Table 1 Patient demographics Number of patients Age (mean±SD) Gender (% female) BMI (mean±SD) Trauma history (%) Hand involvement† (%) NYUHJD 80 65.6±9.9 61.3 26.4 + 3.7 20 53 POP study* 50 61.2±11.9 82 31.1 + 6.3 18 58 *Duke University Medical Center; †by clinical examination. BMI, body mass index; NYUHJD, New York University Hospital for Joint Diseases; POP, Prediction of Osteoarthritis Progression study. response genes, including those for IL1α, IL1β, IL1Ra, TNFα, IL10, oestrogen receptor 1, were genotyped. SNPs genotyped (gene, rs number, frequent nucleotide>less frequent nucleotide, minor allele frequency) were IL1A(+4845), rs17561, G>T, 30.5%; IL1B(−511), rs16944, C>T, 33.4%; IL1B(−1464), rs1143623, G>C, 32.8%; IL1B(−3737), rs4848306, C>T, 44.7%; IL1B(+3954), rs1143634, C>T, 24.2%; IL1B(+3877), rs1143633, A>G, 39.7%; IL1RN(+2018), rs419598, T>C, 27.3%; IL1RN, rs315952, C>T, 25.8%; IL1RN, rs9005, G>A, 30.5%; IL10(−1082), rs1800896, A>G, 46.7%; IL10(−819), rs1800871, T>C, 17.3%; IL10(−592), rs1800872, A>C, 20.2%; TNFA(−308), rs1800629, A>G, 21.7%; ESR1_PvuII, rs2234693, T>C, 44.9%; ESR1_Xbal, rs9340799, A>G, 33.8%. Genotyping was accomplished (Interleukin Genetics Clinical Laboratory, Waltham, Massachusetts, USA; CLIA certi- ﬁed) by PCR targeting the sequence surrounding the SNPs stud- ied. Multiplexed single-base extension reactions were performed. Genotypes were analysed (Beckman Coulter, Brea, California, USA; CEQ8800) and ﬁnal genotypes were scored by laboratory personnel blinded to all patient data. Synovial ﬂuid analysis Synovial ﬂuid (SF) samples were a required component of participation in the POP study, so SF samples were avail- able from all subjects in the POP cohort. Fifty POP subjects met the entrance criteria for this study as described earlier; therefore, the SF samples represented no selection bias. Fluid samples were aspirated directly (n=36) or by lavage (n=14), corrected for dilution by the urea method11 and analysed blinded to the clinical information. Cytokines (pg/ml) were quantiﬁed by multiplex bead assays (Bio-Plex; Bio-Rad, Life Science research, Hercules, California, USA). High-sensitivity C-reactive protein concentrations (mg/l) were measured using the UBI Magiwel Enzyme Immunoassay (United Biotech, Mountain View, California, USA; minimum detectable concen- tration of 0.00035 mg/l, interassay variation ≤7.4%). Cartilage oligomeric matrix protein (COMP) was measured by sandwich ELISA with monoclonal antibodies 17C10 and 16F12 to human COMP (minimum detection 120 ng/ml, intra-assay and inter- assay variation ≤5.8% and 8.7%, respectively). IL1ra was mea- sured using the Quantikine Human IL1Ra Immunoassay from R&D Systems (Minneapolis, Minnesota, USA), with a mini- mum detectable dose of 6.26 pg/ml. Statistical methods Primary analyses evaluated associations between genotypes and radiographic severity, as measured by KL scores. Patients with KL scores of 1, 2 were compared with those with scores of 3, 4. To determine whether results were potentially due to aberrant KL score distributions, a second analysis was performed compar- ing KL1 with KL2–4 (results were unchanged). Genotype devia- tion from Hardy–Weinberg equilibrium was tested (Pearson’s χ² Ann Rheum Dis 2010;69:856–861. doi:10.1136/ard.2009.113043 Extended report test) in the control sample. Genotype associations with radio- graphic severity were determined using χ² statistics or Fisher’s exact test, adjusted for non-genetic risk factors, age, body mass index (BMI) and gender, where appropriate, using multivariate logistic regression analysis. The effect of multiple comparisons was considered in the discovery population (NYUHJD) involv- ing 15 SNPs. Comparisons of JSW between genotypes were made using non-parametric Wilcoxon test and a mixed model analysis of variance for correlated data to adjust for two knees in the same subject. RESULTS The NYUHJD population was analysed ﬁrst. Patients with more severe knee OA (KL 3, 4 vs 1, 2) were older (p=0.013) but did not differ by gender (p=0.96) or BMI (p=0.46). Subsequent analyses were age adjusted. All SNPs were in Hardy–Weinberg equilibrium and were evaluated for association with radiographic severity. After adjustment for multiple comparisons, one SNP in the IL1Ra gene (IL1RN) was signiﬁcantly associated with decreased risk for severe OA and two other IL1RN SNPs showed the same protective trend (table 2). IL10 SNPs, also associated with radio- graphic severity, were not signiﬁcant after adjustment for mul- tiple comparisons. We then tested the Duke POP cohort for IL1RN gene variations, based on association with severity in the NYUHJD population. In the POP study, age, gender or BMI were not signiﬁcant inﬂuences, but two of three IL1RN SNPs were signiﬁcantly associated with knee OA severity (table 2). All other SNPs tested in the NYUHJD population were subse- quently evaluated in the POP patients, and none was signiﬁcant (data not shown). Since all signiﬁcant SNPs were in one gene, we evaluated haplotype effects on severity. Of nine possible haplotypes from three IL1RN SNPs, four had a frequency >1%, and one (rs419598/rs315952/rs9005=CTA) was associated with reduced risk for severity in both populations (table 2). We then combined the two populations for further analy- ses focusing on the IL1RN loci. In the combined dataset, age was associated with severity (p=0.0065) but gender and BMI were not. IL1RN SNPs and haplotypes were associated with decreased risk for severe disease by KL grade and JSW (table 3). The ﬁrst JSW analysis was knee based and included the smaller JSW for all knees (n=251 knees with complete data; 126 patients from NYUHJD and Duke POP). JSW analyses were adjusted for age, gender and BMI, and the knee-based analy- sis used a mixed model analysis of variance for intercorrelated data to adjust for two knees in the same subject. Two IL1RN SNPs were signiﬁcantly associated with greater mean JSW, as was the IL1RN CTA haplotype (mean JSW in patients carrying the CTA haplotype=3.99 mm±1.77, vs reference haplotypes= 3.14 mm+1.93; p=0.0008). In addition to the IL1RN genotype and haplotype effects observed for the signal knees, a similar IL1RN CTA haplotype effect was seen in the contralateral knees when analysed for KL scores of 3–4 (OR=0.065; 95% CI 0.0076 to 0.55; p=0.0024) and JSW (p=0.008). In a person-based analysis of signal knees only, all signal knees (n=126 with complete data) from NYUHJD and Duke, were classiﬁed as to medial (n=95) or lateral-dominant (n=31) disease. The protective effect was apparent for medial knee OA (mean JSW in patients carrying the CTA haplotype=3.37 mm±1.66, vs reference haplotypes =2.29 mm±1.73; p=0.0054). There were no signiﬁcant IL1RN genotype associations for the lateral compartment (mean JSW in patients carrying the 857