Abstract
Cytosolic phospholipase A(2) (cPLA(2)) group IValpha is a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. cPLA(2)(-/-) mice have reduced allergen-induced bronchoconstriction and bronchial hyperresponsiveness. The goal of this study was to investigate polymorphisms of the (CA)(n) and (T)(n) microsatellites and surrounding regions in the cPLA(2)alpha gene promoter. We analysed the cPLA(2) promoter regions containing (CA)(n) and (T)(n) repeats in 87 patients with severe asthma and in 48 control subjects by bidirectional sequencing. Functional studies were performed utilizing reporter genes derived from subjects with varying numbers of these repeats, and on constructs with a series of deletions. We found that the (CA)(n) and (T)(n) regions are polymorphic and that constructs with CA or T repeats or CA and T repeats deleted revealed, respectively, a 41.8 +/- 7%, 22.3 +/- 5% and 100 +/- 20% increase in reporter gene activity. A lower number of CA or T repeats caused higher cPLA(2) promoter luciferase activity. The group of shorter alleles of the (CA)(n) microsatellite region (n = 12-18) (P(cor) = 0.00006), and the group of shorter alleles of (T)(n) repeats region (n = 17-38) (P(cor) = 0.0039) occurred significantly more often in patients with severe asthma. We also found novel SNPs in positions -292 C > G, -185 A > C, -180 T > C and -165 A > C. Two of them were associated with the severe asthma phenotype: -180T allele (P(cor) = 0.03996) and -185 A allele (P(cor) = 0.03966). These results demonstrate that (CA)(n) and (T)(n) repeats may have an influence on cPLA(2) transcription which might play a role in severe asthma pathogenesis.