High-throughput single-nucleotide polymorphism analysis of the IL1RN locus in patients with ankylosing spondylitis by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

OBJECTIVE: To examine single-nucleotide polymorphisms (SNPs) in the 3' region of the IL1RN gene in a large Caucasoid case-control series and in ankylosing spondylitis (AS) families from Western Canada by use of high-throughput MassArray matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry SNP typing. METHODS: An association analysis was performed in a case-control cohort of 394 AS cases and 500 controls. Family-based association analysis was performed in 58 simplex and 13 multiplex families. Three SNPs located in the 3' region of the IL1RN gene (T/C at position 27810 in exon 4, T/C at position 30735 in exon 6, and G/C at position 31017 in exon 6) were examined by high-throughput MassArray MALDI-TOF mass spectrometry. Haplotype inference software programs were used to infer the most likely haplotypes and to compare haplotype frequencies, which were then further analyzed in family-based association studies by transmission disequilibrium tests. RESULTS: The frequency of allele C at SNP position 30735 in exon 6 was significantly increased in AS cases (35.1%) versus controls (27.8%), as was the phenotype frequency (61.7% versus 48.6%). A significantly increased frequency of SNP allele G at position 31017 in exon 6 in cases (32.9%) versus controls (28.3%) was also noted. A highly significant difference in the overall distribution of haplotype frequencies was evident between cases and controls, with significant increases in the frequencies of the 27810C/30735C/31017C and 27810C/30735T/31017G haplotypes, but a significant reduction in the estimated frequency of the 27810C/30735T/31017C haplotype, in the AS cases. Estimation of haplotype frequencies based on 2 SNP markers indicated a highly significant increase in the 30735C/31017C haplotype and a highly significant decrease in the 30735T/31017C haplotype in cases compared with controls. Preliminary evidence for reduced transmission of the 27810C/30735T/31017C 3-marker haplotype was also found in family-based association analyses. CONCLUSION: Our data establish a highly significant disease association with markers in the IL1RN gene. In the absence of nonsynonymous coding sequence substitutions, it is possible that the primary disease-associated locus regulates gene expression. Association with specific haplotypes raises the possibility that the primary disease locus is in linkage disequilibrium with a specific combination(s) of markers in the IL1RN gene.

HLA and cytokine gene polymorphisms in relation to occurrence of palindromic rheumatism and its progression to rheumatoid arthritis.

OBJECTIVE: Palindromic rheumatism (PR) is an episodic arthropathy that may precede typical rheumatoid arthritis (RA), although pathogenetic relationships between these disorders remain unclear. The predictive value for those immunogenetic risk factors implicated in RA for disease progression in PR remains to be established. A previous retrospective analysis from our group has implicated rheumatoid factor in disease progression. Our objective was to determine the contribution of HLA and cytokine gene polymorphisms implicated in RA to predisposition to PR and to progression of PR to chronic joint inflammation. METHODS: We studied 147 patients with PR seen in a tertiary referral center; 87 were selected retrospectively from the period 1986-96 using a structured selection process and 60 were selected prospectively in the period 1997-2001. Comparison groups included 149 patients with RA and 149 ethnically matched controls. Typing for HLA-DRB1 alleles and HLA-DRB1-04 subtypes was performed following polymerase chain reaction (PCR) amplification using sequence-specific primers (SSP). Cytokine genotypes were ascertained following PCR-SSP with and without digestion with restriction enzymes. Time-adjusted survival analysis (Kaplan-Meier) and multivariate logistic regression were used to assess the independent contribution of immunogenetic markers in assessing progression of PR to chronic joint inflammation. RESULTS: Thirty-one percent of patients progressed to connective tissue disease after a mean of 10.6 (retrospective group) and 3.9 (prospective group) years. A significantly increased prevalence of the shared epitope (SE) allele was noted in patients with PR (65%) versus controls (39%) (OR 2.9, 95% CI 1.8-4.6, p < 0.001). This primarily reflected increased prevalence of the DRB1-0401 and 0404 and not DRB1-01 alleles. A weak contribution to disease susceptibility was also noted with carriage of the IL4 promoter -590T (OR 1.8, 95% CI 1.1-3.0, p = 0.02) and IL4 intron 3 RP1 (OR 1.7, 95% CI 1.1-2.9, p = 0.03) alleles. The TNFa +489A allele was associated with RA (OR = 2.7, 95% CI 1.5-5.1, p = 0.001) in both SE+ and SE- patients, but not with PR. Time-adjusted and multivariate Cox regression analysis revealed that only homozygosity for SE alleles was a significant independent risk factor for disease progression to chronicity in PR (hazard ratio 2.9, 95% CI 1.2-6.9, p = 0.02). However, none of 8 patients homozygous for SE- DRB1 XP4n alleles developed chronic disease after 10 years of followup (p = 0.07). CONCLUSION: The immunogenetic risk profile for PR resembles that for RA, indicating that PR is likely not an independent entity. A significant gene dose effect for SE alleles is operative in determining risk for progression from PR to RA.

