Genome-wide association scan identifies a prostaglandin-endoperoxide synthase 2 variant involved in risk of knee osteoarthritis

Abstract

Osteoarthritis (OA), the most prevalent form of arthritis in the elderly, is characterized by the degradation of articular cartilage and has a strong genetic component. Our aim was to identify genetic variants involved in risk of knee OA in women. A pooled genome-wide association scan with the Illumina550 Duo array was performed in 255 controls and 387 cases. Twenty-eight variants with p < 1 x 10(-5) were estimated to have probabilities of being false positives <or=0.5 and were genotyped individually in the original samples and in replication cohorts from the UK and the U.S. (599 and 272 cases, 1530 and 258 controls, respectively). The top seven associations were subsequently tested in samples from the Netherlands (306 cases and 584 controls). rs4140564 on chromosome 1 mapping 5' to both the PTGS2 and PLA2G4A genes was associated with risk of knee OA in all the cohorts studied (overall odds ratio OR(mh) = 1.55 95% C.I. 1.30-1.85, p < 6.9 x 10(-7)). Differential allelic expression analysis of PTGS2 with mRNA extracted from the cartilage of joint-replacement surgery OA patients revealed a significant difference in allelic expression (p < 1.0 x 10(-6)). These results suggest the existence of cis-acting regulatory polymorphisms that are in, or near to, PTGS2 and in modest linkage disequilibrium with rs4140564. Our results and previous studies on the role of the cyclooxygenase 2 enzyme encoded by PTGS2 underscore the importance of this signaling pathway in the pathogenesis of knee OA.

Figure 1

Study Strategy Used for Discovery and Replication

Figure 2

Expression of PLA2G4A and PTGS2 in Chondrocytes PTGS2 and PLA2G4A gene expression was tested by RT-PCR on mRNA obtained from freshly isolated knee-cartilage chondrocytes from five OA patients. The primers amplified a 330 bp PTGS2 PCR product and a 650 bp PLA2G4A PCR product. Human placenta and normal synovium were used as noncartilage control tissues. Water was used as negative control.

Figure 3

Position on Chromosome 1 of the PTGS2 and PLA2G4A Genes, rs4140564 and rs5275 SNPs The LD block for rs4140564 (from Perlegen Genome browser build36) is shown, as are the frequencies of rs5275-rs4140564 haplotypes in UK samples.

Figure 4

Allelic Expression Analysis at PTGS2 SNP rs5275 Expression analysis was carried out for nine UK patients who had severe end-stage OA with RNA extracted from articular cartilage. For each patient, 19 (patient 1) or 20 (patients 2–9) individual cDNA amplifications and SBE reactions were performed. Forty-five individual PCR and SBE reactions were performed for genomic DNA (five reactions per patient). The cDNA allelic ratios were compared with the 45 genomic ratios with a two-tailed Mann-Whitney exact test. Four patients had undergone a THR (H), and five patients had undergone TKR (K). Data shown are the mean +SD, ^∗p < 0.005; ^∗∗∗p < 1.0 × 10⁻⁶.