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© 2002 American Society for Clinical Oncology BRCA1 and BRCA2 Mutation Frequency in Women Evaluated in a Breast Cancer Risk Evaluation ClinicByFrom the Abramson Family Cancer Research Institute and Departments of Biostatistics and Epidemiology and Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA; Mayo Foundation, Rochester, MN; and Institute of Cancer Research, Sutton, United Kingdom. Address reprint requests to Barbara L. Weber, MD, Biomedical Research Building II/III, Rm 514, 421 Curie Blvd, Philadelphia, PA 19104; email: weberb{at}mail.med.upenn.edu
PURPOSE: To determine the prevalence of BRCA1 and BRCA2 mutations in families identified in a breast cancer risk evaluation clinic. PATIENTS AND METHODS: One hundred sixty-four families seeking breast cancer risk evaluation were screened for coding region mutations in BRCA1 and BRCA2 by conformation-sensitive gel electrophoresis and DNA sequencing. RESULTS: Mutations were identified in 37 families (22.6%); 28 (17.1%) had BRCA1 mutations and nine (5.5%) had BRCA2 mutations. The Ashkenazi Jewish founder mutations 185delAG and 5382insC (BRCA1) were found in 10 families (6.1%). However, 6174delT (BRCA2) was found in only one family (0.6%) despite estimates of equal frequency in the Ashkenazi population. In contrast to other series, the average age of breast cancer diagnosis was earlier in BRCA2 mutation carriers (32.1 years) than in women with BRCA1 mutations (37.6 years, P = .028). BRCA1 mutations were detected in 20 (45.5%) of 44 families with ovarian cancer and 12 (75%) of 16 families with both breast and ovarian cancer in a single individual. Significantly fewer BRCA2 mutations (two [4.5%] of 44) were detected in families with ovarian cancer (P = .01). Eight families had male breast cancer; one had a BRCA1 mutation and three had BRCA2 mutations. CONCLUSION: BRCA1 mutations were three times more prevalent than BRCA2 mutations. Breast cancer diagnosis before 50 years of age, ovarian cancer, breast and ovarian cancer in a single individual, and male breast cancer were all significantly more common in families with BRCA1 and BRCA2 mutations, but none of these factors distinguished between BRCA1 and BRCA2 mutations. Evidence for reduced breast cancer penetrance associated with the BRCA2 mutation 6174delT was noted.
MUTATIONS IN BRCA1 and BRCA2 are associated with hereditary susceptibility to breast and ovarian cancer.1,2 The penetrance of BRCA1 mutations is estimated to be 50% to 80% for breast cancer and 20% to 50% for ovarian cancer,3-5 although one hospital-based series of Ashkenazi women with breast cancer analyzed for germline founder mutations in BRCA1 and BRCA2 estimated a breast cancer penetrance of 36% for these mutations.6 For BRCA2 mutation carriers overall, penetrance is estimated to be 30% to 80% for breast cancer and 10% to 20% for ovarian cancer by 70 years of age.3,7 Despite the high cancer risk associated with BRCA1 and BRCA2 mutations in individuals, the population frequency of mutations is low, with mutations in BRCA1 and BRCA2 estimated to be responsible for approximately 6% of all breast cancers diagnosed before 50 years of age,8,9 approximately 1% to 2% of breast cancer diagnosed after 50 years of age,8 and 5% of all ovarian cancers.10 Multiple studies have found common predictors of germline BRCA1 and BRCA2 mutations, including Ashkenazi Jewish descent,11 early-onset breast cancer,12,13 and personal or family history of ovarian cancer.14,15 Women affected with breast and at least one other primary cancer, particularly ovarian cancer, also seem to be at increased risk for carrying a BRCA1 or BRCA2 mutation.16 Male breast cancer has been strongly associated with BRCA2 mutations, with an estimated cumulative risk of 6% by age 70 years.6,17,18 Although numerous studies of BRCA1 mutation prevalence in various cohorts have been published in recent years, few have evaluated the predictors for the occurrence of both BRCA1 and BRCA2 mutations in a clinic-based population. In this study we present such an analysis of 159 women and five men presenting to our risk evaluation clinic. These data are important in defining the likelihood of identifying BRCA1 and BRCA2 mutations in women who present to high-risk cancer evaluation clinics to provide accurate guidance to women and families considering genetic testing.
