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OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine whether peripheral blood mononuclear cells (PBMCs) from children with recent-onset polyarticular juvenile idiopathic arthritis (JIA) exhibit biologically or clinically informative gene expression signatures. METHODS: Peripheral blood samples were obtained from 59 healthy children and 61 children with polyarticular JIA prior to treatment with second-line medications, such as methotrexate or biologic agents. RNA was extracted from isolated mononuclear cells, fluorescence labeled, and hybridized to commercial gene expression microarrays (Affymetrix HG-U133 Plus 2.0). Data were analyzed using analysis of variance at a 5% false discovery rate threshold after robust multichip analysis preprocessing and distance-weighted discrimination normalization. RESULTS: Initial analysis revealed 873 probe sets for genes that were differentially expressed between polyarticular JIA patients and healthy controls. Hierarchical clustering of these probe sets distinguished 3 subgroups within the polyarticular JIA group. Prototypical patients within each subgroup were identified and used to define subgroup-specific gene expression signatures. One of these signatures was associated with monocyte markers, another with transforming growth factor beta-inducible genes, and a third with immediate early genes. Correlation of gene expression signatures with clinical and biologic features of JIA subgroups suggested relevance to aspects of disease activity and supported the division of polyarticular JIA into distinct subsets. CONCLUSION: Gene expression signatures in PBMCs from patients with recent-onset polyarticular JIA reflect discrete disease processes and offer a molecular classification of disease. [ABSTRACT FROM AUTHOR]

OBJECTIVE: 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. METHODS: 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 of >300 children with JIA and >500 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. RESULTS: 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 in >800 JIA cases and >500 controls. The meta-analysis also failed to confirm an association between JIA and CTLA4 variants. CONCLUSION: These results suggest that C-318T, A49G, CT60, and haplotypes tagged by these CTLA4 SNPs are not associated with JIA or major JIA subtypes. [ABSTRACT FROM AUTHOR]

Background: Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children, the etiology of which involves a strong genetic component, but much of the underlying genetic determinants still remain unknown. Our aim was to identify novel genetic variants that predispose to JIA., Methods: We performed a genome-wide association study (GWAS) and replication in a total of 1166 JIA cases and 9500 unrelated controls of European ancestry. Correlation of SNP genotype and gene expression was investigated. Then we conducted targeted resequencing of a candidate locus, among a subset of 480 cases and 480 controls. SUM test was performed to evaluate the association of the identified rare functional variants., Results: The CXCR4 locus on 2q22.1 was found to be significantly associated with JIA, peaking at SNP rs953387. However, this result is subjected to subpopulation stratification within the subjects of European ancestry. After adjusting for principal components, nominal significant association remained (p < 10(-4)). Because of its interesting known function in immune regulation, we carried out further analyses to assess its relationship with JIA. Expression of CXCR4 was correlated with CXCR4 rs953387 genotypes in lymphoblastoid cell lines (p = 0.014) and T-cells (p = 0.0054). In addition, rare non-synonymous and stop-gain sequence variants in CXCR4, putatively damaging for CXCR4 function, were significantly enriched in JIA cases (p = 0.015)., Conclusion: Our results suggest the association of CXCR4 variants with JIA, implicating that this gene may be involved in the pathogenesis of autoimmune disease. However, because this locus is subjected to population stratification within the subjects of European ancestry, additional replication is still necessary for this locus to be considered a true risk locus for JIA. This cell-surface chemokine receptor has already been targeted in other diseases and may serve as a tractable therapeutic target for a specific subset of pediatric arthritis patients with additional replication and functional validation of the locus.

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., (Copyright © 2013 by the American College of Rheumatology.)

We used the Immunochip array to analyze 2,816 individuals with juvenile idiopathic arthritis (JIA), comprising the most common subtypes (oligoarticular and rheumatoid factor-negative polyarticular JIA), and 13,056 controls. We confirmed association of 3 known JIA risk loci (the human leukocyte antigen (HLA) region, PTPN22 and PTPN2) and identified 14 loci reaching genome-wide significance (P < 5 × 10(-8)) for the first time. Eleven additional new regions showed suggestive evidence of association with JIA (P < 1 × 10(-6)). Dense mapping of loci along with bioinformatics analysis refined the associations to one gene in each of eight regions, highlighting crucial pathways, including the interleukin (IL)-2 pathway, in JIA disease pathogenesis. The entire Immunochip content, the HLA region and the top 27 loci (P < 1 × 10(-6)) explain an estimated 18, 13 and 6% of the risk of JIA, respectively. In summary, this is the largest collection of JIA cases investigated so far and provides new insight into the genetic basis of this childhood autoimmune disease.

