Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis

OBJECTIVES: To determine if polymorphisms within the Toll-like receptor 4 (TLR4) gene are associated and linked with juvenile idiopathic arthritis (JIA). To investigate any possible gene-gene (epistatic) interaction between TLR4 and macrophage migration inhibitory factor (MIF) gene polymorphisms. METHODS: 313 simplex families (each containing one affected JIA proband) were genotyped. Two known functionally important single nucleotide polymorphisms (SNPs) within the TLR4 gene (Asp299Gly and Thr399Ile) were typed by SNaPshot ddNTP primer extension and capillary electrophoresis.Single point and multipoint transmission disequilibrium tests (TDT) were carried out through the extended TDT and TDT phase packages for the two TLR4 SNPs. Epistatic interaction between TLR4 haplotypes and the previously JIA associated MIF CATT(7)-MIF-173*C promoter haplotype was investigated by chi(2) test and unconditional logistic regression in Stata version 7. RESULTS: No distortion from random inheritance was observed by single point analysis for TLR4 Asp299Gly (p = 0.89) or TLR4 Thr399Ile (p = 0.40). Similarly, no distortion in transmission was seen when the TLR4 haplotypes were studied (p = 0.54). Additionally, no evidence for gene-gene interaction between TLR4 polymorphisms and the previously associated MIF gene polymorphisms was found (p = 0.40). CONCLUSIONS: No linkage or association was seen for Asp299Gly or Thr399Ile SNPs of TLR4 with JIA susceptibility. No evidence of an epistatic interaction between these TLR4 polymorphisms and MIF polymorphisms was found.     

Mutation screening of the macrophage migration inhibitory factor gene: positive association of a functional polymorphism of macrophage migration inhibitory factor with juvenile idiopathic arthritis

OBJECTIVE: To determine if polymorphisms of the macrophage migration inhibitory factor (MIF) gene are associated with juvenile idiopathic arthritis (JIA). METHODS: Denaturing high-performance liquid chromatography was used to screen the MIF gene in 32 UK Caucasian controls and 88 UK Caucasian JIA patients. Ninety-two healthy UK Caucasian controls were then genotyped for each of the polymorphic positions identified. A panel of 526 UK Caucasian JIA patients and 259 UK Caucasian controls were subsequently genotyped for a single-nucleotide polymorphism (SNP) identified in the 5'-flanking region of the gene, using SNaPshot ddNTP primer extension and capillary electrophoresis. The functional significance of this polymorphism was also studied using luciferase-based reporter gene assays in human T lymphoblast and epithelial cell lines. RESULTS: A tetranucleotide repeat CATT((5-7)) beginning at nucleotide position -794 and 3 SNPs at positions -173 (G to C), +254 (T to C), and +656 (C to G) of the MIF gene were identified. No JIA-specific mutations were found. Allele and genotype frequencies differed significantly between the controls and the JIA patients for the MIF-173 polymorphism. Individuals possessing a MIF-173*C allele had an increased risk of JIA (34.8% versus 21.6%) (odds ratio 1.9, 95% confidence interval 1.4-2.7; P = 0.0002). Furthermore, the MIF-173* G and C variants resulted in altered expression of MIF in a cell type-specific manner. Serum levels of MIF were also significantly higher in individuals who carried a MIF-173*C allele (P = 0.04). CONCLUSION: The -173-MIF*C allele confers increased risk of susceptibility to JIA. Our data suggest a cell type-specific regulation of MIF, which may be central to understanding its role in inflammation.     

Macrophage migration inhibitory factor promoter polymorphisms and the clinical expression of scleroderma

