Family History of Breast and Ovarian Cancers and BRCA1 and BRCA2 Mutations in a Population-Based Series of Early-Onset Breast Cancer

Niklas Loman, Oskar Johannsson, Ulf Kristoffersson, Håkan Olsson, Åke Borg

Affiliations of authors: N. Loman, O. Johannsson, Å. Borg (Department of Oncology), U. Kristoffersson (Department of Clinical Genetics), Lund University Hospital, Sweden.

Correspondence to: Niklas Loman, M.D., Department of Oncology, Lund University Hospital, SE-221 85 Lund, Sweden (e-mail: Niklas.Loman{at}onk.lu.se).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: BRCA1 and BRCA2 are the two major susceptibility genes involved in hereditary breast cancer. This study was undertaken to provide reliable population-based estimates of genetic influence and to characterize the nature and prevalence of BRCA1 and BRCA2 germline mutations in early-onset breast cancer. Methods: In a series comprising all women diagnosed with breast cancer under the age of 41 years in southern Sweden during 1990 through 1995 (n = 262), family history of cancer was evaluated in 95% (n = 250) of the case subjects and germline mutations in BRCA1 and BRCA2 were analyzed in 89% (n = 234). All statistical tests were two-sided. Results: A total of 97 case subjects had at least one first- or second-degree relative with breast or ovarian cancer; 34 (14%; 95% confidence interval [CI] = 9.6% to 18%) cases had at least two first- or second-degree relatives, 22 (8.8%; 95%CI = 5.3% to 12%) had one first-degree relative, and 41 (16%; 95% CI = 12% to 21%) had one second-degree relative with either cancer. If two females affected with breast or ovarian cancer who were related through an unaffected male were also defined as first-degree relatives, then a higher number of case subjects, 120 (48%; 95% CI = 42% to 54%), had at least one first-degree or second-degree relative with breast or ovarian cancer. Sixteen (6.8%; 95% CI = 4.0% to 11%) BRCA1 mutation carriers and five (2.1%; 95% CI = 0.70% to 4.9%) BRCA2 mutation carriers were identified. Among case subjects with one first- or more than one first- or second-degree relative with breast or ovarian cancer, BRCA mutations were more frequent (P<.001) than among the case subjects without this degree of family history. BRCA mutations were also statistically significantly more common among women with bilateral breast cancer than among women with unilateral breast cancer (P = .002). BRCA mutations were more common among younger case subjects than among older ones (P = .0027). Conclusions: Almost half (48%) of women in southern Sweden with early-onset breast cancer have some family history of breast or ovarian cancer, and 9.0% of early-onset breast cancer cases are associated with a germline mutation in BRCA1 or BRCA2. Mutation carriers were more prevalent among young women, women with at least one first- or second-degree relative with breast or ovarian cancer, and women with bilateral breast cancer.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
About one in eight women in the western world will develop breast cancer at some time during their lives (1) (data from the Swedish cancer register). In Sweden, the median age at diagnosis of breast cancer is about 65 years, and 3% of these cancers will occur in women under the age of 40 years (data from the Swedish cancer register). A positive family history of the disease is one of the strongest risk factors for developing breast cancer, especially among young women (1). Segregation analyses of large population-based sets of families performed before the identification of BRCA1 and BRCA2 and not selected for family history suggest that highly penetrant, autosomal dominant genes may be responsible for a number of breast cancer cases (2). In such families, breast cancer risk is positively associated with decreasing age of the index case subject (3). The two major breast cancer susceptibility genes, BRCA1 and BRCA2 (46), were initially considered to account for the majority of all familial breast and breast–ovarian cancers. Subsequent studies from different populations have shown that considerably lower proportions of breast cancer families are associated with these genes and that the proportional contribution of BRCA1 and BRCA2 differs in different populations of the world (7).

The situation among the population in the present study is reflected in a previously published article from our department (8). We have reported BRCA1 and BRCA2 mutation frequencies in 106 Swedish and Danish breast cancer and breast–ovarian cancer families. In this population, BRCA1 mutations were identified in 23% of the families and BRCA2 mutations in 10% (8). The frequency of BRCA1 and BRCA2 mutations among incident cases of breast cancer also varies between different populations, partly because of the variable contribution of founder mutations (Table 1Go). Studies of BRCA1 and BRCA2 germline mutations among incident cases of breast cancer show (a) that germline mutations in these genes are more common among younger breast cancer patients than among older ones, (b) that germline BRCA1 and BRCA2 mutations in populations without strong founders together account for fewer than 10% of the breast cancer cases, and (c) that germline mutations in these genes are more prevalent in populations with strong founder effects (Ashkenazi and Iceland) (911,22) than in populations without such mutations (Table 1Go) (1218). Although some previous population-based studies have included large sample sets, they suffer from either low inclusion rate or incomplete mutation screening.


