Short Communication BRCA1 Susceptibility Markers and Postmenopausal Breast Cancer: The Iowa Women’s Health Study

Much research on early-onset breast cancer families has been performed and has shown that breast cancer in many of these families is linked to either BRCA1 or BRCA2. Fewer studies have examined the role of genetic predisposition in postmenopausal breast cancer. A nested case-control family study of breast cancer was conducted within the Iowa Women’s Health Study, a population-based prospective study of 41,836 postmenopausal women. Probands were 251 incident cases diagnosed between 1988 and 1989. Three-generation pedigrees were developed through mailed questionnaires. From this collection of pedigrees, thirteen were identified for more detailed genetic analysis. Sibling-pair linkage analyses were performed using polymorphic markers in candidate regions in these 13 families with multiple cases of breast and other cancers. Four of the DNA markers are located on chromosome 17, and two of these (D17S579 and THRA1) flank the BRCA1 locus. Significant evidence for linkage to D17S579 was obtained in the total sample, in a model without inclusion of covariates or age at onset (P 5 0.005), and in a model adjusted for five measured covariates and for variable age at onset (P 5 0.008). Complete sequencing of the BRCA1 gene in these families, including all intron/exon boundaries, failed to reveal any mutations in 24 women with breast cancer from the 13 families. These data suggest that in some families identified by postmenopausal breast cancer cases, breast cancer risk may be mediated by a gene (or genes) in the BRCA1 region, but not BRCA1 itself. Introduction A family history of breast cancer has been shown to increase a woman’s risk of the disease. For some women, a family history of breast cancer represents inherited susceptibility, which is estimated to be responsible for approximately 5% of all cases (1). This suggests that about 1 in 160 women will develop breast cancer due to an inherited predisposition. Risk increases as age of onset in the affected family member decreases (2–4), but risk remains elevated in women whose primary relatives were diagnosed with late-onset breast cancer (5–7). In the IWHS, risk to sisters of postmenopausal breast cancer cases was increased for both early-onset (relative risk 5 1.41) and late-onset (relative risk 5 1.81) disease after controlling for measured risk factors (7). Segregation analyses have also supported the role of a major gene in late-onset breast cancer (8, 9). Genetic factors may therefore be relevant to lateonset breast cancer development. Much research has been published on genetic factors in early-onset disease. Recently, there has been considerable focus on the BRCA1 locus, a site at which mutation carriers are predisposed to developing breast and ovarian cancers. Estimates of the frequency of women carrying BRCA1 mutations range from 1 in 200 to 1 in 2000 (10), and the risk of developing breast cancer by age 70 among carriers has been estimated to be up to 80% (11). Although some of the variation in the occurrence of breast cancer associated with BRCA1 mutations may be due to differences in the mutations [with at least 254 mutations identified to date (12)], variable penetrance suggests that other genetic and environmental factors contribute to the development of the disease. One example is HRAS1 variable number tandem repeats that have been shown to modify the risk of ovarian cancer (but not breast cancer) in BRCA1 mutation carriers (13). Narod et al. (14) also reported on risk modifiers of BRCA1 mutations, including reproductive factors such as age at menarche (below age 12), parity (,3), and year of birth (after 1930). These factors could not explain the total variability of expression and penetrance. Beyond the findings of a few reports, it remains uncertain what factors, modifiable or not, may delay or prevent disease onset in BRCA1 mutation carriers. Anthropometric variables (15) and the number of pregnancies have also been shown to affect the risk of breast cancer (15–17). We previously showed in this population of women that the increase in risk of breast cancer associated with a high WHR or low parity is more pronounced among women with a family history of breast cancer compared with those without such a family history (17), especially for breast/ovarian cancer (18). These data suggest that genetic factors may be relevant to late-onset breast cancer and that heterogeneity may be influenced by nongenetic risk factors. Mutations in the BRCA1 gene are estimated to account for nearly one-half of early-onset breast cancer families (19). Most of the studies examining the role of BRCA1 have identified Received 4/30/99; revised 2/2/00; accepted 2/28/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by The Margaret Mitchell Scholarship of the American Cancer Society, Minnesota Division and by grants from the National Cancer Institute (R01-CA39742 and R01-CA55747), the American Cancer Society (IN-13-30-14), and the Biomedical Research Support Grant Program, Division of Research Resources (S07-RR55448 and N01-RR00400). Some of the results were obtained using the program package S.A.G.E., which is supported by Grant RR03655 from the Division of Research Resources. 