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-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 → 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.