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Table 1. Published studies of prevalence of BRCA1 and BRCA2 mutations among cases of female breast cancer*
 
The goals of the current study were to estimate the frequency of genetic factors in a south Swedish population-based set of early-onset (aged <41 years) breast cancer case subjects and to provide a reliable frequency of the contribution of BRCA1 and BRCA2 germline mutations. We collected information about family history of cancer from the women themselves through a questionnaire and their medical records. Whenever possible, we verified data on family history by using independent sources. Mutation analysis of BRCA1 and BRCA2 covered the whole coding regions of BRCA1 and BRCA2.


    SUBJECTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Patients

According to the Regional Cancer Register in Lund, Sweden, which covers the South Swedish Health Care Region (about 1 500 000 inhabitants), a total of 6433 women were diagnosed with breast cancer during the period January 1, 1990, through December 31, 1995; 271 (4.2%) of these cases were registered in women 40 years or younger. Nine of these cases were either recorded twice (six cases) or incorrectly as invasive cancers (three cases) when review of the pathologic examination showed carcinoma in situ or Paget's disease. A total of 259 of these women were 40 years of age or younger at the time of their first invasive breast cancer. In addition, three women were already diagnosed before 1990 with an invasive breast cancer and had had their second invasive breast cancer before the age of 41 years during the time period of the present study. In all, 225 (86%) of the 262 women were alive on September 1, 1996, when this study was initiated. After approval had been obtained from the ethical committee of Lund University Hospital and the clinicians responsible for the treatment and follow-up of patients had been contacted, an invitation was sent to all 225 women asking them to participate. All of the participating women were asked to return a questionnaire covering family history of cancer and other risk factors for breast cancer. Women who had died could be included if samples of their blood or tissue were available in the tissue bank. These women had given their consent and had filled in a similar questionnaire at the time of blood sampling, and the majority of them had already been informed about their mutation status.

For the analysis of family history of cancer, pedigrees were constructed with every index woman as the proband. Information about family history was collected from the questionnaires, and this information was confirmed independently and expanded by means of contacts with the local parish or the local revenue authorities that keep the local census registries. Children, siblings, parents and their siblings, as well as grandparents and, if possible, their siblings were identified. If no other source was available, data about family history were collected from the medical records. By these means, each woman was classified according to her hereditary history of breast and/or ovarian cancer. In a few cases, data were available from only one branch of the family. These cases were considered evaluable and were included in the study. Therefore, the results concerning family history are to be considered a minimum of the true figures in the families. A common criterion for offering clinical mutation screening of BRCA1 and BRCA2 in Sweden is families with at least three first-degree relatives with breast or ovarian cancer, at least one of whom is under age 50 years, and families with two first-degree relatives, at least one of whom is under age 40 years. All case subjects under age 30 years are offered mutation screening as well. According to these criteria, two females affected with breast or ovarian cancer and related to each other through an unaffected male are considered to be first-degree relatives. By this definition, families with a family pattern consistent with dominant inheritance of breast or ovarian cancer are easily identified. If paternal heritage is not accounted for in this way, a proportion of cases with a probable hereditary cancer are overlooked. In this study, we present data both using strict criteria and separately using this wider definition of a first-degree relative.

Data on cancer incidence in the families of the index women were available until December 31, 1998, in the national Swedish cancer register and until July 31, 2000, in the South Swedish cancer register.

Mutation Screening

Screening for BRCA1 and BRCA2 mutations was performed as described previously (8,19,20). Genomic DNA from blood or, in a few subjects, fresh frozen tumor tissue was used as the source for the polymerase chain reaction (PCR)-based assays. BRCA1 exon 11 and BRCA2 exons 10 and 11 were screened with the protein truncation test (PTT). Exon 7 in BRCA1 and exon 3 in BRCA2 were sequenced directly, and the remaining exons were screened with either single-strand conformation polymorphism (SSCP) or denaturing high-performance liquid chromatography (dHPLC) analysis. Samples with autoradiographs or chromatograms indicating infrequent sequence variants were investigated further and characterized with the use of direct sequencing.

