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

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-alpha to this region. Cotransfection experiments with an estrogen receptor-alpha expression vector in MDA-MB-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 3 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 3 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.