions Polymorphic variants in the RAC2 and the nearby IL2RB locus are independent JIA risk loci. Use of the statistical and bioinformatics approach to produce candidates for laboratory validation is an important strategy in defining the function of risk variants in JIA. Disclosure of Interest None declared

Objective To investigate the clinical phenotypes and demographic characteristics of 183 affected sibling pairs (ASPs) with juvenile rheumatoid arthritis (JRA) and to determine whether there are differences between the clinical phenotypes of the ASP cohort compared with patients with sporadic disease and whether there is greater sharing of specific clinical features within versus between sibpairs. Methods Details of the JRA Affected Sibpair Registry operations have been described previously. The frequencies of phenotypes in the 2 cohorts were tabulated, summary statistics were determined, and comparisons were made by chi-square test or t-test. Sibling risk, sibling risk ratios (λs), and odds ratios were calculated to assess familial aggregation of several different clinical manifestations. Results The most common onset type among the 164 nontwin ASPs was pauciarticular (65% overall). Fifty-three percent of the ASPs were concordant for pauciarticular-onset JRA; 19% were concordant for a polyarticular disease onset. Among subjects with polyarticular-onset disease, significantly more joints were involved at onset in simplex patients than in ASPs (P = 0.008). The difference in age at JRA onset within sibpairs (sibling 1 versus sibling 2) was not significantly different. ASPs developed disease at a mean real-time difference of 5.1 years apart. Familial aggregation was found for tenosynovitis (λs 29.5), leukocytosis (λs 25), rheumatoid factor (λs 11.0), anemia (λs 1.7), and antinuclear antibodies (λs 1.3). Conclusion This study confirms the findings of earlier studies showing that a high proportion of ASPs overall show concordance of disease-onset type, except for the subset of patients with systemic disease, and that nontwin ASPs do not develop disease at the same point in real time. We conclude that JRA and its clinical manifestations do not differ substantially between ASPs and the simplex population. The exception is the number of affected joints at JRA onset among patients with polyarticular-onset disease. Familial aggregation of clinical features among ASPs adds strong evidence for a genetic background in this disease.

Background/Purpose: Oligoarticular (oligo) onset juvenile idiopathic arthritis (JIA) is defined as 4 or fewer affected joints within 6 mo of disease onset. This JIA subtype typically involves large joints. Patients who develop arthritis in >4 joints after the first 6 mo are considered to have extended oligo JIA, and have a worse prognosis. Patients with polyarticular (poly) onset JIA, defined 5 or more affected joints within 6 mo of disease onset, overall have a poorer prognosis than either oligo or extended oligo JIA patients. Poly onset JIA typically involves small joints such as joints of the fingers and toes. It has not been previously determined whether oligo onset JIA patients with small joint involvement are more likely to develop extended oligo JIA. The objective of this study was to compare the incidence of extended oligo JIA between oligo JIA patients with only small versus only large joint involvement at disease onset. Methods: We conducted a retrospective cohort study comparing patients with oligo onset JIA with only small joint (n=24) versus only large joint involvement (n= 47). All patients from our clinic from 2006–2013 with only small joint oligo JIA in the first 6 months of disease were included in the cohort. Because of the rarity of such patients, we also included small joint oligo onset JIA patients from a multicenter JIA genomics study based at Cincinnati Children's Hospital Medical Center. The large joint group was randomly selected from our clinic population of oligo JIA patients with only large joint involvement in the first 6 mo. The two groups were compared using the Wilcoxon rank sum test for continuous variables and Pearson's Chi-square test or Fisher's exact test for categorical variables. Results: 9 out of 24 (37.5%) patients with oligo onset JIA involving only small joints developed extended arthritis (>4 joints), compared to 3 out of 47 (6.38%) of patients with only large joint oligo onset JIA [p = 0.002]. Although not statistically significant, there was a trend towards female gender in the small joint group [79% females in the small joint only group, and 57% females in the large joint only group; P = 0.07], and in length of follow up with a mean of 71 months in the small joint group (median: 65, range 12–183 months) and 53 months in the large joint group (median: 45, range 9–165 months) [P = 0.07]. There was no statistically significant difference in percent of patients with positive ANA or mean age of onset between the two groups Conclusion: Patients with oligo onset JIA involving only small joints are more likely to develop extended arthritis. These patients may warrant more aggressive early treatment compared to their peers with large joint oligo onset JIA. Further studies with a larger cohort are needed before changes to treatment approaches can be recommended. The heterogeneity demonstrated within oligo onset JIA highlights the limitations of the current JIA subtype classification criteria.