Statistical Methods

Differences in age between groups of case subjects were analyzed with the use of the Wilcoxon rank-sum test. Differences in dichotomous variables were analyzed with the use of the chi-squared test. Differences in the age of mutation carriers compared with that of noncarriers were analyzed with the use of logistic regression. For this analysis, index case subjects were grouped into three age groups: younger than 31 years, 31–35 years old, and 36–40 years old. P values less than .05 were considered to be statistically significant. All statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The study consisted of two parts: the epidemiologic study and the molecular genetic study. In the epidemiologic study, family history of cancer was investigated in 250 (95%) of the 262 index case subjects. The number of case subjects evaluated for family history in each age group were as follows: three (100%) of three aged less than 26 years, 21 (100%) of 21 aged 26–30 years, 59 (95%) of 62 aged 31–35 years, and 167 (95%) of 176 aged 36–40 years.

In the molecular genetic study, the relative contributions of BRCA1 and BRCA2 germline mutations were investigated in 234 (89%) of the 262 case subjects. Similarly, case subjects investigated in this part of the study consisted of three (100%) of three aged less than 26 years, 20 (95%) of 21 aged 26–30 years, 58 (94%) of 62 aged 31–35 years, and 153 (87%) of 176 aged 36–40 years.

Information on history of bilateral breast cancer and other malignancies in index case subjects was available for all 262 index case subjects through follow-up in the central Swedish cancer registry; no index women emigrated during the follow-up period. Information about family history of cancer was not available for 12 women; in 11 of these case subjects, this lack of information was due to the fact that the women were immigrants, and data on family history were not available in their medical records. Of the 250 families who were studied in the epidemiologic study, diagnoses of malignancy were independently confirmed in 211 (84%).

Bilateral Breast Cancer and Ovarian Cancer Among Index Case Subjects

Bilateral breast cancer. After a median follow-up of 5.5 years, 18 (6.9%) of the 262 women were diagnosed with bilateral breast cancer. Sixteen of these women were diagnosed with both cancers before the age of 41 years. Five of the case subjects with bilateral breast cancer were BRCA1 mutation carriers, and one was a BRCA2 mutation carrier. Thus, of 18 subjects with bilateral disease, six (33%; 95% confidence interval [CI] = 13% to 59%) were carriers of a BRCA1 or BRCA2 mutation. Mutations in either gene occurred statistically significantly more often in subjects with bilateral breast cancer than in subjects with unilateral breast cancer, where 15 (6.9%; 95% CI = 3.9% to 11%) of 216 were mutation carriers (P = .002). Five (83%) of the six mutation-positive case subjects with bilateral disease had at least one first-degree relative with breast or ovarian cancer.

Ovarian cancer. Ovarian cancer was recorded in two index case subjects—one at age 38 years after having had breast cancer at age 35 years and the other at age 33 years before subsequently developing breast cancer at age 35 years. Surprisingly enough, no BRCA1 or BRCA2 mutations were identified in these two subjects. However, in the first case, the ovarian cancer diagnosis can be questioned. According to a revision of the microscopic slides during her treatment planning, the abdominal manifestations of cancer that were detected at 38 years of age may actually have been metastases from her previously diagnosed breast cancer rather than a primary ovarian cancer. Neither of the two women had any family history of breast or ovarian cancer.

