David N. Glass

Efficacy of Low-Dose Methotrexate in Rheumatoid Arthritis

Twenty-eight patients with refractory rheumatoid arthritis completed a randomized 24-week double-blind crossover trial comparing oral methotrexate (2.5 to 5 mg every 12 hours for three doses weekly) with placebo. The methotrexate group had significant reductions (P less than 0.01 as compared with the placebo group) in the number of tender or painful joints, the duration of morning stiffness, and disease activity according to physician and patient assessments at the 12-week crossover visit; reductions in the number of swollen joints (P less than 0.05) and 15-m walking time (P less than 0.03) also occurred. These variables, as well as the grip strength and erythrocyte sedimentation rate, showed significant (P less than 0.01) improvement at 24 weeks in the population crossed over to methotrexate. A significantly increased frequency (P less than 0.03) of the HLA-DR2 haplotype occurred in the eight patients with the most substantial response to methotrexate. Adverse reactions during methotrexate therapy included transaminase elevation (21 per cent), nausea (18 per cent), and diarrhea (12 per cent); one patient was withdrawn from the trial because of diarrhea. One patient died while receiving the placebo. Methotrexate did not affect measures of humoral or cellular immunity. We conclude that this trial provides evidence of the short-term efficacy of methotrexate in rheumatoid arthritis, but the mechanism of action is unknown. Longer trials will be required to determine the ultimate safety and effectiveness of this drug.

Dense genotyping of immune-related disease regions identifies 14 new susceptibility loci for juvenile idiopathic arthritis

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.

Inherited deficiency of the second component of complement. Rheumatic disease associations.

The prevalence of homozygous and heterozygous deficiency of the second component of complement (C2) was determined in patients with rheumatic disease including 137 with systemic lupus erythematosus (SLE), 274 with juvenile rheumatoid arthritis, and 134 with rheumatoid arthritis. 1 C2 homozygous deficient and 19 possible heterozygous deficient individuals were identified by using both immunochemical and functional assays to determine C2 levels. Of the 20, 8 had SLE (5.9%), 10 had juvenile rheumatoid arthritis (3.7%), and 2 had rheumatoid arthritis (1.4%), the homozygous deficient individual having SLE. The prevalence of C2 deficiency in the SLE and juvenile rheumatoid arthritis patients was significantly increased (P = 0.0009 and P = 0.02, respectively) when compared with controls, 6 (1.2%) of 509 blood donors having C2 levels consistent with heterozygous deficiency. 15 of the 20 C2 deficient patients were HLA typed and found to have antigens A10(Aw25), B18, or both. The patients with C2 deficiency and SLE had earlier age of onset of disease and less antinuclear antibody when compared with the C2 normal SLE patients. 11 families of the propositi were studied and found to have one or more C2 heterozygous deficient individuals. The family members had an equal distribution of rheumatic disease and antinuclear antibody in the C2 deficient and C2 normal groups. C2 deficient individuals were found to have significantly lower levels of properdin Factor B (242 mug/ml+/-54) when compared with the non-C2 deficient family members (282 mug/ml+/-73). These data support the concept that inherited deficiency of C2 is significantly associated with both SLE and juvenile rheumatoid arthritis.

Subtype-specific peripheral blood gene expression profiles in recent onset juvenile idiopathic arthritis

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.

Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC13-4 polymorphisms.

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.

A comprehensive review of the genetics of juvenile idiopathic arthritis

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.

Age-specific effects of juvenile rheumatoid arthritis-associated HLA alleles

OBJECTIVE To define the onset and duration of effect of the HLA alleles that are associated with disease susceptibility and protection in juvenile rheumatoid arthritis (JRA) and 2 of its subtypes. METHODS We typed 680 patients with JRA and 254 ethnically matched unrelated controls for HLA class I and II genes. The frequency of each allele was calculated for each of the age-at-onset, onset type, and sex categories and plotted against the allele frequency in the control population. Survival analysis (with onset of disease as the terminating event) was used to calculate the age by which 50% (St0.5) and 80% (St0.2) of the children with particular alleles and combinations of alleles develop disease. This allele-specific survival analysis also allowed for the comparison of the overall survival functions for the various JRA subtype and sex categories. RESULTS Certain alleles are strongly associated with early susceptibility to pauciarticular JRA, including HLA-A2, DR8, DR5, and DPB1*0201. Fifty percent of the children carrying at least 1 of these alleles had disease onset prior to their third birthday. Among children who carried HLA-A2 and any 2 HLA-DR alleles (DR3, DR5, DR6, or DR8), the median age at the onset of pauciarticular disease was 2.7 years. Combinations of A2 and DPB1*0201 and one DR allele narrowed the window further to a median age at onset of 2.4 years. B27 and DR4 were associated with protection early in life but with increased risk later in childhood, with St0.5 values of 7.3 and 6.6 years, respectively, for pauciarticular JRA and St0.5 values of 10.2 and 10.7 years, respectively, for polyarticular JRA. Sex strongly influenced the age at which many of the alleles have their effect. CONCLUSION These data define at what age and for how long various HLA alleles influence susceptibility and protection (window-of-effect) in patients with JRA. In addition, these data establish more clearly the boundaries of ages-at-onset for 2 of the subtypes of the disease.

The susceptibility loci Juvenile Idiopathic Arthritis shares with other autoimmune diseases extend to PTPN2, COG6 and ANGPT1

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, Crohns 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.

Juvenile idiopathic arthritis and HLA Class I and Class II interactions and age-at-onset effects

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.

Juvenile rheumatoid arthritis: linkage to HLA demonstrated by allele sharing in affected sibpairs.

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 Fishers 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.