Objective: Rheumatoid arthritis is associated with an excess of agalactosylated (G0) IgG that is considered relatively proinflammatory. Assessment of this association in juvenile idiopathic arthritis (JIA) is complicated by age-dependent IgG glycan variation. The aim of this study was to conduct the first large-scale survey of IgG glycans in healthy children and patients with JIA, with a focus on early childhood, the time of peak JIA incidence., Methods: IgG glycans from healthy children and disease-modifying antirheumatic drug-naive patients with JIA were characterized using high-performance liquid chromatography. Agalactosylated glycans were quantitated with reference to monogalactosylated (G1) species. Associations were sought between the G0:G1 ratio and disease characteristics., Results: Among healthy children ages 9 months to 16 years (n = 165), the G0:G1 ratio was highly age dependent, with the ratio peaking to 1.19 in children younger than age 3 years and declining to a nadir of 0.83 after age 10 years (Spearman's ρ = 0.60, P < 0.0001). In patients with JIA (n = 141), the G0:G1 ratio was elevated compared with that in control subjects (1.32 versus 1.02; P < 0.0001). The G0:G1 ratio corrected for age was abnormally high in all JIA subtypes (enthesitis-related arthritis was not assessed), most strikingly in systemic JIA. Glycosylation aberrancy was comparable in patients with and those without antinuclear antibodies and in both early- and late-onset disease and exhibited at most a weak correlation with markers of inflammation., Conclusion: IgG glycosylation is skewed toward proinflammatory G0 variants in healthy children, in particular during the first few years of life. This deviation is exaggerated in patients with JIA. The role for IgG glycan variation in immune function in children, including the predilection of JIA for early childhood, remains to be defined., (Copyright © 2012 by the American College of Rheumatology.)

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., (Copyright © 2012 by the American College of Rheumatology.)

Objective: Associations between shared epitope (SE)-encoding HLA-DRB1 alleles and rheumatoid arthritis (RA) are well established. However, only a limited number of studies have investigated these alleles in patients with childhood-onset RA, which is defined as rheumatoid factor- and/or anti-citrullinated protein antibody-positive juvenile idiopathic arthritis. The aims of this study were to investigate the largest cohort of patients with childhood-onset RA for association with SE alleles and to determine whether there is a hierarchy of risk based on the amino acid sequence of the SE., Methods: High-resolution HLA-DRB1 genotypes were obtained for 204 patients with childhood-onset RA and 373 healthy control subjects. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for different SE-encoding HLA-DRB1 alleles. In addition, genotype ORs were calculated for combinations of SE alleles classified into S(2) , S(3P) , or L alleles, based on amino acid sequences in position 70-74 of the DRβ1 chain, as proposed by Tezenas du Montcel et al., Results: We confirmed associations between HLA-DRB1 SE alleles and childhood-onset RA (76% of patients carried 1 or 2 SE alleles compared with 46% of control subjects; OR 3.81, 95% CI 2.4-6.0, P < 1 × 10(-7) ). We also observed associations between individual SE alleles (HLA-DRB1*0101, *0401, *0404, *0405, *0408, and *1001) and childhood-onset RA. Genotype-specific risk estimates suggested a hierarchy of risk, with the highest risk among individuals heterozygous for S(2) /S(3P) (OR 22.3, 95% CI 9.9-50.5, P < 0.0001)., Conclusion: We confirm the association between SE-encoding HLA-DRB1 alleles and susceptibility to childhood-onset RA. The excess risk conferred by carriage of the combination of S(2) and S(3P) risk alleles suggests that children with DRβ1 chains containing the KRAA and QRRAA or RRRAA sequences are especially susceptible to RA., (Copyright © 2012 by the American College of Rheumatology.)