Patient Population All families were recruited from clinics at the University of Michigan between 1993 and 1995 and the University of Pennsylvania in 1995. During this period, participants were predominantly white (95% white, 2% African-American, and 2% Hispanic) and highly educated (69% had completed college). The mean age of participants was 44.1 years, with a range from 18 to 78 years. Twenty-seven percent of participants were of Ashkenazi Jewish heritage. Fifty-three percent of participants were self-referred, 40% were referred by a physician, 5% were referred by a family member, and 2% were referred by a cancer-related foundation. All had the perception of being at elevated risk of inherited susceptibility to breast cancer. Families were eligible if the proband was affected with breast or ovarian cancer and at least one additional family member was affected with breast or ovarian cancer. A total of 164 participants were enrolled (159 women and five men). All participants consented to BRCA1 and BRCA2 testing for clinical or research purposes. All patients were offered the opportunity to learn their mutation status and received genetic counseling concerning the impact of such information. Personal and family histories of all cancers were recorded, including ages of diagnosis of all cancers as well as the number of related women in each family at risk for breast cancer (age  20 years). Patients were questioned for self-reported ethnicity and were grouped as either of Ashkenazi Jewish descent or other, which also included families with unknown ethnic background. Pathology reports were obtained on all probands and on other family members when available. The testing protocol was approved by duly constituted institutional review boards at both the University of Michigan and the University of Pennsylvania.
Mutation Analysis Sequence variants selected by atypical banding patterns on CSGE gels were reamplified from the original genomic DNA and purified with PCR select II (5'3') purification columns. Products were manually sequenced using a PCR sequencing kit (Promega, Madison, WI) following the manufacturer’s instructions or using an ABI 377 and dye terminators. Each product was sequenced in both forward and reverse directions.
Statistical Analysis The analyses were used to describe the specific characteristics (phenotypes) of the families that were found to carry mutations in BRCA1, BRCA2, or either gene (genotype). Characteristics included the following: presence of ovarian cancer, presence of a breast and ovarian cancer multiple primary case, presence of male breast cancer, average breast cancer age of diagnosis in the family, age of breast cancer diagnosis in the proband, and Ashkenazi Jewish ancestry. All age variables were analyzed as continuous variables. The cancer diagnosis descriptors were analyzed as dichotomous variables (presence v absence). Contingency tables and Fisher’s exact test analyses were used to evaluate differences in the frequency of BRCA1 or BRCA2 mutations across groups. For analyses of continuous variables, such as mean age of diagnosis of breast cancer, analysis of variance was used to evaluate differences between the families carrying each genotype (BRCA1, BRCA2, either mutation, or neither mutation).
Overall, ninety percent of clinical patients had a family history of breast cancer, with a mean predicted lifetime breast cancer risk of 20% (range, 8% to 48%).20 The mean predicted risk of a BRCA1 mutation was 18%, with a range from 0.01% to 95%. Fifty-eight percent of clinical patients had a predicted risk of carrying a BRCA1 mutation of higher than 5%. Slightly less than half of patients (47%) completed BRCA1/2 testing after counseling. The factors affecting the decision to undergo testing among this cohort have been previously reported.21 Of the clinic patients eligible for this study, 155 of 159 had been previously diagnosed with breast cancer; the remaining four women had a diagnosis of ovarian cancer and a family history of breast cancer. Three families of female probands had at least one case of male breast cancer. All five male probands had both a personal diagnosis of breast cancer and a family history of female breast cancer; thus, a total of eight families with male breast cancer were analyzed. All families had at least two individuals diagnosed with breast cancer (range, two to 11), with an average of 3.85 breast cancer cases per family (median, three). The 44 families with a history of both breast and ovarian cancer had an average of 1.6 cases of ovarian cancer (range, one to seven; median, one). Of the 164 families studied, 37 germline coding region mutations of known functional significance were detected in either BRCA1 or BRCA2 (Table 1). Twenty-eight (76%) of these mutations were found in BRCA1, and the remaining nine (24%) mutations were in BRCA2. Families with and without mutations had the same percentage of women affected with breast cancer (41% and 42% of women over 21 years of age, respectively). Six (24.0%) of 25 known Ashkenazi Jewish families had BRCA1 mutations. No BRCA2 mutations were identified in individuals of known Ashkenazi Jewish heritage; however, the BRCA2 founder mutation (6174delT) was found once in a family of uncertain ancestry.