OBJECTIVE: To investigate the potential association between functional polymorphisms in the gene for the innate mediator, macrophage migration inhibitory factor (MIF), and the clinical expression of systemic sclerosis (SSc). METHODS: Genomic DNA samples and clinical data were collected from the Scleroderma Family Registry and DNA Repository at the University of Texas Health Science Center at Houston. A total of 740 subjects were studied; 203 of them had diffuse cutaneous SSc (dcSSc), 283 had limited cutaneous SSc (lcSSc), and the remaining 254 healthy subjects served as controls. Association analyses were performed on the whole data set and on patient and sex subsets. Significant relationships were determined between clinical variables and MIF polymorphisms for each disease subtype in the studied groups. RESULTS: The frequency of the -173*C MIF allele, which was previously reported to be associated with high production of MIF, was lower in the lcSSc group (12.6%) than in the dcSSc (19.2%) or control (18.5%) groups (P = 0.010 and P = 0.011, respectively). Haplotype analysis for 2 closely linked polymorphisms in the MIF promoter showed that in white subjects with lcSSc or dcSSc, the lcSSc population had a significantly lower representation of the high-expression MIF haplotype defined by -173*C and -794 with 7 CATT repeats (C7) (P = 0.015, odds ratio 1.94 [95% confidence interval 1.14-3.32]). Fibroblasts encoding the C7 MIF haplotype were observed to produce more MIF upon in vitro stimulation than those with a non-C7 haplotype. CONCLUSION: Functional promoter polymorphisms in the MIF gene affect the clinical presentation of SSc. The proinflammatory haplotype defined by C7 is underrepresented in patients with lcSSc.     

A gene in the telomeric HLA complex distinct from HLA-A is involved in predisposition to juvenile idiopathic arthritis

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is associated with particular alleles at 3 different HLA loci: HLA-A, HLA-DR/DQ, and HLA-DP. These associations are independent of each other (i.e., they cannot be explained by the known linkage disequilibrium between alleles at these loci). The purpose of this study was to look for additional JIA susceptibility genes in the HLA complex. METHODS: One hundred two Norwegian JIA patients and 270 healthy individuals, all carrying the DQ4;DR8 haplotype, were investigated by scanning approximately 10 megabases of DNA covering the HLA complex with microsatellite polymorphisms. An expectation-maximization algorithm was used to estimate haplotype frequencies, and the distribution of microsatellite alleles on the high-risk DQ4;DR8 haplotype was compared between patients and controls, to exclude effects secondary to linkage disequilibrium with these susceptibility genes. RESULTS: Allele 5 at the microsatellite locus D6S265 (D6S265* 5), 100 kb centromeric of HLA-A, showed a strong positive association with the disease (odds ratio 4.7, corrected P < 10(-6)). Haplotype analysis demonstrated that the D6S265*5 association was not caused by linkage disequilibrium to the gene encoding HLA-A*02, which has previously been reported to be associated with JIA. Instead, our data suggested that a gene in linkage disequilibrium with D6S265*5, but distinct from HLA-A*02, is involved in the predisposition to JIA. CONCLUSION: We found that D6S265*5 could be a marker for an additional susceptibility gene in JIA which is distinct from A*02, adding to the risk conferred by DQ4;DR8.     

Macrophage migration inhibitory factor (MIF) and oligoarticular juvenile idiopathic arthritis (o-JIA): association of MIF promoter polymorphisms with response to intra-articular glucocorticoids

OBJECTIVES: To address the clinical relevance of macrophage migration inhibitory factor (MIF) promoter polymorphisms in oligoarticular juvenile idiopathic arthritis (o-JIA) by evaluating their associations with serum and SF MIF levels, with response to intra-articular glucocorticoid injections and with outcome of the disease. METHODS: Seventy-five Caucasian patients with o-JIA were studied. Alleles of the -794 CATT variable number of tandem repeats (VNTR) and of the -173 G/C single nucleotide polymorphism (SNP) were identified by capillary electrophoresis following fluorescently labelled PCR and by allelic discrimination assay, respectively. MIF levels were measured by ELISA. The association of MIF promoter polymorphisms with polyarticular extension, Childhood Health Assessment Questionnaire (CHAQ) score at the last follow-up visit and occurrence of chronic anterior uveitis was evaluated only in patients with a follow up > 5 years. RESULTS: Neither of the MIF promoter polymorphisms was associated with serum MIF levels, nor with the long-term outcome of o-JIA. The -173 G/C SNP was significantly associated with both SF MIF levels and duration of response to intra-articular glucocorticoid injection. Carriers of a MIF -173 C allele were 4 times more likely to relapse within 3 months. No association was found between the different MIF CATT alleles and both SF MIF levels and duration of response to intra-articular glucocorticoids. CONCLUSION: Our study shows the clinical relevance of the MIF -173 G/C SNP in o-JIA and suggests that the -173 C allele may represent a predictor of poor response to intra-articular glucocorticoid treatment.     