Objectives: Methotrexate (MTX) is the mainstay treatment for juvenile idiopathic arthritis (JIA), however approximately 30% of children will fail to respond to the drug. Identification of genetic predictors of response to MTX would be invaluable in developing optimal treatment strategies for JIA. Using a candidate gene approach, single nucleotide polymorphisms (SNPs) within genes in the metabolic pathway of MTX, were investigated for association with response to treatment in JIA cases., Methods: Tagging SNPs were selected across 13 MTX metabolic pathway genes and were genotyped using Sequenom genotyping technology in subjects recruited from the Sparks Childhood Arthritis Response to Medication Study. Response to MTX was defined using the American College of Rheumatology (ACR) paediatric response criteria and SNP genotype frequencies were compared between the worst and best responders (ACR-Ped70) to MTX. An independent cohort of US JIA cases was available for validation of initial findings., Results: One SNP within the inosine triphosphate pyrophosphatase gene (ITPA) and two SNPs within 5-aminoimidazole-4-carboxamide ribonucleotide transformylase gene (ATIC) were significantly associated with a poor response to MTX. One of the ATIC SNPs showed a trend towards association with MTX response in an independent cohort of US JIA cases. Meta-analysis of the two studies strengthened this association (combined p value=0.002)., Conclusions: This study presents association of a SNP in the ATIC gene with response to MTX in JIA. There is now growing evidence to support a role of the ATIC gene with response to MTX treatment. These results could contribute towards a better understanding of and ability to predict MTX response in JIA.

Objective: To test for associations between non-major histocompatibility complex susceptibility loci previously reported in autoimmune diseases and juvenile idiopathic arthritis (JIA)., Methods: Published autoimmune disease genome-wide association studies were reviewed, and 519 single-nucleotide polymorphisms (SNPs) were selected for association testing. The initial cohort included 809 JIA cases and 3,535 controls of non-Hispanic, European ancestry. Of the SNPs, 257 were successfully genotyped, while 168 were imputed with quality. Based on findings in the initial cohort, replication was sought for 21 SNPs in a second cohort of 1,015 JIA cases and 1,569 controls collected in the US and Germany. For the initial cohort, tests for association were adjusted for potential confounding effects of population structure by including principal components derived from a genome-wide association study as covariates in logistic regression models. Odds ratios (ORs) and 95% confidence intervals were calculated., Results: Testing for association of previously reported autoimmune disease genetic associations in the initial cohort suggested associations with JIA in 13 distinct loci. Of these, 7 were validated in the replication cohort. Meta-analysis results for the replicating loci included PTPN22 (rs6679677 [OR 1.58, P = 1.98 × 10(-12) ], rs2476601 [OR 1.64, P = 1.90 × 10(-13) ], and rs2488457 [OR 1.32, P = 6.74 × 10(-8) ]), PTPN2 (rs1893217 [OR = 1.33, P = 1.60 × 10(-9) ] and rs7234029 [OR 1.35, P = 1.86 × 10(-10) ]), ADAD1-IL2-IL21 (rs17388568 [OR 1.24, P = 1.13 × 10(-6) ] and rs13143866 [OR 0.83, P = 1.95 × 10(-4) ]), STAT4 (rs3821236 [OR = 1.27, P = 2.36 × 10(-6) ] and rs7574865 [OR = 1.31, P = 2.21 × 10(-6) ]), C12orf30 (rs17696736 [OR = 1.19, P = 2.59 × 10(-5) ]), COG6 (rs7993214 [OR = 0.76, P = 1.10 × 10(-5) ]), and ANGPT1 (rs1010824 [OR = 0.79, P = 2.91 × 10(-4) ]). These polymorphisms have been reported in diseases such as rheumatoid arthritis, type 1 diabetes mellitus, Crohn's disease, and multiple sclerosis., Conclusion: General susceptibility loci for autoimmunity are shared across diseases, including JIA, suggesting the potential for common therapeutic targets and mechanisms., (Copyright © 2010 by the American College of Rheumatology.)

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., (Copyright © 2010 by the American College of Rheumatology.)

