Association of two functional polymorphisms in the CCR5 gene with juvenile rheumatoid arthritis.

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.

Impaired reproductive fitness in mothers of children with juvenile autoimmune arthropathies.

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.

Susceptibility to JRA/JIA: complementing general autoimmune and arthritis traits.

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.

Variability in TRBV haplotype frequency and composition in Caucasian, African American, Western African and Chinese populations.

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.

Gene expression in juvenile arthritis and spondyloarthropathy: pro-angiogenic ELR+ chemokine genes relate to course of arthritis.

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.

Polymorphism in the MHC-encoded LMP7 gene: association with JRA without functional significance for immunoproteasome assembly.

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.

A novel polymorphism in the pseudogene TCRBV5S5 combines with TCRBV6S1 to define three haplotypes.

In the current study, we report a G to A single nucleotide polymorphism at base pair 396 of the TCRBV5S5P gene. This polymorphism has a frequency of 0.20 in a cohort of Caucasian controls. In addition, we provide evidence for linkage disequilibrium between TCRBV5S5P and the TCRBV6S1 gene.

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios.

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.

Expression of angiogenic factors in juvenile rheumatoid arthritis: correlation with revascularization of human synovium engrafted into SCID mice.

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.

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 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.

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.

Transmission disequilibrium as a test of linkage and association between HLA alleles and pauciarticular-onset juvenile rheumatoid arthritis.

OBJECTIVE: To determine if HLA class I and II alleles previously found to be associated with (or protective against) pauciarticular-onset juvenile rheumatoid arthritis (pauci-onset JRA) in population-association studies are transmitted from heterozygous parents to an extent different from the expected 50%. METHODS: One hundred one Caucasian North American families that had a child with pauci-onset JRA and at least 1 parent who was heterozygous for the allele of interest were available for analysis. Both biologic parents and all children (affected and unaffected) were typed for HLA class I and II alleles. The transmission disequilibrium test (TDT) was used to determine if affected offspring received the disease-associated (or protective) allele more (or less) frequently than its alternate allele. In families in which an unaffected sibling was available, the unmatched chi-square test was used to determine if a meiotic segregation distortion bias existed. RESULTS: HLA class I alleles A2, B27, and B35 showed a significantly higher than expected frequency of transmission to affected offspring, as did class II alleles DR5 and DR8. HLA-DR4 was found to be transmitted significantly less frequently than expected to affected, but not unaffected, offspring. All alleles that showed an excess transmission to affected offspring were transmitted to unaffected offspring at expected rates. When the data were stratified by age and sex, the likelihood of transmission of some of the alleles was strongly influenced by these variables. For example, excess transmission of HLA-DR5 was found exclusively in female patients who were younger at the time of disease onset. CONCLUSION: Results from these family-based studies rule out the possibility that HLA disease associations found in earlier studies were a result of population stratification and establish linkage and association between the major histocompatibility complex and pauci-onset JRA.

Contrasting cytokine profiles in the synovium of different forms of juvenile rheumatoid arthritis and juvenile spondyloarthropathy: prominence of interleukin 4 in restricted disease.

OBJECTIVE: To determine the pattern of expression of Type 1 and Type 2 cytokines in synovial tissues and fluids (SF) of patients with different forms of juvenile rheumatoid arthritis (JRA) and juvenile spondyloarthropathy (JSpA), and to contrast these with findings in adult patients with RA. METHODS: Sixty-three SF mononuclear cell preparations and synovial tissue samples from 50 patients with JRA or JSpA and 7 synovial tissues from patients with adult onset RA were analyzed by reverse transcription polymerase chain reaction for the presence or absence of interleukin 2 (IL-2), tumor necrosis factor-beta (TNF-beta), interferon-gamma (IFN-gamma), and IL-10 and IL-4 mRNA. RESULTS: IL-4 mRNA was identified significantly more often in the synovial compartment of patients with pauciarticular onset disease (JRA or JSpA) compared with polyarticular onset JRA (58 vs 14%; p < 0.01) or RA (29%). Similarly, IL-4 mRNA was detected more often in those with a persistently pauciarticular disease course compared to those with a polyarticular course (68 vs 30%; p < 0.01). Furthermore, the combination of IL-4 and IL-10 mRNA was found more frequently in nonerosive compared with erosive disease (38 vs 15%; p < 0.05). IL-2 and TNF-beta mRNA were found in all groups. IFN-gamma mRNA was detected in 33% of those with systemic onset JRA compared with 85% of other types of JRA (p < 0.01). CONCLUSION: This study provides further evidence of immunopathological differences between chronic forms of arthritis with childhood onset, and highlights similarities with and differences from adult RA. Our findings suggest that IL-4, possibly in combination with IL-10, has an antiinflammatory or disease restricting role.