Promoter polymorphism in the macrophage migration inhibitory factor gene is associated with obesity

OBJECTIVE: To explore the association of promoter polymorphisms of macrophage migration inhibitory factor (MIF) gene with obesity.Subjects:In total, 213 nondiabetic Japanese subjects. They were divided into three groups according to World Health Organization definitions: lean (body mass index (BMI) <25 kg/m2), overweight (25 < or = BMI < 30 kg/m2) and obese (BMI> or = 30 kg/m2). METHODS:We examined two polymorphic loci in the MIF gene in the subjects: a single-nucleotide polymorphism at position -173 (G/C) and a CATT-tetranucleotide repeat polymorphism at position -794, which both can affect promoter activity in different cells. RESULTS: We detected four alleles: 5-, 6-, 7- and 8-CATT at position -794. Genotypes without the 5-CATT allele (X/X, X refers to 6-, 7- or 8-CATT alleles) were more common in obese subjects than in lean or overweight groups (P = 0.013). The X-CATT allele was more frequent in obese subjects than in lean or overweight subjects (P = 0.030). In contrast, -173G/C was not associated with obesity. Among the haplotypes of the two promoter polymorphisms, G/5-CATT ((-173G/C)/(-794[CATT](5-8))) was associated with a decreased risk of obesity (P = 0.025) and G/6-CATT with an increased risk of overweight (P=0.028).Conclusion:Promoter polymorphism in the MIF gene is linked with obesity.     

Wnt-1-inducible signaling pathway protein 3 and susceptibility to juvenile idiopathic arthritis

OBJECTIVE: To determine whether Wnt-1-inducible signaling pathway protein 3 (WISP3) polymorphisms are associated with susceptibility to juvenile idiopathic arthritis (JIA). METHODS: The exons and the intron/exon boundaries of the WISP3 gene were mutation-screened by denaturing high-performance liquid chromatography in 86 patients with polyarticular-course JIA (>/=5 joints affected) and 15 controls. Seven single-nucleotide polymorphisms (SNPs) were genotyped, using allelic discrimination, in a case-control study. Initially, 159 patients with polyarticular-course JIA and 263 controls were studied, followed by study of a replication cohort of 181 patients with polyarticular-course JIA and 355 controls. Available parents of patients with polyarticular-course JIA were also genotyped. Finally, other JIA subgroups were studied (initial cohort, n = 218; replication cohort, n = 213). Single-point and haplotype analysis was carried out. RESULTS: Positive association with SNP WISP3*G84A was observed and replicated in 2 cohorts of patients with polyarticular-course JIA. Specifically, homozygosity of the mutant allele (WISP3*84AA) conferred a 2-fold increased risk of disease susceptibility (for the initial cohort, odds ratio [OR] 2.1, 95% confidence interval [95% CI] 1.1-4.2, P = 0.03; for the replication cohort, OR 2.0, 95% CI 1.0-4.3, P = 0.05). Strong linkage disequilibrium was observed between SNPs; however, no haplotypic effect of an order of magnitude greater than the single-point WISP3*G84A association was observed. Using the transmission disequilibrium test, a trend toward overtransmission of the WISP3*84A allele was observed in patients with polyarticular-course JIA. No association of any WISP3 polymorphism was observed in the other JIA subgroups. CONCLUSION: Association and replication of a polymorphism within the first intron of the WISP3 gene have been shown in patients with polyarticular-course JIA. The functional significance of the WISP3*G84A SNP is being determined.     

Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor

OBJECTIVE: To study whether genetic variants of macrophage migration inhibitory factor (MIF), the MIF -173G>C and CATT(5-8) alleles, are associated with disease severity and levels of circulating MIF in patients with rheumatoid arthritis (RA). METHODS: Genotyping was performed in patients with early RA and in healthy controls. Demographic data, disease activity, and outcome measurements were compared between patients with and without the MIF variants. MIF -173G>C and CATT(5-8) polymorphisms were genotyped, and a newly developed enzyme-linked immunosorbent assay for human MIF was used. Allele and genotype distributions of the MIF -173G>C and CATT(5-8) polymorphisms were compared between patients and controls by chi-square test. Multiple regression analysis was performed to assess the independence of the MIF functional genetic variants as risk factors for radiologic joint damage. RESULTS: Genotyping of the -173G>C and CATT(5-8) polymorphisms of MIF in RA patients and healthy individuals (n = 277 each) revealed similar frequencies of genotypes and haplotypes in both groups. No significant differences in demographic or clinical features were observed between RA patients carrying the MIF -173C allele or the MIF CATT7 allele or both and non-carrier RA patients. Radiologic joint damage was significantly higher in patients carrying risk alleles of the MIF -173G>C or the MIF CATT(5-8) functional variants. No synergistic effects between both genetic variants were observed. Multivariate regression analysis revealed that presence of the MIF -173C/C and MIF CATT(7/7) genotypes and having 1 MIF -173C allele were independent prognostic variables. Carriership of the MIF -173C allele (P = 0.002) or MIF CATT7 allele (P = 0.004) was associated with significantly higher circulating MIF levels compared with those in subjects having none of the risk-conferring alleles, and greater circulating MIF levels correlated with more severe radiologic joint damage (r = 0.64, P = 0.001). CONCLUSION: The MIF polymorphisms are not associated with RA susceptibility but are associated with high levels of radiologic joint damage. High circulating MIF levels were shown to correlate strongly with radiologic joint damage, suggesting that MIF expression is genetically determined and can be used as a novel prognostic tool in RA.     

Functional and prognostic relevance of the -173 polymorphism of the macrophage migration inhibitory factor gene in systemic-onset juvenile idiopathic arthritis

OBJECTIVE: To address the functional and prognostic relevance of the -173 single-nucleotide G-to-C polymorphism of the macrophage migration inhibitory factor (MIF) gene in patients with systemic-onset juvenile idiopathic arthritis (systemic-onset JIA) by evaluating its association with serum and synovial fluid levels of MIF, with glucocorticoid requirement, and with the outcome of the disease. METHODS: A total of 136 patients with systemic-onset JIA were studied, including 98 patients from the British Paediatric Rheumatology Study Group's National Repository for JIA and 38 patients who were followed up at the IRCCS Policlinico San Matteo (Pavia, Italy) and the IRCCS G. Gaslini (Genoa, Italy). The MIF-173 polymorphism was genotyped using SnaPshot ddNTP primer extension and capillary electrophoresis. MIF levels were measured by enzyme-linked immunosorbent assay. The evaluation of the association of the MIF-173 polymorphism with outcome was performed only in Italian patients who were followed up for >5 years, by analyzing retrospectively 1) the number of joints with active arthritis and the number of joints with limited range of motion; 2) the score, at the last visit, on the Italian version of the Childhood Health Assessment Questionnaire (C-HAQ); and 3) data concerning the treatment regimens during the disease course. RESULTS: Systemic-onset JIA patients carrying a MIF-173*C allele had serum and synovial fluid levels of MIF significantly higher than those in patients with the GG genotype. The duration of glucocorticoid treatment on a daily regimen was significantly longer in patients carrying a MIF-173*C allele than in MIF-173 GG homozygous patients. Moreover, the duration of clinical response to intraarticular injection of triamcinolone hexacetonide was significantly shorter in patients carrying a MIF-173*C allele. At the last visit, the numbers of joints with active arthritis, the C-HAQ scores, and the numbers of joints with limited range of motion were significantly higher in patients carrying the MIF-173*C allele. CONCLUSION: Our study shows the functional relevance of the MIF-173 polymorphism and suggests that the MIF-173*C allele is a predictor of poor outcome in systemic-onset JIA.     