Objectives: Little is known about the mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment., Methods: Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after the treatment were selected for single nucleotide polymorphism (SNP) genotyping. Genotype frequencies were compared between nonresponders and responders (ACR-Ped70). An independent cohort was available for validation., Results: Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, P<0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analyzed for genetic association in response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort., Conclusion: This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyze genetic variation in differentially expressed genes. We have identified a gene, which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes that are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis.

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: 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.

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.

The immune response HLA class II DRB1 gene provides the major genetic contribution to Juvenile Idiopathic Arthritis (JIA), with a hierarchy of predisposing through intermediate to protective effects. With JIA, and the many other HLA associated diseases, it is difficult to identify the combinations of biologically relevant amino acid (AA) residues directly involved in disease due to the high level of HLA polymorphism, the pattern of AA variability, including varying degrees of linkage disequilibrium (LD), and the fact that most HLA variation occurs at functionally important sites. In a subset of JIA patients with the clinical phenotype oligoarticular-persistent (OP), we have applied a recently developed novel approach to genetic association analyses with genes/proteins sub-divided into biologically relevant smaller sequence features (SFs), and their "alleles" which are called variant types (VTs). With SFVT analysis, association tests are performed on variation at biologically relevant SFs based on structural (e.g., beta-strand 1) and functional (e.g., peptide binding site) features of the protein. We have extended the SFVT analysis pipeline to additionally include pairwise comparisons of DRB1 alleles within serogroup classes, our extension of the Salamon Unique Combinations algorithm, and LD patterns of AA variability to evaluate the SFVT results; all of which contributed additional complementary information. With JIA-OP, we identified a set of single AA SFs, and SFs in which they occur, particularly pockets of the peptide binding site, that account for the major disease risk attributable to HLA DRB1. These are (in numeric order): AAs 13 (pockets 4 and 6), 37 and 57 (both pocket 9), 67 (pocket 7), 74 (pocket 4), and 86 (pocket 1), and to a lesser extent 30 (pockets 6 and 7) and 71 (pockets 4, 5, and 7).

Objective: IL2RA/CD25, the gene for interleukin-2 receptor alpha, is emerging as a general susceptibility gene for autoimmune diseases because of its role in the development and function of regulatory T cells and the association of single-nucleotide polymorphisms (SNPs) within this gene with type 1 diabetes mellitus (DM), Graves' disease, rheumatoid arthritis (RA), and multiple sclerosis (MS). The aim of this study was to determine whether SNPs within the IL2RA/CD25 gene are associated with juvenile idiopathic arthritis (JIA)., Methods: Three SNPs within the IL2RA/CD25 gene, that previously showed evidence of an association with either RA, MS, or type 1 DM, were selected for genotyping in UK JIA cases (n=654) and controls (n=3,849). Data for 1 SNP (rs2104286) were also available from North American JIA cases (n=747) and controls (n=1,161). Association analyses were performed using Plink software. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated., Results: SNP rs2104286 within the IL2RA/CD25 gene was significantly associated with UK JIA cases (OR for the allele 0.76 [95% CI 0.66-0.88], P for trend=0.0002). A second SNP (rs41295061) also showed modest evidence for association with JIA (OR 0.80 [95% CI 0.63-1.0], P=0.05). Association with rs2104286 was convincingly replicated in the North American JIA cohort (OR 0.84 [95% CI 0.65-0.99], P for trend=0.05). Meta-analysis of the 2 cohorts yielded highly significant evidence of association with JIA (OR 0.76 [95% CI 0.62-0.88], P=4.9x10(-5))., Conclusion: These results provide strong evidence that the IL2RA/CD25 gene represents a JIA susceptibility locus. Further investigation of the gene using both genetic and functional approaches is now required.