Most (23 of 28, 82%) of the BRCA1 mutations were frameshift mutations (Table 2). In addition, one missense, one splice-site, and three nonsense mutations were found in BRCA1. Of the BRCA2 mutations, six were frameshift and three were nonsense mutations. Five additional sequence variants were detected in BRCA2 but were excluded from analyses, because they were likely polymorphisms (and are not included in Table 2). Although four of the variants involve an amino acid charge gain or loss, which increases the likelihood of mutation significance, two were detected in families with known truncating mutations and one did not track with disease in the family. The one missense mutation that does not encode an amino acid charge change has been previously characterized as a polymorphism based on population frequency. Including the only BRCA2 missense mutation of potential significance (K314T), the maximum possible BRCA2 mutation frequency in this population is 6.1%, with a total of 23.2% of families having either a BRCA1 or BRCA2 mutation.
The average age of breast cancer diagnosis in female probands and of all the women affected with breast cancer in each family was compared based on mutation status (Table 3). The average age of breast cancer diagnosis was significantly younger in the families with BRCA1 (41.4 years) or BRCA2 mutations (40.1 years) than in families without detectable mutations (50.1 years) (P < .0001). Probands with BRCA1 and BRCA2 mutations were diagnosed with breast cancer at an average age of 37.6 and 32.1 years, respectively (P = .028).
Forty-four families had at least one case of ovarian cancer, of which 22 (50%) had either a BRCA1 or BRCA2 mutation (Table 4). Twenty mutations were found in BRCA1, and two mutations were found in BRCA2. Sixteen of 44 breast-ovarian cancer families had at least one family member with both primary breast and ovarian cancer. Twelve (75%) of these 16 families had a BRCA1 mutation; no BRCA2 mutations were found in this group. Twenty (71%) of 28 families with BRCA1 mutations had at least one case of ovarian cancer in the family compared with 22 (17%) of 127 families without detectable mutations (P = .001). However, there was no difference between the number of BRCA2 mutation–positive and BRCA2 mutation–negative breast-ovarian families (two of nine v 22 of 127, respectively).
Eight of the 164 families screened had at least one case of male breast cancer. Among these families, one mutation in BRCA1 and three mutations in BRCA2 were found. Three (33%) of the nine families with BRCA2 mutations had at least one case of male breast cancer compared with four (3.2%) of 127 male breast cancer families with no detectable mutation (P = .001). Only one of 28 families with BRCA1 mutations had a case of male breast cancer, not significantly different from families without a mutation (P = .91).
Identification of BRCA1 and BRCA2 mutation carriers is an important focus in prevention and early detection of breast and ovarian cancer risk. Many medical centers providing cancer care in the United States and Western Europe now provide this clinical service. However, few data are available to estimate the proportion of positive and negative tests that might be expected in a referral risk evaluation clinic, which impacts provider recommendations, patient decisions, and cost estimates for these services. In our analysis, 22% of families seeking genetic testing in our high-risk breast cancer clinic had a detectable mutation in BRCA1 or BRCA2. These data indicate that referral for cancer risk evaluation effectively selects for a reasonable percentage of mutation carriers but also highlight the fact that most women evaluated even at large regional and national referral centers will not have detectable mutations. In this series in the United States, BRCA1 mutations were three times more common that BRCA2 mutations. The ratio of BRCA1 to BRCA2 mutations varies widely between subpopulations globally, with Icelandic sister pairs with breast cancer having almost exclusively BRCA2 mutations22 and roughly equal numbers of BRCA1 and BRCA2 mutations in French breast cancer families22 and in British women with early-onset breast cancer.9 One reason for this variability is population founder effects, which is clearly the case in Iceland. The increased frequency of BRCA1 mutations in this series is consistent with other studies based in the United States; Frank et al24 found twice as many BRCA1 mutations as BRCA2 mutations in women younger than 50 years of age with breast cancer. However, given that a selection bias exists in families seeking risk evaluation, the variable ratio also could be explained by differences in the penetrance of BRCA1 