Comparative sequence analysis of the human T cell receptor beta chain in juvenile rheumatoid arthritis and juvenile spondylarthropathies: evidence for antigenic selection of T cells in the synovium.

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.

Juvenile rheumatoid arthritis in affected sibpairs.

OBJECTIVE: To describe the demographics and clinical disease in affected sibpairs (ASPs) with juvenile rheumatoid arthritis (JRA), and to compare JRA as it occurs in ASPs with that from non-ASP JRA populations described in the literature. METHODS: A rare disease research registry was established with a focus on JRA ASPs to facilitate accrual of patients for genetic, epidemiologic, and clinical studies. Physicians likely to care for patients with JRA were made aware of the registry and its goals by a variety of methods and asked to refer patients for entry. RESULTS: To date, 71 ASPs have been registered and complete information has been obtained. These affected sibs differed in age by a mean of 4.1 years (SD 3.4) and in age at disease onset by 2.8 years (SD 3.0). The actual time difference between onset in sib 1 versus sib 2 averaged 4.4 years (SD 4.2). Sixty-three percent of the sibpairs were concordant for sex, and 76% for JRA onset type. Onset type within sibpairs did not appear to be random, based upon comparisons with non-ASP populations. Greater than expected concordance was seen among those with pauciarticular-onset and polyarticular-onset JRA. Seventy-nine percent of the pairs were concordant for course type. Seven sets of twins were included (approximately 10% of the total), all were concordant for onset and course type (6 sets with pauciarticular, 1 set with polyarticular), and disease onset was separated by a mean of only 3.3 months. Within the onset and course types, the clinical disease, such as the female:male ratio, age at onset, and serologic findings, in ASPs resembled that which has been described in the literature. CONCLUSION: A higher than expected degree of concordance for onset type of JRA exists between sibpairs, indicating that genetic influences play a role. Affected sibs do not tend to develop their disease at approximately the same point in time, except for the twin sets. Clinical features of the disease within the various subtypes appear similar to those in non-ASP populations.

Long-term health outcomes and quality of life in American and Italian inception cohorts of patients with juvenile rheumatoid arthritis. II. Early predictors of outcome.

OBJECTIVE: To determine whether demographic, clinical, and immunogenetic variables measurable during the first 6 months of illness long-term health outcomes and quality of life in patients with juvenile rheumatoid arthritis (JRA). METHODS: Patient eligibility criteria: (1) first examined in our units between 1958 and 1990 within 6 months of onset of symptoms; (2) diagnosis of JRA by American College of Rheumatology criteria; (3) disease duration of at least 5 years at the time of assessment of outcome. Instruments used: (1) the Health Assessment Questionnaire (HAQ, short form), or Childhood HAQ (CHAQ) to measure disability (0-3 scale), (2) pain, and (3) parental assessment of overall well being, each scored on a 15 cm visual analog scale; (4) the Quality of Life Scales (QOLS) (adults only). Independent variables that showed significant results using univariate tests underwent multiple logistic regression analysis. RESULTS: 227 patients were available for analysis. Mean duration of disease at time of assessment of outcome was 15 years (range 5.3-36.1). Univariate tests allowed 11 variables for disability, 9 for pain, 7 for overall well being, and 4 for QOL into the multivariate analysis. The best predictor of higher disability was the articular severity score (odds ratio, OR, 5.69) while antinuclear antibody positivity foretold less disability (OR 0.29). HLA-DR5 positivity conferred the greatest risk for pain (OR 3.34), while HLA-B5, DR3, and C3 were protective (OR 0.25, 0.28, 0.33, respectively). Early hand involvement was the strongest predictor of poorer overall well being (OR 8.75). Only the erythrocyte sedimentation rate was predictive of future QOL, but the model yielded a low C statistic (< 70%) and the OR 95% confidence limits were extreme (OR 9.77; 95% confidence interval, 1.22-77.8). CONCLUSION: Clinical and immunogenetic variables measurable within 6 months of onset of JRA can be used to predict future disability, pain, and well being. QOL appears more difficult to forecast, perhaps due to the multiple domains that make up this outcome. Further study is needed to identify other genetic and laboratory factors that predict outcome in JRA with greater precision.