Background/Purpose: Autoimmune disorders affect 5 to 10% of the population. Clinically distinct autoimmune phenotypes often share genetic susceptibility factors. Relatives of children with juvenile idiopathic arthritis (JIA) have increased prevalence of autoimmunity. We sought to investigate the prevalence of familial autoimmunity among subjects with JIA, systemic lupus erythematosus (SLE) and juvenile dermatomyositis (JDM) in the CARRA Registry, the largest multicenter observational Registry for pediatric rheumatic disease. Methods: Children with JIA, SLE and JDM from pediatric rheumatology clinics in the US were enrolled in the CARRA Registry between May 2010 and May 2013. Demographic and disease-related data were collected from time of diagnosis to enrollment visit. We compared if subjects with JIA, SLE and JDM had different proportions of autoimmunity among first-degree relatives (FDR) using Chi-square tests. If a significant difference was detected, pairwise comparisons, adjusted for multiple comparisons, were made using a significance level of 0.017. Results: The sample consisted of 4677 JIA, 639 SLE and 440 JDM subjects. The proportion of subjects having FDR with any autoimmune disease in the JDM group (20.5%) was significantly less compared to subjects with JIA (31.8%, p < 0.001) or SLE (31.9%; p < 0.001). Compared to subjects with JDM, subjects with JIA had a greater proportion of FDR with JIA (p < 0.001), and RA (p = 0.002), and compared to subjects with SLE, those with JIA had a significantly greater proportion of FDR with psoriasis (p < 0.001), JIA (p = 0.001), ankylosing spondylitis (p = 0.016), Crohn's disease/ulcerative colitis (p = 0.002), and autoimmune thyroiditis (p = 0.005). Compared to those with JIA, subjects with SLE had a significantly higher proportion of FDR with SLE (p < 0.001) and type-I diabetes (p < 0.001), and compared to subjects with JDM, those with SLE had a significantly higher proportion of FDR with SLE (p < 0.001), RA (p = 0.003), and type-I diabetes (p < 0.001).1 Table 1. Comparison of familial autoimmunity among subjects with JIA, SLE and JDM Disease Group Family History of Disease JIA N = 4,677 SLE N = 639 JDM N = 440 P-value a All values expressed as percentages. b Significantly different from JIA (p < 0.017) c Significantly different from SLE (p < 0.017) Any autoimmune diseases 31.8 31.9 20.5b, c < 0.001 Inflammatory Arthritis-AS, SpA, JIA, RA 13.0 9.2b 4.3b, c < 0.001 - Ankylosing spondylitis 1.3 0.2b 0.5 0.014 - Spondyloarthopathy 0.8 0.0 0.0 0.015 - JIA 5.2 2.2b 0.9b < 0.001 - RA 7.3 7.8 3.4b, c 0.007 SLE 1.7 11.1b 1.1c < 0.001 Psoriasis 8.0 2.5b 5.0 < 0.001 Crohn's disease or ulcerative colitis 2.7 0.6b 1.1 0.002 Multiple Sclerosis 1.0 1.1 1.1 0.905 Autoimmune thyroiditis 5.3 2.7b 5.5 0.015 Celiac disease 0.8 0.8 1.4 0.439 Type 1 Diabetes 3.1 7.4b 3.0c < 0.001 Other autoimmune 4.6 5.0 4.6 0.893 Conclusion: Children with AAV had predominantly renal and constitutional manifestations. Younger age and a more severe renal disease phenotype may characterize MPA with patients requiring additional treatment for the consequences of kidney disease. The wide variations in time to diagnosis continue to suggest that pediatric AAV is poorly recognized. Ongoing biomarker-driven studies may complement systems for subclassifying patients with AAV.

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-γ mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4− cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-γ than CD4+/CCR4− cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.