and BRCA2 mutations, as has been demonstrated for the Ashkenazi Jewish founder mutations.25 Selection for or against families with ovarian cancer or male breast cancer also are likely to be particularly influential. Among the 37 unequivocal disease-associated mutations detected, only two mutations, 185delAG (six mutations) and 5382insC (four mutations) were detected more than twice. Both of these mutations are well characterized as founder mutations in the Ashkenazi Jewish population. Interestingly, 5382insC is present at approximately one tenth the frequency of 185delAG in the Ashkenazi Jewish population but is detected with almost equal frequency in this tested cohort.26 This observation may be a function of higher penetrance, resulting in greater proportion of families with the 5382insC mutation coming to medical attention, or, alternatively, these data may reflect subgroups of the Ashkenazi Jewish population referred to our clinic. Also of note, whereas previous studies suggest that the BRCA2 mutation 6174delT is more prevalent than the sum of both BRCA1 founder mutations (185delAG and 5382insC), a significant deviation from these ratios was seen here (9:1 BRCA1:BRCA2). 6174delT is present in 1.4% of Ashkenazi Jews,11,24 1.3 times greater the population frequency of 185delAG and more than 12 times the frequency of 5382insC. Thus, this clinic-based study supports other work suggesting that 6174delT is associated with significantly lower breast cancer risk than either 185delAG or 5382insC.24,27,28 These data are consistent with the hypothesis that the ovarian cancer cluster region in BRCA2, which includes 6174delT, represents a region of decreased breast cancer penetrance. This region also is associated with an increased ovarian cancer penetrance in other studies,7 but the small number of ovarian cancers in this study precluded an analysis of this assocation. Notably, almost half of the at-risk women in families without detectable BRCA1 or BRCA2 mutations were affected with breast cancer. These data support our previous finding that the number of breast cancers per family is not a sensitive means for identifying families with BRCA1 or BRCA2 mutations. These data also support the hypothesis that there are additional breast cancer susceptibility genes that remain to be identified, because the pattern of breast cancer in some of these families is consistent with the presence of a highly penetrant autosomal-dominant susceptibility allele. Undetected BRCA1 and BRCA2 mutations may explain some of these families; however, linkage analysis and mutation detection sensitivity estimates suggest that undetected mutations are unlikely to explain all of the families in this study without mutations. Large genomic deletions, a known source of BRCA1 mutations, do escape detection by both CSGE and direct sequencing. However, in a recently published series also derived from this clinic population, BRCA1 genomic rearrangements accounted for only 12% of mutation-negative breast and ovarian cancer families, with most of those rearrangements found in families with multiple primary breast and ovarian cancer cases.29 This study reaffirms several previous predictors for the presence of BRCA1 and BRCA2 mutations, such as ovarian cancer, breast and ovarian cancer in a single individual, male breast cancer, and early average of breast cancer diagnosis. It also provides a combined analysis of both genes in a single cohort that is likely to be similar to the families seen in other risk evaluation clinics in the United States. Finally, this study provides an example in which the selection bias inherent in clinic-based cohorts provides information that may be useful in defining penetrance (6174delT, with fewer than expected mutations) and demonstrates that findings from population-based cohorts may not be reflected in clinic populations (in this case, low average age of BRCA2-related breast cancer diagnosis). Although rigorously designed case-control studies and population-based cohorts are invaluable in defining the prevalence, mutational spectrum, and penetrance of cancer susceptibility genes, it is important to remember that these data are ultimately put to use in the clinic, where inherent selection biases may produce a different picture, one that should be considered when counseling patients about undergoing genetic testing and, in particular, when considering management strategies for women with documented mutations.
Supported by grant nos. CA57601 (B.L.W.) and CA84030 (K.L.N.) from the National Cancer Institute and grants from the Susan G. Koman Foundation (M.A.B. and B.L.W.), Breast Cancer Research Foundation (B.L.W.), and Institute for Cancer Research (M.R.S.).