Macrophage migration inhibitory factor polymorphisms do not predict therapeutic response to glucocorticoids or to tumour necrosis factor alpha-neutralising treatments in rheumatoid arthritis

BACKGROUND: Macrophage migration inhibitory factor (MIF) is an inflammatory mediator associated with RA severity. In various diseases, MIF polymorphisms are associated with clinical response glucocorticoid (GC) treatment. It is unclear whether MIF polymorphisms determine GC response in rheumatoid arthritis (RA) and to other RA treatments. Therefore, the question of whether two functional variants in MIF are associated with the response to tumour necrosis factor (TNF)alpha-neutralising and GC treatments in RA was investigated. METHODS: Data from two cohorts of an RA registry were used. For patients who started with TNFalpha-neutralising (infliximab) or GC treatment, courses with a duration of at least 3 months were included and response to TNFalpha blockers or GC was calculated according to the European League Against Rheumatism response criteria. MIF -173G-->C genotyping was achieved using an assay-on-demand allelic discrimination assay, and alleles of the CATT repeat element were identified using a fluorescently labelled PCR primer and capillary electrophoresis. Logistic-regression modelling was used for the statistical analysis. RESULTS: In total, 192 courses of oral prednisone or methylprednisolone injections in 98 patients with RA and 90 patients with RA who were on TNFalpha-neutralising treatments were documented. In all, 27% of the patients with RA were found to be heterozygous for seven CATT repeats (CATT(7)) and 31% were heterozygous for -173C. Respectively, 4% and 6% of the patients with RA were homozygous for the MIF CATT(7) repeat or the MIF -173C allele. Carrier status and homozygosity for CATT(7 )repeat and the MIF -173C allele were not associated with response to GC (odds ratios (ORs) close to 1) or to TNFalpha-neutralising treatment (ORs close to 2). CONCLUSION: The MIF-CATT(7) repeat and the MIF-173G-->C functional variant are not strongly associated with a decreased clinical response to TNFalpha-neutralising or GC treatment in RA.     

A gene in the telomeric HLA complex distinct from HLA–A is involved in predisposition to juvenile idiopathic arthritis

Objective

Juvenile idiopathic arthritis (JIA) is associated with particular alleles at 3 different HLA loci: HLA–A, HLA–DR/DQ, and HLA–DP. These associations are independent of each other (i.e., they cannot be explained by the known linkage disequilibrium between alleles at these loci). The purpose of this study was to look for additional JIA susceptibility genes in the HLA complex.

Methods

One hundred two Norwegian JIA patients and 270 healthy individuals, all carrying the DQ4;DR8 haplotype, were investigated by scanning ∼10 megabases of DNA covering the HLA complex with microsatellite polymorphisms. An expectation‐maximization algorithm was used to estimate haplotype frequencies, and the distribution of microsatellite alleles on the high‐risk DQ4;DR8 haplotype was compared between patients and controls, to exclude effects secondary to linkage disequilibrium with these susceptibility genes.

Results

Allele 5 at the microsatellite locus D6S265 (D6S265* 5), 100 kb centromeric of HLA–A, showed a strong positive association with the disease (odds ratio 4.7, corrected P < 10⁻⁶). Haplotype analysis demonstrated that the D6S265*5 association was not caused by linkage disequilibrium to the gene encoding HLA–A*02, which has previously been reported to be associated with JIA. Instead, our data suggested that a gene in linkage disequilibrium with D6S265*5, but distinct from HLA–A*02, is involved in the predisposition to JIA.

Conclusion

We found that D6S265*5 could be a marker for an additional susceptibility gene in JIA which is distinct from A*02, adding to the risk conferred by DQ4;DR8.

     

Functional and prognostic relevance of the −173 polymorphism of the macrophage migration inhibitory factor gene in systemic‐onset juvenile idiopathic arthritis

Objective

To address the functional and prognostic relevance of the −173 single‐nucleotide G‐to‐C polymorphism of the macrophage migration inhibitory factor (MIF) gene in patients with systemic‐onset juvenile idiopathic arthritis (systemic‐onset JIA) by evaluating its association with serum and synovial fluid levels of MIF, with glucocorticoid requirement, and with the outcome of the disease.

