Subjects
The Australian Breast Cancer Family Study, a population-based, age-stratified, case–control–family study of first primary invasive breast cancer in women younger than age 40 years was carried out in Melbourne and Sydney from 1992 to 1995 [9–11]. The study was extended from 1996 to 2000 to also include women up to age 59 years [10]. Cases were women with a diagnosis of a first primary breast cancer identified through the Victorian and New South Wales cancer registries. Controls were women without breast cancer selected from the electoral roll (adult registration for voting is compulsory in Australia) using stratified random sampling, frequency-matched for age.
Cases and controls were administered a questionnaire to record the family history of cancer and other known or potential risk factors for breast cancer. With the subjects' permission, all living parents, aunts, grandparents, and adult siblings were asked to participate, and were administered the same risk-factor questionnaire [10, 11]. Ancestry was assessed by an open-ended question, and from the country of birth of the respondents, their parents and their grandparents. The great majority of the subjects' parents and grandparents were born in Australia, the British Isles or Western Europe. In subanalyses restricted to Caucasian women, subjects either with any Australian aboriginal, Torres Strait Islander or Maori heritage or with any country of birth in the South Pacific, Indian Ocean, Caribbean islands or Asia were excluded.
The family history of cancers was systematically collected from each case and each control, and included the cancer history of all of their first-degree and second-degree relatives. This history was subsequently checked with each living relative at the time of their interview. A family history of breast cancer was defined as having at least one first-degree or second-degree relative with breast cancer. Verification of all cancers reported by subjects and their relatives was sought through personal interview, cancer registries, pathology reports, hospital records, clinicians and death certificates.
Subjects participating in the study conducted from 1996 to 2000 were asked for information on the number of X-ray examinations or radiation treatments they had undergone, and their age at first X-ray examination or radiation treatment. Exposure to X-rays/ionizing radiation was considered as a yes/no variable, irrespective of age at first exposure.
Interviews were conducted for 1579 of 2304 eligible cases (68.5%) and for 1021 of 1531 eligible controls (66.7%). Attrition of cases was due to death (1.8%), to refusal by the surgeon (8.5%), to refusal by the proband (16.4%), to nonresponse by the surgeon (1.3%), to non-response by the proband (1.2%) and to failure to locate the proband (2.3%). Attrition of controls was due to refusal (28.2%) and to nonresponse (5.1%). Not all interviewed cases and controls elected to donate a blood sample for DNA studies. Genotyping for the T2119C variant was carried out on the 1331 cases (84% of those participating) and 649 controls (64% of those participating) with DNA available at the time of analysis, and subsequently for the C3161G variant on 1453 cases (92% of those participating) and 793 controls (78% of those participating). PCR success rates were greater than 99%. The average age ± standard deviation of cases and controls was 39.6 ± 9.0 and 42.0 ± 8.8 years, respectively, for individuals genotyped for the T2119C variant, and was 41.7 ± 8.7 and 40.1 ± 8.9 years, respectively, for individuals genotyped for the C3161G variant.
Approval of this study was obtained from the ethics committees of The University of Melbourne, the New South Wales Cancer Council, The Anti-Cancer Council of Victoria, and The Queensland Institute of Medical Research.
Molecular analysis
Collection of peripheral blood and DNA extraction have been described previously [12]. The ATM variants were detected using the ABI Prism 7700 Sequence Detection System 5' exonuclease assay (Perkin-Elmer Corp., Foster City, USA), using the methodology as described previously [13]. The T2119C variant was detected using the forward and reverse primers 5'-CGCTGTCTTCTGGGTTTATCAG-3' and 5'-CCTTCCTAACAGTTTACCAAAGTTGA-3', and the probes 5'-FAM-CTGAATAATTACTCAtCTGAGGTGAGAT-TAMRA-3' (T allele) and 5'-VIC-TCT-GAATAATTACTCAcCTGAGGTGA-TAMRA-3' (C allele). The C3161G variant was detected using the forward and reverse primers 5'-CTCTATTTCATATTTAACCACAGTTCTTTTC-3' and 5'-GTCTTTTCCCATTACATTAAGAATGG-3', and the probes designed to the complement strand 5'-FAM-CCCATTTTGAATAAgGATCAGCCTACGG-TAMRA-3' (C allele) and 5'-VIC-CCCATTTTGAATAAcGATCAGCCTACGG-TAMRA-3' (G allele).
Statistical methods
Allele frequencies were estimated and compared assuming that alleles within an individual were independent binomial variables. The Hardy-Weinberg equilibrium assumption was assessed for defined groups using maximum likelihood methods by comparing the observed numbers of different genotypes with those expected under Hardy-Weinberg equilibrium in that group. The odds ratio (OR) and the 95% confidence interval (CI) were calculated using unconditional logistical regression, with and without adjustment for measured risk factors. We used stratified analysis to determine whether the genotype-breast cancer association varied by age <40 or ≥ 40 years, by family history, by menopausal status and by chest exposure to X-rays and/or ionizing radiation. All statistical tests and P values were two tailed and, following convention, statistical significance was taken as a nominal P < 0.05. SPSS (version 10.0; SPSS Australia Pty Ltd), Epi-Info 6 (freeware; http://www.cdc.gov/epiinfo/ei6.htm) and Ottutil software (freeware; http://linkage.rockefeller.edu/ott/linkutil.htm) were used for the statistical analyses.