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Struewing JP, Hartge P, Wacholder S, et al: The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N Engl J Med 336: 1401-1408, 1997 4. Easton DF, Ford D, Bishop DT: Breast and ovarian cancer incidence in BRCA1-mutation carriers: Breast Cancer Linkage Consortium. Am J Hum Genet 56: 265-271, 1995[Medline] 5. Ford D, Easton DF, Bishop DT, et al: Risks of cancer in BRCA1-mutation carriers: Breast Cancer Linkage Consortium. Lancet 343: 692-695, 1994[CrossRef][Medline] 6. Fodor FH, Weston A, Bleiweiss IJ, et al: Frequency and carrier associated with common BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer patients. Am J Hum Genet 63: 45-51, 1998[CrossRef][Medline] 7. Thompson D, Easton D, Breast Cancer Linkage Consortium: Variation in cancer risks, by mutation position, in BRCA2 mutation carriers. Am J Hum Genet 68: 410-419, 2001[CrossRef][Medline]
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Stratton JF, Gayther SA, Russell P, et al: Contribution of BRCA1 mutations to ovarian cancer. N Engl J Med 336: 1125-1130, 1997 11. Oddoux C, Struewing JP, Clayton CM, et al: The carrier frequency of the BRCA2 6174delT mutation among Ashkenazi Jewish individuals is approximately 1%. Nat Genet 14: 188-190, 1996[CrossRef][Medline]
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Hall JM, Lee MK, Newman B, et al: Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250: 1684-1689, 1990 13. Frank TS: Identifying women with inherited risk for ovarian cancer: Who and why? Contemp OB/GYN 43: 2-11, 1998 14. Narod SA, Feunteun J, Lynch HT, et al: Familial breast-ovarian cancer locus on chromosome 17q12-q23. Lancet 338: 82-83, 1991[CrossRef][Medline]
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Couch FJ, DeShano ML, Blackwood MA, et al: BRCA1 mutations in women attending clinics that evaluate the risk of breast cancer. N Engl J Med 336: 1409-1415, 1997 16. Shih HA, Nathanson KL, Seal S, et al: BRCA1 and BRCA2 mutations in patients with multiple primary cancers. Clin Cancer Res 11: 4259-4264, 2000
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Wooster R, Neuhausen SL, Mangion J, et al: Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13. Science 265: 2088-2090, 1994 18. Easton DF, Steele L, Fields P, et al: Cancer risks in two large breast cancer families linked to BRCA2 on chromosome 13q12-13. Am J Hum Genet 61: 120-128, 1997[Medline]
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Ganguly A, Rock MJ, Prockop DJ: Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 90: 10325-10329, 1993 20. Claus EB, Risch N, Thompson WD: Genetic analysis of breast cancer in the cancer and steroid hormone study. Am J Hum 48: 232-242, 1991
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Armstrong K, Calzone K, Stopfer J, et al: Factors associated with decisions about clinical BRCA1/2 testing. Cancer Epidemiol Biomarkers Prev 9: 1251-1254, 2000 22. Arason A, Jonasdottir A, Barkardottir RB, et al: A population study of mutations and LOH at breast cancer gene loci in tumours from sister pairs: Two recurrent mutations seem to account for all BRCA1/BRCA2 linked breast cancer in Iceland. J Med Genet 35: 446-449, 1998[Abstract] 23. Serova OM, Boutrand L, Stoppa-Lyonnet D, et al: BRCA2 mutations in hereditary breast and ovarian cancer in France. Am J Hum Genet 60: 1236-1239, 1997 (letter)[Medline] 24. Frank TS, Manley SA, Olopade OI, et al: Sequence analysis of BRCA1 and BRCA2: Correlation of mutations with family history and ovarian cancer risk. J Clin Oncol 16: 2417-2425, 1998[Abstract]
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Satagopan JM, Offit K, Foulkes W, et al: The lifetime risks of breast cancer in Ashkenazi Jewish carriers of BRCA1 and BRCA2 mutations. Cancer Epidemiol Biomarkers Prev 10: 467-473, 2001 26. Roa BB, Boyd AA, Volcik K, et al: Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2. Nat Genet 14: 185-187, 1996[CrossRef][Medline] 27. Moslehi R, Chu W, Karlan B, et al: BRCA1 and BRCA2 mutation analysis of 208 Ashkenazi Jewish women with ovarian cancer. Am J Hum Genet 66: 1259-1272, 2000[CrossRef][Medline]
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Warner E, Foulkes W, Goodwin P, et al: Prevalence and penetrance of BRCA1 and BRCA2 gene mutations in unselected Ashkenazi Jewish women with breast cancer. J Natl Cancer Inst 91: 1241-1247, 1999 29. Unger MA, Nathanson KL, Calzone K, et al: Screening for genomic rearrangements in families with breast and ovarian cancer identifies BRCA1 mutations previously missed by conformation-sensitive gel electrophoresis or sequencing. Am J Hum Genet 67: 841-850, 2000[CrossRef][Medline] Submitted June 7, 2001; accepted October 5, 2001. This article has been cited by other articles:
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
