BMC Cancer 2009, 9:438 http://www.biomedcentral.com/1471-2407/9/438 advanced breast cancer from the Multiethnic Cohort (Afri- can Americans, Latinos, Japanese, Native Hawaiians, and European Americans) to identify novel SNPs and deter- mine their contribution to breast cancer risk in the Multi- ethnic Cohort (cases: 1612, controls: 1961). Two SNPs were significantly associated with breast cancer risk in this study (one in each of SMRT and CALCOCO1). These SNPs, however, are found exclusively or nearly exclusively in African Americans and therefore cannot be feasibly tested in DNA banks derived from European individuals. One SRC-3 SNP previously identified to be protective in our study [15] was genotyped (rs2230782) and found not to be associated with altered breast cancer risk in the Haiman study [16]. The other SNP we reported to be pro- tective (rs2076546) was not genotyped in this study since it focused on non-synonymous SNPs. Here we report an extension of our previous study that identified two SNPs within SRC-3 associated with reduced breast cancer risk [15]. We followed a similar approach by genotyping candidate SNPs for associations with breast cancer risk in a high-risk, BRCA1/2 mutation-negative case-control study; however, the original study was extended in three ways. First, three additional coregulators were examined. Second, we sequenced 96 apparently nor- mal individuals from two populations (48 Caucasian Americans and 48 African Americans) to discover novel SNPs and to confirm or reveal SNP frequency information in different populations. Third, a larger population was examined, almost doubling the number of cases and sig- nificantly improving our statistical power. The association studies allowed us to strengthen the significance of the protective effect previously reported for a SNP in SRC-3 while extending it to a rare two-SNP haplotype that is highly protective for breast cancer risk. Methods SNP Discovery Target sequence obtained from NCBI consisting of all exons, 500 bp of proximal promoter, and 25 bp of flank- ing introns from SRC-1, SRC-3, NCoR, and SMRT was submitted for primer design and Sanger sequencing to Polymorphic DNA Technologies Inc. (Alameda CA). DNA from 96 samples (48 Caucasian American, 48 African American) obtained from the Coriell Institute (Camden, NJ, USA) (sample sets: HD100CAU and HD100AA) was sequenced in both directions and aligned to NCBI refer- ence sequence and previously reported SNPs in dbSNP. These samples had been collected and anonymized by the National Institute of General Medical Sciences. Visual inspection of chromatograms was conducted for hetero- zygous calls. Genotyping Cohort A case-control study was conducted investigating a Ger- man familial breast cancer study cohort. Unrelated, Ger- man, female BRCA1/2 mutation negative index cases from breast cancer families were used in this study. The samples, all of Caucasian origin, were collected during the years 1997-2005 by six centers of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC: centers of Heidelberg, Würzburg, Cologne, Kiel, Düssel- dorf and Munich, see authors affiliations). Familial cases were identified based on (A1) families with two or more breast cancer cases including at least two cases with onset below the age of 50 years; (A2) families with at least one male breast cancer case; (B) families with at least one breast cancer and one ovarian cancer case; (C) families with at least two breast cancer cases including one case diagnosed before the age of 50 years; (D) families with at least two breast cancer cases diagnosed after the age of 50 years; (E) single cases of breast cancer with age of diagno- sis before 35 years. These selection criteria which have pre- viously been reported [17] enrich for cases caused by genetic factor(s). The control population included healthy and unrelated female blood donors collected by the Insti- tute of Transfusion Medicine and Immunology (Man- nheim), sharing the ethnic background and sex with the breast cancer patients. The age distribution in the controls and cases was similar (controls: mean age 45.6 years, median age 46 years, age range from 18 to 68 years old; cases: mean age 45.1 years, median age 45 years, age range from 19 to 87 years old). According to the German guide- lines for blood donation, all blood donors were examined by a standard questionnaire and gave their informed con- sent. They were randomly selected during the years 2004- 2007 for this study and no further inclusion criteria were applied during recruitment. The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany). Genotyping Genotyping was conducted using TaqMan allelic discrim- ination assays. Primers and TaqMan MGB probes were purchased from Applied Biosystems (Foster City, CA). SRC-3 Q586H: 5'-CTGGGCTTTTATTGCGACCAAA-3V, reverse 5VGCTCTCCTTACTTTCTTTGTCACTGA-3'; Taq- Man probes: forward 5'-TTCAATGTGTCACTCAAAT-3'- VIC, reverse 5'-CAATGTGTCAGTCAAAT-3'-FAM. SRC-3 T960T: forward 5'-CCTGCACTGGGTGGCT-3', reverse 5'-CTCGCACCTGGTATGCTATTAGAC-3'; Taq- Man probes: forward 5'-CTATTCCCACATTGCCTC-3'- VIC, reverse 5'-TTCCCACGTTGCCTC-3'-FAM. SRC-3 C218R: forward 5'-AGACATAAACGCCAGTCCT- GAAATG-3', reverse 5'-GCCAGAGATATGAAACAATGC AGTG-3'; TaqMan probes: forward 5'-TGAAATGCG CCA- GAG-3'-VIC, reverse 5'-TGAAATGTGCCAGAG-3'-FAM. Page 3 of 9 (page number not for citation purposes)