Objective: Systemic juvenile idiopathic arthritis (JIA) is associated with macrophage activation syndrome. Macrophage activation syndrome bears a close resemblance to familial hemophagocytic lymphohistiocytosis (HLH). The development of familial HLH has been recently associated with mutations in MUNC13-4. The purpose of this study was to assess for possible sequence alterations in MUNC13-4 in patients with systemic JIA/macrophage activation syndrome., Methods: The MUNC13-4 sequence was analyzed in 18 unrelated patients with systemic JIA/macrophage activation syndrome, using 32 primer pair sets designed to amplify the 32 exons and at least 100 basepairs of the adjacent intronic regions. DNA samples obtained from 73 unrelated patients with systemic JIA and no history of macrophage activation syndrome and 229 unrelated healthy individuals were used as controls., Results: The biallelic sequence variants in MUNC13-4 reported in familial HLH were present in 2 of the 18 patients with JIA/macrophage activation syndrome. Further analysis of the MUNC13-4 sequences revealed an identical combination of 12 single-nucleotide polymorphisms (SNPs) in 9 of the remaining 16 patients with systemic JIA/macrophage activation syndrome (56%). Additional analysis suggested that these 12 SNPs (154[-19] g>a, 261[+26] c>g, 388[+81] g>a, 388[+122] c>t, 570[-60] t>g, 888 G>C, 1389[+36] g>a, 1992[+5] g>a, 2447[+144] c>t, 2599 A>G, 2830[+37] c>g, 3198 A>G) were inherited as an extended haplotype. In several patients, in addition to the described haplotype, there were other SNPs in the second allele of MUNC13-4. Moreover, 1 patient had a complex mutation with 2 changes, 2542 A>C and 2943 G>C, in a cis configuration. The haplotype was present in only 27 (12%) of 229 healthy control subjects (chi(2) = 23.5) and in 6 (8.2%) of 73 patients with systemic JIA and no history of macrophage activation syndrome., Conclusion: The data suggest an association between MUNC13-4 polymorphisms and macrophage activation syndrome in patients with systemic JIA.

Juvenile idiopathic arthritis (JIA) is the most common chronic arthropathy of childhood which is believed to be influenced by both genetic and environmental factors. The progress in identifying genes underlying JIA susceptibility using candidate gene association studies has been slow. Several associations between JIA and variants in the genes encoding the human leukocyte antigens (HLA) have been confirmed and replicated in independent cohorts. However it is clear that genetic variants outside the HLA also influence susceptibility to JIA. While a large number of non-HLA candidate genes have been tested for associations, only a handful of reported associations such as PTPN22 have been validated. In this review we discuss the principles behind genetic studies of complex traits like JIA, and comprehensively catalogue non-HLA candidate-gene association studies performed in JIA to date and review several validated associations. Most candidate gene studies are underpowered and do not detect associations, and those that do are often not replicated. We also discuss the principles behind genome-wide association studies and discuss possible implications for identifying genes underlying JIA. Finally we discuss several genetic variants underlying multiple clinically distinct autoimmune phenotypes.

Juvenile idiopathic arthritis (JIA) is an autoimmune (AI) disease characterized by chronic arthritis in children. Children with JIA have increased prevalence of other AI diseases. Furthermore, relatives of children with JIA have been shown to have an increased prevalence of AI diseases. Our objective was to determine if there were differences in the prevalence of AI diseases among maternal and paternal relatives of children with JIA. Information about AI diseases among all living first- and second-degree relatives was collected by structured interviews with families of 121 simplex JIA families, 23 multiplex JIA families, and 45 control families. Overall, the prevalence of AI diseases was significantly increased among maternal second-degree relatives of cases compared to that of maternal second-degree relatives of controls [14% vs. 4.3%; p < 0.001]. The prevalence of AI diseases among mothers of JIA cases was three times that of fathers [32.3% vs. 11.4%; p < 0.0001]. The prevalence of AI diseases among all maternal second-degree relatives of children with JIA was significantly higher than that of all paternal second-degree relatives [14% vs. 7.9%; p < 0.004]. Although additional paternal effects cannot be excluded, together these results demonstrate that maternal relatives of children with JIA have an increased prevalence of autoimmunity compared to paternal relatives, suggesting that there might be a maternal parent-of-origin effect in JIA.