Methods

A total of 136 patients with systemic‐onset JIA were studied, including 98 patients from the British Paediatric Rheumatology Study Group's National Repository for JIA and 38 patients who were followed up at the IRCCS Policlinico San Matteo (Pavia, Italy) and the IRCCS G. Gaslini (Genoa, Italy). The MIF‐173 polymorphism was genotyped using SnaPshot ddNTP primer extension and capillary electrophoresis. MIF levels were measured by enzyme‐linked immunosorbent assay. The evaluation of the association of the MIF‐173 polymorphism with outcome was performed only in Italian patients who were followed up for >5 years, by analyzing retrospectively 1) the number of joints with active arthritis and the number of joints with limited range of motion; 2) the score, at the last visit, on the Italian version of the Childhood Health Assessment Questionnaire (C‐HAQ); and 3) data concerning the treatment regimens during the disease course.

Results

Systemic‐onset JIA patients carrying a MIF‐173*C allele had serum and synovial fluid levels of MIF significantly higher than those in patients with the GG genotype. The duration of glucocorticoid treatment on a daily regimen was significantly longer in patients carrying a MIF‐173*C allele than in MIF‐173 GG homozygous patients. Moreover, the duration of clinical response to intraarticular injection of triamcinolone hexacetonide was significantly shorter in patients carrying a MIF‐173*C allele. At the last visit, the numbers of joints with active arthritis, the C‐HAQ scores, and the numbers of joints with limited range of motion were significantly higher in patients carrying the MIF‐173*C allele.

Conclusion

Our study shows the functional relevance of the MIF‐173 polymorphism and suggests that the MIF‐173*C allele is a predictor of poor outcome in systemic‐onset JIA.

     

A polymorphic variant inside the osteopontin gene shows association with disease course in oligoarticular juvenile idiopathic arthritis

BACKGROUND: Oligoarticular onset juvenile idiopathic arthritis (JIA) has a variable disease course. In some patients the disease remains confined to a few joints (persistent oligoarticular) while in others it extends to affect more joints (oligoarticular extended). Osteopontin is thought to play a role in the pathogenesis. OBJECTIVE: To investigate whether a polymorphic variant in the human osteopontin gene, which is in linkage disequilibrium with recently characterised promoter variants, is associated with the disease course in oligoarticular JIA. METHODS: Genotyping of the two base pair insertion/deletion variant at +245 in the first intron was undertaken by polymerase chain reaction (PCR) amplification of DNA fragments, using a fluorescently labelled primer, followed by allele detection after rapid separation of PCR products on an automated DNA sequencer. RESULTS: Allele 2 of the polymorphic variant in the osteopontin first intron was significantly associated with the persistent oligoarticular form rather than the extended form of JIA. This was verified at the level of genotype and allele frequencies. CONCLUSIONS: The results suggest that osteopontin gene polymorphism is associated with the disease course in oligoarticular JIA and might therefore represent a useful genetic marker to characterise patients with oligoarticular JIA who are at risk of a worse outcome.     

HLA and susceptibility to juvenile idiopathic arthritis: a study of affected sibpairs in an isolated Finnish population

OBJECTIVE: To determine the effects of class I (A, B, and C) and II (DRB1 and DQB1) HLA loci alleles and DRB1-DQB1 haplotypes on genetic susceptibility to juvenile idiopathic arthritis (JIA) in families with 2 or more affected siblings. METHODS: A total of 83 affected siblings belonging to 38 families and corresponding to 50 affected sibpairs, their parents, and 45 healthy sibs were typed for HLA in A, C, B, DRB1, and DQB1 loci. Two study designs were used to explore linkage and association: a case-population control design and a family design using the linkage method: identical-by-descent (IBD) allele-sharing and the association analysis methods. Associations in family data were analyzed using the independent transmission disequilibrium test (TDT) for linkage in the presence of association. This was supplemented by the family-based association test (FBAT) to look for association in the presence of linkage, and is robust for population stratification and phenotype-based selection of data. RESULTS: Significantly increased HLA allele frequencies among the affected siblings compared to Finnish bone marrow donors were observed for HLA alleles Cw4 (odds ratio, OR, 1.7), B27 (1.8), B35 (1.7), and DR8 (3.7). The observed ratio of sharing 0, 1, and 2 HLA haplotypes (A, C, B, DRB1, and DQB1) among affected sibpairs (ASP) was 10:23:17, significantly different from expected (p < 0.001), using a formula that takes into account disease prevalence and the sibling recurrence risk. In the univariate association analysis, both independent TDT and FBAT found significantly increased transmission of the DRB1*0801 and DQB1*0402 alleles and Cw*0401. Independent positive allele effects of Cw*0401, DRB1*0801, and DQB1*0402 as well as negative effects of Cw*0701 and DQB1*0302 were shown by the family-based association analysis of the joint allele main effects. Multi-allelic test for association of each locus confirmed significant associations of the DRB1 and DQB1 loci in the risk of JIA. We found DRB1*0801/DQB1*0402 haplotype to be strongly associated (p < 0.001) with JIA, supporting findings of the haplotype associations-based ASP design. CONCLUSION: Both linkage analysis of the affected sibpairs and association analysis of nuclear families with JIA provided overwhelming evidence of the major contribution of HLA to genetic susceptibility to JIA. The association analysis of HLA-A, C, B, DRB1, and DQB1 alleles by both TDT and FBAT tests confirmed in the Finnish population that the most significant associations prevailed for DRB1*0801, DQB1*0402, as expected from previous observations, and supported the independent role of Cw*0401.     