Infliximab in ankylosing spondylitis: a prospective observational inception cohort analysis of efficacy and safety.

OBJECTIVE: Infliximab, a neutralizing antibody to tumor necrosis factor-alpha, appears to be effective therapy in ankylosing spondylitis (AS), although treatment is costly and serious infections are an increasing concern. We investigated the efficacy and tolerability of infliximab in a prospective observational inception cohort of patients with nonsteroidal antiinflammatory drug-refractory AS seen in both university and community based practice. We also used a lower dose, 3 mg/kg, than has been evaluated to date in AS. METHODS: We included all consecutive patients with AS starting infliximab therapy 3 mg/kg i.v. at 0, 2, and 6 weeks and q 2 months between April 2000 and October 2001. Data were systematically collected at baseline, 14 weeks, and 1 year, or at withdrawal, and included demographic characteristics, Bath AS indexes (BASDAI, BASFI, BASGI, BASMI), adverse events, and reasons for withdrawal. Laboratory measures included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), serum matrix metalloproteinases (MMP) 1 and 3, and serum human cartilage glycoprotein-39 (YLK-40). The first 6 consecutive patients were also studied by several magnetic resonance sequences, including dynamic MRI with gadolinium augmentation of affected joints. Maximal rate of augmentation was determined at baseline and 84 days. Analysis was by intention-to-treat. RESULTS: Twenty-one patients (m:f = 17:4), mean age 42.5 years (range 24-66), mean disease duration 13.8 years (range 3-26), were studied: 13 had active peripheral synovitis at baseline. Mean followup was 47.5 weeks (range 10-77). Four patients withdrew, 2 for serious adverse events (septic osteomyelitis and severe hypersensitivity after 3 and 2 infusions, respectively), one for lack of efficacy, and one lost to followup. Three patients required an increased dose to 5 mg/kg after 14 weeks. Efficacy data were available on 17 patients at 14 weeks; mean BASDAI improved significantly from baseline (6.2) to 14 weeks (2.8) (p < 0.001), with 10 patients (58.8%) showing at least 50% improvement (range 0-99.6%). Significant reduction in mean BASFI (43.4%; p < 0.001), BASGI (44%; p = 0.001), ESR (55%; p < 0.001), and CRP (63.5%; p = 0.01) was evident. Complete remission of peripheral joint disease was seen in 5 of 11 (45.4%) patients evaluated at 14 weeks and maximal rate of MRI defined gadolinium augmentation was significantly decreased (p = 0.04). Reductions in serum YKL-40 and MMP-1 and 3 were nonsignificant, but significant correlations were observed between changes in BASDAI, ESR, CRP, and changes in serum levels of MMP-3 and YKL-40 (p < 0.005 to p < 0.05). Followup data on 8 patients completing 1 year of therapy revealed continued efficacy at a dose of 3 mg/kg every 8 weeks. CONCLUSION: Infliximab appears to be effective and well tolerated for both axial and peripheral joint disease in AS even at lower doses than those examined to date. Suppression of markers of cartilage degradation/turnover commensurate with reductions in clinical and laboratory measures of disease activity suggests that these markers should be further validated as surrogates for structural damage in AS. Controlled trials are warranted to further assess the potential of this agent in ameliorating structural damage.