Identification and functional characterization of polymorphisms in human cyclooxygenase-1 (PTGS1)

Abstract

Objective-Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H 2 , which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1.Methods-Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin.Results-Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35 ± 5%), R78W (36 ± 4%), K185T (59 ± 6%), G230S (57 ± 4%), and L237M (51 ± 3%) variant proteins had significantly lower metabolic activity relative to wild-type (100 ± 7%), while no significant differences were observed with the R8W (104 ± 10%), P17L (113 ± 7%), and V481I (121 ± 10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC 50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1.Conclusions-Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.

Heritability of Bronchopulmonary Dysplasia, Defined According to the Consensus Statement of the National Institutes of Health

“…32 In humans, several COX genetic variants have been described, which potentially may account for inter-individual differences in pharmacological response to COX inhibitors. 33 A limitation to all retrospective twin analyses, including ours, is the potential for disease misclassification. This can significantly affect heritability estimates (see 13 for a review).…”

Influence of Cyclooxygenase-1 Genotype on ex vivo Aspirin Response in Patients at Risk for Stroke

“…Because of reported linkage disequilibrium between the PTGS1 A–707G and P17L polymorphisms, we also examined whether PTGS1 diplotype contributed to aspirin response [13]. Thirty-nine (66%) subjects had the PTGS1 homozygous wild-type diplotype (–707AA and 17PP), 16 (27%) were heterozygous at either position –707 or 17 (–707AG or 17PL), and 4 (7%) were heterozygous at both positions (–707AG and 17PL).…”

“…Point mutations in the signal peptide region have been reported to disrupt the translocation and processing of various proteins [12]. In vitro studies have shown that platelets expressing the 17L variant are more susceptible to inhibition by indomethacin, another nonsteroidal anti-inflammatory drug [13]. The P17L SNP is in significant linkage disequilibrium with the A–707G SNP in the gene’s promoter region, and there are some data supporting an association between the A–707G SNP and antiplatelet response to aspirin [13,14,15].…”

“…In vitro studies have shown that platelets expressing the 17L variant are more susceptible to inhibition by indomethacin, another nonsteroidal anti-inflammatory drug [13]. The P17L SNP is in significant linkage disequilibrium with the A–707G SNP in the gene’s promoter region, and there are some data supporting an association between the A–707G SNP and antiplatelet response to aspirin [13,14,15]. Other studies, using various methods of measuring aspirin response, have yielded conflicting data on the effects of PTGS1 genotype on aspirin response, and few studies have assessed both the PTGS1 and ITGB3 polymorphisms [14, 16, 17].…”

Novel non-synonymous polymorphisms in the COX-1 gene in Turkish pediatric patients with cardiovascular anomalies

“…Thus, it is believed that the differences seen in the function of COX-1 most likely result from genetic variation in the regulatory or signal peptide region of the gene or from variation in platelet development and aging. Effects of such variation would hypothetically affect either the amount of COX-1 protein or the location of COX-1 protein in the cell (Halushka et al, 2003;Lee et al, 2007). COX-1 structure-function studies suggest that several amino acid substitutions with modest influence on arachidonic acid binding to the COX-1 active site can alter the catalytic efficiency and metabolite profile of COX-1-mediated arachidonic acid metabolism (Thuresson et al, 2001).…”