Background One of the principal findings of genome wide association studies in autoimmune diseases has been the substantial overlap of genetic susceptibility loci identified. This has underpinned fine mapping initiatives such as the Immunochip (IC) project in which custom chips were designed to fine-map regions of associations common to a number of autoimmune diseases. Samples from patients with rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA) and psoriatic arthritis (PsA) have been genotyped using IC; given that all are types of inflammatory arthritis (IA) which exhibit common pathology and some overlap of associated loci, the data emerging from IC provides a unique opportunity to identify novel overlapping regions and determine whether the causal variants within overlapping loci are the same or different for each disease. Objectives To investigate IC fine mapping data to identify genetic regions which are associated with susceptibility to more than one type of IA. Methods As part of the IC project, genotyping, quality control and association analysis was performed for RA (14056 cases and 9583 controls), JIA (1614 polygoarticular and oligoarticular JIA cases and 7153 controls) and PsA (929 cases and 4537 controls). Some controls were shared between cohorts. For this analysis all common SNPs (MAF>0.05) with p -3 in any of the 3 diseases were first selected. IC regions were defined as including all SNPs from the 1000 Genomes Project CEU population (September 2009 release) that were in 0.1-cM (HapMap3 CEU) recombination blocks around each GWAS region lead marker. IC Regions which contained SNPs associated with more than one type of IA were considered overlapping. Results 26 regions showed association at p -3 with more than one type of IA, of these, in RA and JIA two loci ( PTPN22 and STAT4 ) reached genome wide significance (5×10 -8 ), with a further 8 associated at p -4 in both diseases. Interestingly 2 of these loci are also associated with PsA at p -3 ( RUNX1 and PTPN2 ). 4 of these loci have been previously associated with both RA and JIA ( PTPN22, STAT4, PTPN2, and IL2RA ) whilst 6 are novel overlapping regions ( ANKRD55, RUNX1, IL2RB, DNASE1L3, UBE2L3 and COG6 ). For the PTPN22, STAT4, ANKRD55, DNASE1L3, PTPN2 and UBE2L3 regions the RA and JIA associated SNPs are either identical or highly correlated (r 2 >0.8), the direction of effect is the same in both diseases and odds ratios similar. For RUNX1 and COG6 the associated SNPs are different and weakly correlated (r 2 >0.4 IL2RA and IL2RB the pattern of association in the region is completely different. Conclusions These findings add to the body of evidence that there are shared susceptibility genes for inflammatory arthritis. Further investigation is required to fully explore whether the regions contain the same or different causal effect, including functional studies to determine whether, in overlapping loci with the same causal variant, the variant predisposes to disease by the same mechanism. Disclosure of Interest None Declared

Background Genome wide association studies (GWAS) have been hugely successful in identifying susceptibility loci for autoimmune diseases. One interesting outcome of GWAS is the observation that many of the loci are shared across these diseases. The regions identified now require more detailed fine-mapping to localize the association signal and identify putative functional variants. The Immunochip consortium was established to pool confirmed loci from 12 diseases onto a custom genotyping chip investigating ∼200 established autoimmune susceptibility loci. For each locus, all known genetic variation from multiple sources was included. Juvenile idiopathic arthritis (JIA) is the most common arthritic disease of childhood. Candidate gene studies have identified a number of common autoimmune genes that confer susceptibility to JIA, however JIA has been less well studied using large-scale approaches. Objectives To use the Immunochip to fine-map previously associated regions and to identifying novel loci for JIA. Methods Genotyping was performed using the Immunochip, in a large cohort from the UK, US and Germany comprising 1749 JIA oligoarthritis and RF negative polyarthritis cases and 8854 controls. All raw genotyping data was combined for clustering and QC. SNPs failed QC based on a call rate Results Initial analysis has not only confirmed previously associated JIA loci (HLA, PTPN22, IL2, STAT4, PTPN2 and SH2B3/ATXN2) but has strengthened their association, such that all now reach genome-wide significance. A number of novel loci have been identified, some of which showed weak evidence previously, such as IL2RA, IL7R and IRF1, and others which have never been associated with JIA, such as RUNX1, FAS and ANKRD55. These will require validation in independent cohorts. Conclusions The Immunochip project enables cost-effective fine-mapping of autoimmune loci in diseases such as JIA. This preliminary analysis has confirmed and strengthened the association of a number of previously associated genes as well as the identification of novel susceptibility loci for JIA. Further analysis of this data will help characterize all associated variants and identify the likely causal variants for future functional studies. Acknowledgements:Childhood Arthritis Prospective Study (CAPS), Childhood Arthritis Response to Medication Study (SPARKS-CHARMS), BSPAR study group, UK JIA Genetics Consortium (UKJIAGC), Cincinnati Registry for Juvenile Arthritis Genetics (CRJAG), Consortium for Juvenile Arthritis Genetics (CJAG), and USA-Juvenile Arthritis Genetics Cohort (USA-JAGC). Disclosure of Interest None Declared

Purpose The structure of human IgG includes 2 internal glycans that modulate the conformation and function of the Fc region. Adult-type rheumatoid arthritis (RA) has been associated with an elevated proportion of hypogalactosylated (G0) IgG that exhibits enhanced fixation of complement and greater pathogenicity in experimental murine arthritis. We aimed to determine the profile of IgG glycans in normal children and in those with juvenile idiopathic arthritis (JIA), for comparison with healthy adults and adult RA controls.

JIA affected sibling pairs present high correlation for ANA and ILAR category G Filocamo, C Malattia, I Foeldvari, V Stanevicha, S Nielsen, T Herlin, C Pruunsild, F Zulian, Z Balogh, F Dressler, I Rumba, MG Alpigiani, E Cortis, F Falcini, R Trauzeddel, G Calcagno, L Lepore, M Alessio, DN Glass, SD Thompson, A Martini, N Ruperto, for the Paediatric Rheumatology International Trials Organisation (PRINTO)