Abstract Mammographic density (MD) is a strong predictor of breast cancer risk and is highly correlated with the hormone exposure profiles of women. It is also a highly heritable risk factor (heritable component ∼67%). As such, many efforts have been made to identify underlying genetic determinants of MD within pathways related to steroid hormone biosynthesis and metabolism. Several associations with genetic polymorphisms in genes involved in estrogen metabolism have been previously described (e.g. COMT and HSD3B1). Still, there is controversy among the conclusions of these studies. In addition, only a few candidate genes have been interrogated at a time. In this study, we assess associations of genetic variants with MD using the most extensive estrogen metabolic pathway coverage to date.Materials and methods:Cases were 891 Swedish-born women who were 50-74 years of age at diagnosis and diagnosed with breast cancer between October 1993 and March 1995. 840 controls were population and frequency matched to the expected age distribution of the cases.MD was measured using a computer-assisted technique (Cumulus, University of Toronto, Canada) and evaluated by comparing the dense area by the total breast area.Genotyping was performed on a total of 239 SNPs in 34 estrogen metabolic genes using the Sequenom (San Diego, California) primer extension-based assay. SNPs with a call rate <85%, minor allele frequency <1% or out of Hardy-Weinberg Equilibrium (p<0.05/239) were excluded from further analysis.The Cochran-Armitage trend test was carried out for all SNPs with adjustments for age, body mass index, menopausal status and hormone replacement therapy usage.Results:We report a novel association finding between MD and the gene CYP11A1. 5 out of 6 SNPs within the gene were found to be significant after adjustment. The average increase in MD attributed to these SNPs is 5.4%. Other genes with significant associations include HSD17B3, STS, and NQO1.Discussion:A high level of endogenous estrogen is well-known to be correlated with an increased risk of breast cancer. CYP11A1 encodes a member of the cytochrome P450 superfamily of enzymes and is of interest as it represents is the first as well as a rate-limiting step in steroid hormone biosynthesis.In addition, a role for CYP11A1 in the metabolism of vitamin D3 has been reported recently. High vitamin D levels have been associated with a decrease in both breast cancer risk and MD. Although the active compound most commonly implicated is 1,25-dihydroxyvitamin D3 derived from a more extensively studied pathway, new studies have shown that a by-product of CYP11A1 exhibits similar functions in the regulation of cell proliferation and differentiation.Although CYP11A1 has been previously reported to be associated with breast cancer, it has never been studied in relation to MD. This novel finding suggests that CYP11A1 exerts an influence on MD through two different pathways linked to breast cancer and affirms MD as an intermediate phenotype for the disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5161.