Supplementary Methods s8

SUPPLEMENTARY METHODS

Carolina Breast Cancer Study. The Carolina Breast Cancer Study (CBCS) is a population-based, case-control study of breast cancer conducted in 24 counties of central and eastern North Carolina between 1993 and 2001 (Newman et al., 1995;

Millikan et al., 2003). Incident cases of primary invasive and in situ breast cancer were identified using the North Carolina Central Cancer Registry, with over-sampling of

African-American and younger women. Controls were frequency-matched to cases based upon age (± 5 years) and self-reported race using randomized recruitment.

Cases were subtyped using a panel of immunohistochemistry markers that identified the

"intrinsic" breast cancer subtypes (Carey et al., 2006; Millikan et al., 2008). Definitions of subtypes were luminal A (ER+ PR+ HER2-), luminal B (ER+PR+HER2+), basal-like

(ER-PR-HER2-, ck5/6+ and/or HER1+), HER2+ (ER-PR-HER2+), and unclassified

(negative for all five markers). Procedures for recruiting and enrolling study participants and obtaining biologic samples were approved by the Institutional Review Board of the

UNC School of Medicine, and informed consent was obtained from each participant.

Genotyping of B-Myb. Genotyping for the B-Myb polymorphism at codon 427, Serine

(AGC) to Glycine (GGC) (rs2070235), was conducted using DNA extracted from peripheral blood lymphocytes. The ABI 7700 Sequence Detection System, or

"Taqman"™ assay (Applied Biosystems, ABI, Foster City, CA, USA) was employed, with polymerase chain reaction (PCR) primers and probes designed using Primer

Express TM software (ABI) and assay conditions based on the allelic discrimination protocol from Applied Biosystems. PCR primer and probe sequences are available upon request. Out of a total of 3862 samples genotyped, the failure rate was 0.4%, and complete agreement was obtained on a 10% random sample.

There were no significant departures from Hardy-Weinberg equilibrium among

African-American cases (p=0.80), African-American controls (p=0.77), Caucasian cases

(p=0.27), or Caucasian controls (p=0.51).

Cell line genotyping was performed by Polymorphic DNA Technologies, Inc

(Alameda, CA, USA).

Genotype Statistical Analysis

B-Myb genotypes were determined for 1256 cases with subtype information (500

African-American, 756 Caucasian) and 1814 controls (679 African-American, 1135

Caucasian). Odds ratios (ORs) and 95% Confidence Intervals (CIs) for each breast cancer subtype versus all controls were calculated using unconditional logistic regression. The PROC GENMOD statement in SAS (version 8.2; SAS Institute, Cary,

NC, USA) was used to incorporate offsets derived from sampling probabilities used to identify eligible study participants, race (African-American, Caucasian), and age (11- level ordinal variable representing 5-year age categories). Trend tests were conducted by calculating the p-value for B-Myb genotype coded as an ordinal variable. Likelihood ratio tests (LRTs) were used to test for modification of ORs by race.