Juvenile idiopathic arthritis (JIA) is primarily associated with HLA-DR8 but not DQ4 on the DR8-DQ4 haplotype

BACKGROUND: Juvenile idiopathic arthritis (JIA) is strongly associated with the DR8-DQ4 haplotype. The genes encoding DR8 and DQ4 are in strong linkage disequilibrium (LD) and occur together on the same HLA haplotype in almost all patients and controls. Because of the strong LD it is not clear whether DR8, DQ4, or both, are primarily associated with JIA. OBJECTIVE: To unveil the primary association of JIA--that is, with DR8 or DQ4. METHODS: DRB1, DQA1, and DQB1 alleles of 585 Norwegian and 47 Polish unrelated patients with JIA (categorised as pauciarticular and rheumatoid factor negative polyarticular JIA), and of 3155 Norwegian and 158 Polish unrelated controls, were typed using a polymerase chain reaction or oligonucleotide hybridisation and sequence-specific primers method. RESULTS: Several haplotypes which encoded DR8 (that is, carried DRB1*08) and which did not encode DQ4 (that is, did not carry DQA1*0401) were found. Such haplotypes were found in three Norwegian patients and two controls (p=0.029). In the Polish population such haplotypes were found among four patients with JIA and two controls (p=0.025). No haplotypes which carried DQA1*0401 and DQB1*0402 in the absence of DRB1*08 were found, either among patients with JIA (Polish and Norwegian) or among the controls (Polish). CONCLUSION: On the DR8-DQ4 haplotype the DRB1*08 allele is primarily associated with JIA.     

Frequency and nature of cytokine gene polymorphisms in type 1 autoimmune hepatitis.

Genetic involvement in type 1 autoimmune hepatitis (AIH) is indicated by a marked female preponderance and strong, well-established, human leukocyte antigen (HLA) associations. These associations, however, are not universal and a number of genes outside the major histocompatibility complex may also play a role in susceptibility to type 1 AIH. Prime candidates at present are those polymorphic genes encoding the proinflammatory and immunoregulatory cytokines. The aim of this study was to investigate, for the first time, 2 members of the interleukin-1 (IL-1) family (IL-1B and IL-1RN), 3 polymorphic sites in the interleukin-10 (IL-10) gene promoter (positions -1082, -819, and -592), and 2 polymorphisms in the tumor necrosis factor-alpha (TNF-alpha) promoter (positions -308 and -238) in type 1 AIH. The study was performed on 2 independently collected DNA banks, each with appropriate controls, and throughout the analysis associations described in the first set were confirmed in the second set. Standard polymerase chain reaction (PCR)-based genotyping techniques were used. Overall there were no significant differences in the distributions of the IL-1B and IL-10 alleles, genotypes, or haplotypes in either study set. In contrast we report a significant association between type 1 AIH and TNF*2 (first set: 34% of controls vs. 49% of patients, Pc =.014 and second set: 26% vs. 56%, P =.00008). However, TNF*2 is found in strong linkage disequilibrium with the HLA A1-B8-DR3 haplotype and stratification analysis indicates that the association with TNF*2 is interdependent with HLA DRB1*0301. This is an indication that there is more than one susceptibility allele for type 1 AIH on chromosome 6p21.3.     