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: To assess the reproductive fitness of mothers of children with juvenile idiopathic arthritis (JIA)., Methods: A mail survey assessing pregnancy outcome was carried out among mothers of children with JIA (JIA mothers) treated at a tertiary paediatric rheumatology centre. The best friends of the JIA mothers served as controls. Besides family history, sociodemographics and reproductive outcomes were measured, including the number of pregnancies, pregnancy complications and gestational age at the time of delivery., Results: JIA mothers (n = 227) and controls (n = 235) had similar sociodemographics and racial backgrounds. On average, JIA mothers reported a greater number of conceptions than controls (3.5 vs 3.1; P = 0.01) but had significantly higher rates of pregnancy complications (25% vs 15%; P<0.001). Corrected for differences in the absolute number of pregnancies between groups, the chances of having a miscarriage [mean (s.d.), 0.12 (0.18) vs 0.09 (0.16); P = 0.02] or preterm delivery [0.08 (0.21) vs 0.04 (0.15); P<0.02] were significantly greater among JIA mothers than controls., Conclusions: Mothers of children with JIA have impaired reproductive fitness. This phenomenon is unlikely to be the result of difficulty with conception but rather to be due to higher rates of pregnancy loss and premature delivery.

Juvenile rheumatoid arthritis (JRA) is mediated by Th1-immune responses. In children with JRA, synovial T cells express high levels of the Th1-chemokine receptor CC chemokine receptor 5 (CCR5), which has been implicated in susceptibility to rheumatoid arthritis. To test the hypothesis that genetic variation in CCR5 is associated with susceptibility to JRA, we analyzed patterns of variation in the 5'cis-regulatory region of CCR5 in 124 multiplex families from a JRA-affected sibpair registry. After sequencing the upstream region of CCR5, variants were tested for association with JRA by transmission disequilibrium testing. A single nucleotide polymorphism, C-1835T, was significantly undertransmitted to children with early-onset JRA (P<0.01). C-1835T was genotyped in 424 additional simplex and multiplex families. CCR5-1835T allele was undertransmitted in the cohort of all probands with JRA (P<0.02), as well as in those with early-onset (P<0.01) or pauciarticular JRA (P<0.05). Another variant, a 32-bp deletion in the open reading frame of CCR5 (CCR5-Delta32) was also tested in approximately 700 simplex and multiplex families. CCR5-Delta32 was also significantly undertransmitted to probands with early-onset JRA (P<0.05). Both variants are in regions under natural selection, and result in functional consequences. Our results suggest these CCR5 variants are protective against early-onset JRA.

Juvenile rheumatoid arthritis (JRA), also known as juvenile idiopathic arthritis (JIA), includes the most common chronic autoimmune arthropathies of childhood. These two nomenclatures for classification include components representing the major subclasses of disease. The chromosomal regions and the genes involved in these complex genetic traits are being elucidated, with findings often specific for a particular disease subtype. With the advent of new SNP technologies, progress is being made at an ever-increasing pace. This review discusses the difficulties of deciphering the genetic components in complex disorders, while demonstrating the similarities that JRA shares with other autoimmune disorders. Particular emphasis has been placed on positive findings either for candidate genes that have been replicated independently in JRA/JIA, or findings in JRA for which consistent results have been reported in other forms of autoimmunity.

The polymorphic T-cell receptor Vbeta (TRBV) genes encode much of the variable region of the T-cell receptor beta chain. Analysis of allele frequencies of three closely linked polymorphic TRBV genes, TRBV7-3, TRBV9 and TRBV6-4, was undertaken in several populations. The frequencies of these alleles are not significantly different in populations of Caucasians, African Americans and Western Africans. However, Chinese population is extremely homogenous at all three loci. The current study identifies the existence of haplotypic relationships between alleles of these genes in the Caucasian population. The ORF allele TRBV7-3*A3 is found exclusively on chromosomes bearing TRBV9*A2 and TRBV6-4*A2 in this cohort. In contrast, TRBV7-3*A1 and the null allele TRBV7-3*A2 are associated only with TRBV9*A1 and TRBV6-4*A1. This pattern of linkage disequilibrium (LD) is altered in the African American and Western African populations. In these cohorts, there is a marked reduction in LD between alleles of TRBV7-3 and TRBV9. This study is consistent with previous population genetic studies wherein African-derived samples have a greater level of genetic diversity compared to Caucasians. These data also demonstrate that patterns of LD are not consistent across the entire TRBV locus.

