Linked common polymorphisms in the gelatinase a promoter are associated with diminished transcriptional response to estrogen and genetic fitness

Abstract

Gelatinase A (matrix metalloproteinase-2) plays a prominent role in multiple biologic processes. Prior studies have established critical roles for gelatinase A transcriptional regulation by defined enhancer elements. To determine possible functional single nucleotide polymorphisms within these elements, we determined the single nucleotide polymorphism distribution within 1,665 bp of the gelatinase A 5′-flanking region, using a healthy homogeneous Caucasian study group of 463 individuals. Among the polymorphisms detected, a G → A transition at bp - 1575 was located immediately 5′ to a half-palindromic potential estrogen receptor binding site. In estrogen receptor-positive MCF-7 cells the -1575G allele functioned as an enhancer, whereas the -1575A allele reduced transcription activity significantly. Gel shift assays confirmed that the differences in allelic expression affected binding of the estrogen receptor-α to this region. Cotransfection experiments with an estrogen receptor-α expression vector in MDA-ME-231 cells, which do not constitutively express an estrogen receptor, revealed that estrogen receptor is absolutely required for enhancing activity. Allelic distribution analysis indicated that a previously reported C → T transition within an Sp1 binding site at -1306 was in linkage disequilibrium with the -1575G → A transition. Luciferase reporter studies of the linked variant -1575A -1306T allele versus the wild type -1575G -1306C allele demonstrated an additive reduction in estrogen-dependent reporter activity. The frequency of the -1575G → A transition deviated significantly from the expected Hardy-Weinberg distribution in two independently assembled study populations consisting of healthy adult blood donors and newborns of Caucasian origin, both with a calculated 21% reduction in genetic fitness. Gelatinase A is a known estrogen-responsive gene and the demonstration of a loss of function polymorphism within an operational estrogen receptor binding site associated with a decrease in genetic fitness underscores the biologic significance of promoter polymorphism analyses.