2 To whom requests for reprints should be addressed, at Department of Health Sciences Research, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN 55905. 3 The abbreviations used are: IWHS, Iowa Women’s Health Study; IBD, identical by descent; BMI, body mass index; WHR, waist:hip ratio. 507 Vol. 9, 507–511, May 2000 Cancer Epidemiology, Biomarkers & Prevention on August 15, 2017. © 2000 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from linkage and mutations in families in premenopausal cases (20, 21). There have been few linkage studies in families with late-onset disease, and those results have not provided significant evidence for linkage to BRCA1 (22). Barker et al. (23), however, reported a BRCA1 mutation identified from a population-based set of families selected without respect to age at onset. Three breast cancer cases showed strong family histories of breast cancers, with ages of onset similar to cases considered “sporadic.” The BRCA1 mutation (R841W) found in these families is thought to exert less dramatic effects on protein structure than other mutations and thus may exhibit a phenotype that has a later age at onset of breast cancer. These results suggest that when only families with early-onset disease are chosen for BRCA1 screening studies, the older onset families that may carry less severe but important mutations are missed, perhaps reaching 1% of cases in the population. Indeed, it has been suggested that many of the pedigrees studied in BRCA1 research have not been typical and that mutations with milder effects (24) that include cases with older ages of onset (25) may be more common. Other genes implicated for increased risk of breast cancer include INT2, coding for a fibroblast growth factor (chromosome 11q), the estrogen receptor locus (ESR; 6q) and p53, a tumor suppressor gene (17p). MLH1 (3p) and MSH2 (2p) may also be relevant in breast cancer because inherited mutations in MSH2 are associated with the Muir Torre syndrome (26), which has breast cancer as part of the phenotype. In an effort to determine the importance of these various genes in breast cancer, the current study evaluated evidence for linkage of 12 polymorphic markers in six breast cancer candidate regions (2p, 3p, 6q, 11q, 17p, and 17q). Families selected for analysis represent a subset of a populationbased cohort. We evaluated evidence for linkage at these loci in 13 three-generation families with multiple cases of postmenopausal breast cancer. Age at onset and selected environmental covariates were included in the genetic analyses. Materials and Methods Study Population. The IWHS is a population-based cohort study designed to identify risk factors for postmenopausal cancer (27). In 1986, a questionnaire was mailed to approximately 100,000 female Iowa residents between the ages of 55 and 69 years with a valid driver’s license. Women reporting at baseline previous cancers other than skin cancer, a prior mastectomy, or a menstrual period within the last year were excluded. The total cohort at risk for incident breast cancer was 37,105 women. Incident breast cancer cases were identified through the Health Registry of Iowa, part of the National Cancer Institute’s Surveillance, Epidemiology, and End Results program. In 1988 and 1989, there were 265 incident breast cancers in this cohort. A pedigree development form was mailed to index cases to identify first-degree female relatives (mother, sisters, and daughters). First-degree relatives received a mailed health history questionnaire to ascertain risk factor information: (a) alcohol use; (b) body measurements; (c) history of benign breast disease; (d) education level; (e) menstrual factors; (f) oral contraceptive use; and (g) pregnancy history. To assess body fat distribution, a paper tape measure was enclosed along with detailed instructions for measuring waist and hip circumferences. These measurements were used to calculate WHR. Reliability and accuracy of self-measurements of waist and hip in the IWHS are good (28). Self-reports of cancer in first-degree relatives were confirmed by medical record review. Pedigree Extension and Blood Collection. Thirteen families with at least two first-degree relatives with breast cancer were identified. Twelve of these families were ascertained through index cases (probands) with postmenopausal breast cancer. One family was identified through a study participant who did not have breast cancer but had a family history of breast cancer. A genetic counselor extended each pedigree using standard protocols. Updated information was obtained on name, address, age, vital status, and cancer status of index cases’ seconddegree female and male relatives and their spouses. The phenotype for analysis was defined as breast