Juvenile rheumatoid arthritis (JRA) comprises a group of chronic systemic inflammatory disorders that primarily affect joints and can cause long-term disability. JRA is likely to be a complex genetic trait, or a series of such traits, with both genetic and environmental factors contributing to the risk for developing the disease and to its progression. The HLA region on the short arm of chromosome 6 has been intensively evaluated for genetic contributors to JRA, and multiple associations, and more recently linkage, has been detected. Other genes involved in innate and acquired immunity also map to near the HLA cluster on 6p, and it is possible that variation within these genes also confers risk for developing JRA. We examined the TPSN gene, which encodes tapasin, an endoplasmic reticulum chaperone that is involved in antigen processing, to elucidate its involvement, if any, in JRA. We employed both a case-control approach and the transmission disequilibrium test, and found linkage and association between the TPSN allele (Arg260) and the systemic onset subtype of JRA. Two independent JRA cohorts were used, one recruited from the Rheumatology Clinic at Cincinnati Children's Hospital Medical Center (82 simplex families) and one collected by the British Paediatric Rheumatology Group in London, England (74 simplex families). The transmission disequilibrium test for these cohorts combined was statistically significant (chi2 = 4.2, one degree of freedom; P = 0.04). Linkage disequilibrium testing between the HLA alleles that are known to be associated with systemic onset JRA did not reveal linkage disequilibrium with the Arg260 allele, either in the Cincinnati systemic onset JRA cohort or in 113 Caucasian healthy individuals. These results suggest that there is a weak association between systemic onset JRA and the TPSN polymorphism, possibly due to linkage disequilibrium with an as yet unknown susceptibility allele in the centromeric part of chromosome 6.

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: To evaluate the ability of microarray-based methods to identify genes with disease-specific expression patterns in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) of juvenile arthritis patients and healthy controls., Methods: Microarray data (Affymetrix U95Av2) from 26 PBMC and 20 SFMC samples collected from patients with active disease (classified by course according to ACR criteria) were analysed for expression patterns that correlated with disease characteristics. For comparison, PBMC gene expression profiles were obtained from 15 healthy controls. Real-time PCR was used for confirmation of gene expression differences., Results: Statistical analysis of gene expression patterns in PBMC identified 378 probe sets corresponding to 342 unique genes with differing expression levels between polyarticular course patients and controls (t test, P<0.0001). The genes represented by these probe sets were enriched for functions related to regulation of immune cell functions, receptor signalling as well as protein metabolism and degradation. Included in these probe sets were a group of CXCL chemokines with functions related to angiogenesis. Further analysis showed that, whereas angiogenic CXCL (ELR+) gene expression was elevated in polyarticular PBMC, expression of angiostatic CXCL (ELR-) chemokines was lower in polyarticular SFMC compared with corresponding pauciarticular samples (t test, P<0.05)., Conclusions: This pilot study demonstrates that juvenile arthritis patients exhibit complex patterns of gene expression in PBMC and SFMC. The presence of disease-correlated biologically relevant gene expression patterns suggests that the power of this approach will allow better understanding of disease mechanisms, identify distinct clinical phenotypes in disease subtypes, and suggest new therapeutic approaches.

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 (lambda(s)), 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 (lambda(s) 29.5), leukocytosis (lambda(s) 25), rheumatoid factor (lambda(s) 11.0), anemia (lambda(s) 1.7), and antinuclear antibodies (lambda(s) 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.

Previously, 4 children with deletion of the long arm of chromosome 18 and chronic arthritis were reported. We present an 8-year-old girl with arthritis, atrial septal defect, external auditory canal atresia, and developmental delay. She is the fifth child reported with 18q- syndrome and juvenile rheumatoid arthritis. Evidence is mounting that genetic loci on chromosome 18 may play a role in the expression of complex autoimmune diseases. Idiopathic arthritis should be considered as a potential additional feature in 18q- syndrome.