DR- and DQ-associated protection from type 1A diabetes: comparison of DRB1*1401 and DQA1*0102-DQB1*0602*

The transmission disequilibrium test was used to analyze haplotypes for association and linkage to diabetes within families from the Human Biological Data Interchange type 1 diabetes repository (n = 1371 subjects) and from the Norwegian Type 1 Diabetes Simplex Families study (n = 2441 subjects). DQA1*0102-DQB1*0602 was transmitted to 2 of 313 (0.6%) affected offspring (P < 0.001, vs. the expected 50% transmission). Protection was associated with the DQ alleles rather than DRB1*1501 in linkage disequilibrium with DQA1*0102-DQB1*0602: rare DRB1*1501 haplotypes without DQA1*0102-DQB1*0602 were transmitted to 5 of 11 affected offspring, whereas DQA1*0102-DQB1*0602 was transmitted to 2 of 313 affected offspring (P < 0.0001). Rare DQA1*0102-DQB1*0602 haplotypes without DRB1*1501 were never transmitted to affected offspring (n = 6). The DQA1*0101-DQB1*0503 haplotype was transmitted to 2 of 42 (4.8%) affected offspring (P < 0.001, vs. 50% expected transmission). Although DRB1*1401 is in linkage disequilibrium with DQB1*0503, neither of the two affected children who carried DQA1*0101-DQB1*0503 had DRB1*1401. However, all 13 nonaffected children who inherited DQA1*0101-DQB1*0503 had DRB1*1401. In a case-control comparison of patients from the Barbara Davis Center, DQA1*0101-DQB1*0503 was found in 5 of 110 (4.5%) controls compared with 3 of 728 (0.4%) patients (P < 0.005). Of the three patients with DQB1*0503, only one had DRB1*1401. Our data suggest that both DR and DQ molecules (the DRB1*1401 and DQA1*0102-DQB1*0602 alleles) can provide protection from type 1A diabetes.     

Wnt‐1–inducible signaling pathway protein 3 and susceptibility to juvenile idiopathic arthritis

Objective

To determine whether Wnt‐1–inducible signaling pathway protein 3 (WISP3) polymorphisms are associated with susceptibility to juvenile idiopathic arthritis (JIA).

Methods

The exons and the intron/exon boundaries of the WISP3 gene were mutation‐screened by denaturing high‐performance liquid chromatography in 86 patients with polyarticular‐course JIA (≥5 joints affected) and 15 controls. Seven single‐nucleotide polymorphisms (SNPs) were genotyped, using allelic discrimination, in a case–control study. Initially, 159 patients with polyarticular‐course JIA and 263 controls were studied, followed by study of a replication cohort of 181 patients with polyarticular‐course JIA and 355 controls. Available parents of patients with polyarticular‐course JIA were also genotyped. Finally, other JIA subgroups were studied (initial cohort, n = 218; replication cohort, n = 213). Single‐point and haplotype analysis was carried out.

Results

Positive association with SNP WISP3*G84A was observed and replicated in 2 cohorts of patients with polyarticular‐course JIA. Specifically, homozygosity of the mutant allele (WISP3*84AA) conferred a 2‐fold increased risk of disease susceptibility (for the initial cohort, odds ratio [OR] 2.1, 95% confidence interval [95% CI] 1.1–4.2, P = 0.03; for the replication cohort, OR 2.0, 95% CI 1.0–4.3, P = 0.05). Strong linkage disequilibrium was observed between SNPs; however, no haplotypic effect of an order of magnitude greater than the single‐point WISP3*G84A association was observed. Using the transmission disequilibrium test, a trend toward overtransmission of the WISP3*84A allele was observed in patients with polyarticular‐course JIA. No association of any WISP3 polymorphism was observed in the other JIA subgroups.

Conclusion

Association and replication of a polymorphism within the first intron of the WISP3 gene have been shown in patients with polyarticular‐course JIA. The functional significance of the WISP3*G84A SNP is being determined.