cancer, but cancers of the endometrium, cervix, ovaries, prostate, and colon were observed in some of these families. Potentially informative individuals were invited to provide blood samples for genetic analysis. Informative persons included the index case and her mother, sisters, and daughters. If sisters or daughters with breast cancer were deceased, their available spouses and offspring were invited to have blood drawn to permit inference of their genotype. A blood kit that contained a cover letter, consent form, instructions for sample shipment, blood collection tubes, and a protective Styrofoam container was mailed to each individual. Subjects took the blood kits to their personal physicians for venipuncture and return shipment. Genotyping. Genomic DNA was isolated from whole blood for genetic analysis using standard procedures. Genotypes were determined for the following microsatellite genetic markers: (a) chromosome 17q [THRA1 (29), D17S250 (30), D17S579, and D17S588 (31)]; (b) 17p [two markers for p53 (32)]; (c) 11q [INT2 (33)]; (d) 6q [ESR (34)]; (e) 3p [D3S1277 and D3S1611 (35)]; and (f) 2p [D2S123 and D2S119 (36)]. Amplification of the microsatellite markers was performed using 10 ng of genomic DNA, 2 mM MgCl2, 100 mN deoxynucleotide triphosphates, 13 buffer (Promega), 0.5–2.0 pmol of the sense and antisense primers, 0.1 pmol of the fluoridated sense primer for each pair, and 0.5 unit of Taq polymerase (Promega) in a total volume of 5 ml. The PCR cycles were 3 min at 95°C for initial denaturation, followed by 30 s each at 95°C, 55°C, and 72°C and a final cycle of 3 min at 72°C for the final extension. The amplified products were separated on a 6% denaturing polyacrylamide gel on an automated DNA sequencer (Model 4000; LI-COR, Inc. Lincoln, NE) using a fluorescence detection system (37). The bands were visually inspected and analyzed densitometrically. DNA Sequencing. When preliminary results suggested evidence of linkage to markers near the BRCA1 locus, we then examined the BRCA1 gene for inherited mutations in the 24 women with breast cancer available from the 13 families, including 11 index cases and 13 first-degree relatives (Table 1). This included all exons and intron/exon boundaries. Sequencing reactions were done on PCRamplified templates (38). Individual exons were amplified using genomic DNA with the exception of exons 10, 11, 14, 16, 18, and 27, in which multiple amplification and sequencing reactions were required. Amplification primers for BRCA1 were from Friedman et al. (39) and this report. The forward primer for each PCR reaction contains an M13 tail on the 59 end of the primer (40). Sequencing was done using fluorescence-based DNA sequencing, and the banding pattern was visualized using a LI-COR model 4200 Automated DNA sequencer (37). Statistical Analysis. Descriptive characteristics of each family member (vital status, age at death, age at onset, age at exam, and number of relatives) were determined. We performed sibling-pair linkage analysis on these families using the SIBPAL program in the S.A.G.E. computer package (41), an implementation of the Haseman-Elston approach to detection of linkage for both qualitative and quantitative traits (42, 43). This method permits investigation of the genetic component of a trait or disease without specification of the underlying mode 508 Short Communication: BRCA1 Markers and Postmenopausal Breast Cancer on August 15, 2017. © 2000 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from of transmission. It is based on the principle that although siblings share on average 50% of their genes IBD at a given genetic locus, sibling pairs who are concordant on disease status would be expected to share more alleles IBD at the disease-predisposing locus than siblings discordant on disease status. A test for genetic linkage is thereby given by a regression of the sibling-pair squared trait difference (trait 5 breast cancer status) on the estimated proportion of alleles at a particular marker locus shared IBD. The statistic for testing linkage is a one-sided t test of the estimated regression coefficient divided by its SE (42, 43). In addition to simple models including only disease status and alleles IBD, models with adjustment for age at onset of breast cancer and nine breast cancer risk factors (covariates) were also fitted: (a) model 1, no adjustment for variable age at onset or covariates; (b) model 2, adjustment for age at onset; (c) model 3, adjustment for nine selected covariates; and (d) model 4, adjustment for age at onset and five covariates identified as significant in logistic regression. The risk factors included alcohol use (g/day), age at first pregnancy (years), BMI (kg/m), history of benign breast disease (ever/never), education level (grade school, less than high school, high school, some college, college graduate, or graduate school), age at menarche (years), menstrual status (premenopausal/postmenopausal), number of pregnancies, use of oral contraceptives (ever/never), and WHR.