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 A(n)T(n) 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 A(n)T(n) 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 A(n)T(n) alleles or -174/A(n)T(n) 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 < or =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 compare synovial tissue cytokine mRNA expression between patients with juvenile rheumatoid arthritis (JRA) and a heterogeneous group of non-autoimmune arthropathies (controls) with respect to type 1/type 2 balance., Methods: Thirty-five JRA (average 9.1 years' disease duration) and 13 control synovial tissues were studied. As a measure of the type 1/type 2 cytokine balance in a subset of the JRA and control tissues, interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) mRNA levels were measured by competitive fragment reverse transcription-polymerase chain reaction. To quantitate additional cytokines relevant to this balance, multiprobe ribonuclease protection assays were employed measuring IL-5, IL-10, IL-13, IL-15, IL-18, and IL-12 (p35 and p40 subunits). Immunohistochemistry was performed on JRA tissues using antibodies specific for IL-15 and IL-18., Results: A higher IFN-gamma:IL-4 ratio (p = 0.034) was found in JRA tissues compared to controls. JRA tissues also displayed higher mRNA levels of IL-12p35 (p = 0.021), IL-15 (p = 0.002), and IL-18 (p = 0.017), but not IL-4 and IL-10. IFN-gamma expression in JRA, but not controls, correlated strongly with IL-12p35 (r = 0.63) and IL-12p40 (r = 0.73) levels. A subset of IL-15+ and IL-18+ cells was detected in JRA synovial tissues, largely within perivascular aggregates., Conclusion: JRA synovial tissue cytokine expression patterns indicate a type 1 bias, even in the later stages of disease. The strong correlation between IFN-gamma and IL-12 in JRA suggests a prominent role for IL-12 in promoting the type I bias, while IL-15 and IL-18 may also indirectly increase IFN-gamma expression and further bias the immune response.

Objective: To determine if a polymorphism in the immunoproteasome subunit LMP7 was associated with juvenile rheumatoid arthritis (JRA) and had functional significance., Methods: The frequency of LMP7QQ+ vs QQ- (QK and KK genotypes) among 207 patients with JRA and 50 controls was determined. JRA subtypes were pauciarticular (53%), polyarticular (33%), and systemic (14%). Onset was before age 6 (early onset) in 60% of patients. The functional significance of the LMP7 polymorphism was determined by comparing incorporation of LMP7Q vs LMP7K into proteasomes., Results: There was an increased frequency of LMP7QQ in patients vs controls (73 vs 56%; p = 0.016), mainly due to the pauciarticular and systemic JRA subtypes (p = 0.037), and more pronounced in early onset disease (77 vs 56%; p = 0.006). The association persisted with stratification for HLA-DR5(11) and -DPB 1 *0201 (p = 0.002 and 0.013). We found no difference in the relative incorporation of LMP7Q and LMP7K into proteasomes., Conclusions: These results support an association between LMP7QQ homozygosity and JRA, particularly early onset disease. The difference persists with stratification, at least for DR5(11) and DPB1*0201, suggesting that this effect is unlikely to be due to linkage disequilibrium with HLA alleles known to be associated with early onset pauciarticular JRA. Importantly, as there does not appear to be functional significance associated with the LMP7 polymorphism, this may be a marker for another as yet unidentified susceptibility locus.

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-gamma 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-gamma 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.

Objective: Although increased vascularity was noted in early histopathologic studies of juvenile rheumatoid arthritis (JRA) synovium, the available data on angiogenesis in JRA are very limited. The main purposes of this study were to assess expression of the key angiogenic factors in JRA synovium, and to evaluate a SCID mouse model of JRA as an approach to study in vivo regulation of the expression of these factors in JRA., Methods: RNase protection assay was used to assess the expression of the key angiogenic factors in fresh JRA synovium and in JRA synovial tissue fragments that had been minced and then implanted into SCID mice. Vascularity of the samples was assessed by immunohistochemical staining for von Willebrand factor. Synovial specimens obtained from patients with osteoarthritis (OA) or other noninflammatory arthropathies were used as controls., Results: Detectable levels of messenger RNA for vascular endothelial growth factor and angiopoietin 1 and their respective receptors, as well as endoglin and thrombin receptors, were present in all JRA tissue specimens studied. The levels of expression of these factors in JRA tissues were significantly higher than those in tissues obtained from patients with OA or other noninflammatory arthropathies. Furthermore, increased expression of the key angiogenic factors in the fresh JRA tissues correlated with the exuberant revascularization of JRA minced tissue fragments implanted into SCID mice. This was in sharp contrast to the poor revascularization of implanted OA tissues., Conclusion: JRA synovium is characterized by high angiogenic activity. SCID mouse-human JRA synovium chimeras may provide a good approach to study the in vivo regulation of angiogenesis in JRA.