BMC Cancer 2004, 4 http://www.biomedcentral.com/1471-2407/4/9 detailed questions related to work history as a radiologic technologist, lifestyle characteristics, other cancer risk fac- tors, and health outcomes, including breast cancer. Approximately 90,000 technologists responded to each survey. All female technologists reporting a primary breast cancer that was subsequently confirmed based on medical records (pathology report, physician's notes, hospital dis- charge summary or physician correspondence) were eligi- ble for inclusion if still living. In December 1999, when biospecimen collection began, there were 1345 living breast cancer cases with diagnosis years ranging from 1955 to 1998. By the end of December 2001, 748 breast cancer cases had provided informed consent, a blood sample, and responded to a telephone interview that col- lected updated cancer risk factor and family cancer history information and selected work history data. Another 143 cases could not be located or had an unlisted telephone number and did not respond to repeated correspondence inviting participation, 29 were too ill, 238 refused, 21 could not arrange a blood draw or the draw was unsuc- cessful, and 166 were still in process. This study has been approved annually by the human subjects review boards of the National Cancer Institute and the University of Minnesota. Birth and death (if applicable) dates and breast cancer diagnosis dates were obtained for all first-degree female relatives. Data were evaluated for inconsistencies in age, reported generational intervals (all mothers had to be at least 11 years old before the birth of a child), and all breast cancers must have occurred at an age younger than current age or age at death. Initially, there were 2497 rela- tives in the data set and 194 of these relatives were reported to have breast cancer. For a total of 16 of the breast cancers reported in female relatives, the age at diag- nosis was unknown and was imputed using the median age of breast cancer onset in all the relatives for which age was known. There were 60 half-sisters and seven relatives with unknown or un-imputable ages at last observation who were subsequently excluded. After these exclusions, 2430 relatives were retained in the analyses consisting of 190 with breast cancer and 2240 without. Genotyping Approximately 10 nanograms of genomic DNA extracted from peripheral lymphocytes were used as template in Taqman 5'-nuclease assays for all SNPs except for TGFß1 P25R, for which a PCR-RFLP assay was used. Taqman assays were performed using 450 nanomolar primer con- centrations and 100 nanomolar probe concentrations and Universal Master Mix (Applied Biosystems). Probes spe- cific for each SNP were designed with Primer Express soft- ware (Applied Biosystems) and labeled with either 6- FAM, TET, or VIC as reporter dyes and either Black Hole Quencher-1 (IDT, Inc.) or MGB-NFQ (Applied Biosys- tems) as quenchers. The primer and probe sequences and PCR conditions for each SNP are available (on request from JPS at http://lpg.nci.nih.gov/LPG/struewing/pubs). Most assays were performed in 20 microliter reactions in 96-well trays using a 7700 instrument (Applied Biosys- tems), but some were performed in 5 microliter reactions in 384-well trays using a 7900HT instrument (Applied Biosystems). Subjects with each of the three possible genotypes (unless no homozygous variant subjects have ever been identi- fied) for each SNP were confirmed by sequencing and included on each genotyping tray. In addition, approxi- mately 5% of samples (41 samples, distributed as 2 to 7 aliquots of DNA from 8 different anonymous subjects) were included to monitor quality control (QC), with lab- oratory personnel blinded as to which were the QC sam- ples. The genotypes for each of the duplicate QC samples from a subject matched exactly for all SNPs except one, and in this case, uncovered a systematic error in coding the results. This assay was repeated entirely and the QC samples then matched exactly. Statistical methods The analysis was based on a cohort of first degree female relatives of case probands, i.e., breast cancer patients, who were followed retrospectively over time for breast cancer incidence. Although the relatives' genotypes were not observed, the probability distribution could be inferred from the observed genotype of the corresponding case probands and Mendelian inheritance patterns. A marginal likelihood approach [15] was used to estimate age-specific cumulative risks associated with different genotypes while accounting for the uncertainty introduced by using indi- rect information about the relatives' genotype. Separate analyses were performed for each locus. For loci with rare variant frequencies and therefore low power to discern risk differences, we grouped heterozygote and homozy- gote variant genotypes together when the prevalence was less than 10%. For loci with common variants, we first estimated cumulative risks associated with the three gen- otypes separately. For two such loci (IGFBP3 -202A>C and TGFß1 L10P), visual inspection of the age-specific cumu- lative risk graphs revealed no difference in risk between homozygotes and heterozygous variant carriers and thus variant genotypes were combined. As a summary measure for risk associated with variant genotypes, we obtained cumulative risk ratios (RR) at ages 50 and 70 with the homozygous wild type genotype con- sidered the referent category. (Relative risks for breast can- cer up to any age can be calculated using the kin-cohort method. We chose to graphically display risks up to age 80 to represent "lifetime" risk. In the tables we chose to pro- vide risks with confidence intervals (CI) up to ages 50 and Page 3 of 12 (page number not for citation purposes)