Pyrosequencing of polymorphisms in the COX-2 gene (PTGS2) with reported clinical relevance

Genetic variants in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which codes for COX-2, have been identified to modulate the response to COX-2-inhibiting drugs and to be possible risk factors for the incidence or prognosis of cardiovascular or neoplastic diseases, Alzheimer's disease, multiple sclerosis, asthma or osteoarthritis. Clinical evidence thus suggests a clinical importance of COX-2 genetics reaching from disease risk or prognostics up to a personalized therapy with COX-2 inhibitors. The aim of this study was to develop rapid and reliable screening assays for PTGS2 mutations with reported clinical consequences.

SNPs (dbSNP-IDs rs689465, rs689466, rs3918304, rs20415, rs20417, rs5270, rs2745557, rs5277, rs2066826, rs4648276, rs5273, rs5275, rs4648298, rs689469) and a nucleotide-deletion variant (rs20431) were chosen according to reported functional associations. For this selection of variants spanning the whole PTGS2 gene range, Pyrosequencing assays were established in DNA from 350 healthy unrelated Caucasians.

In all 350 DNA samples, the 15 PTGS2 polymorphisms were identified correctly as verified by control samples obtained by conventional sequencing. In silico haplotype analysis based on ten SNPs of greater than 1% observed frequencies identified two haploblocks with a linkage disequilibrium of D' = 0.59. Approximately 50% of the reconstructed haplotypes consisted of non-mutated alleles.

The presently developed Pyrosequencing assays allow for quick and reliable detection of PTGS2 genotypes and may promote further research toward personalized approaches to pathophysiological conditions involving COX-2.