Family History of Breast and Ovarian Cancers Among Index Case Subjects

Table 2 shows the family history of breast and ovarian cancers among index case subjects. Two hundred fifty index women were evaluated for hereditary history. One hundred fifty-three (61%; 95% CI = 55% to 67%) had fewer than one second-degree relative with breast or ovarian cancer (Table 2, AGo). Forty-one (16%; 95% CI = 12% to 21%) of them had one second-degree relative with breast or ovarian cancer. Twenty-two (8.8%; 95% CI = 5.3% to 12%) had one first-degree relative, 26 (10%; 95% CI = 6.6% to 14%) had two first- or second-degree relatives, and eight of them (3.2%; 95% CI = 1.4% to 6.2%) had three first- or second-degree relatives with breast or ovarian cancer. Thus, 34 (14%; 95% CI = 9.6% to 18%) index individuals had at least two first- or second-degree relatives with breast or ovarian cancer, and 97 (39%; 95% CI = 33% to 45%) had some family history of breast or ovarian cancer among first- or second-degree relatives. Twenty women (8.0%; 95% CI = 5.0% to 12%) had at least one first- or second-degree relative with ovarian cancer. If the possibility of male inheritance was accounted for, higher rates of positive family history were recorded (Table 2, BGo). If a wider definition of a first-degree relative (including females affected with breast or ovarian cancer related through a male) was used, 75 (30%; 95% CI = 24% to 36%) case subjects had at least one first-degree relative with breast or ovarian cancer, and an additional 45 (18%; 95% CI = 13% to 23%) subjects had second-degree relative(s) with breast or ovarian cancer. Thus, 120 (48%; 95% CI = 42% to 54%) of the 250 index case subjects presented with some kind of family history of breast or ovarian cancer. Among subjects with bilateral breast cancer, 10 (56%; 95% CI = 31% to 78%) of 18 index case subjects had at least one first-degree relative with breast or ovarian cancer, if male gene transmission is accounted for, as compared with 64 (28%; 95% CI = 22% to 34%) of 232 case subjects with unilateral disease (P = .012). This difference was even more pronounced (P<.001) when we compared the number of case subjects with at least two first-degree relatives in the bilateral disease group (eight of 18; 44% [95% CI = 22% to 69%]) and the unilateral disease group (25 of 232; 11% [95% CI = 7.1% to 15%]).


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Table 2, A. Family history of breast or ovarian cancer among index case subjects with early-onset breast cancer*
 

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Table 2, B. Family history among index cases; male gene transmission accounted for by considering two affected females related through a man to be first-degree relatives*
 
Two index case subjects had a male relative with breast cancer. The paternal grandfather of one 36-year-old woman with breast cancer had had breast cancer diagnosed at age 76 years. Two more breast cancer cases were seen in this family—i.e., in a sister (aged 51 years) of the index patient and in a sister (aged 59 years) of her paternal grandmother (thus not related to the male breast cancer case subject). The other male breast cancer case subject (aged 88 years) was a maternal great-grandfather to an index case subject with breast cancer diagnosed at age 40 years. One of his daughters had had bilateral breast cancer diagnosed at ages 69 and 81 years, respectively, and one of his sons had had a malignant lymphoma diagnosed at age 60 years. The index case subjects in both of these families were BRCA1 and BRCA2 mutation negative.

Analysis of BRCA1 and BRCA2

Analysis of BRCA1 and BRCA2 mutations was performed in 234 (89%) of the 262 case subjects. The 28 dropouts consisted of 24 women who did not wish to participate in the study or who did not reply to the invitation letter as well as four deceased women from whom no tissue was available for analysis. Among the 28 dropouts, data on family history of breast and ovarian cancers were available for 21 (75%). Fourteen of them (67%) did not have any family history if the possibility of male inheritance was accounted for, three of them (14%) had a second-degree relative with breast or ovarian cancer, and four of them (19%) had at least one first-degree relative with breast or ovarian cancer.

Among the 234 index case subjects, BRCA1 mutations were detected in 16 (6.8%; 95% CI = 4.0% to 11%) and BRCA2 mutations were detected in five (2.1%; 95% CI = 0.70% to 4.9%). Eleven (69%) of the 16 BRCA1 mutations were nonsense or frameshift mutations, predicted to result in truncated gene products. Five BRCA1 mutations were missense mutations located in exons 5, 18, and 20, reported previously to the mutation database, i.e., http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/, and by all appearances were associated with disease on the basis of functional studies (25) and evidence of segregation in families. Four of the five BRCA2 mutations were nonsense or frameshift mutations predicted to result in protein truncation, and the fifth mutation was a splice-site mutation of exon 24 (Table 3Go).


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Table 3. Germline BRCA1 and BRCA2 mutations among 234 case subjects with breast cancer who were <41 years of age*
 
Six of the 16 BRCA1 mutations were one of four major Swedish BRCA1 founder mutations (1201del11, 1806C->T, 2594delC, and 3172ins5) identified previously in at least 10 independent families (8) (Borg Å: unpublished results). Two of the BRCA1 mutations (3829delT and 300T->G) have also been identified previously in Scandinavia but in fewer families; the latter of the two mutations was a central European founder mutation. The remaining eight BRCA1 mutations were not identified previously in our population. One (4486delG) of the five BRCA2 mutations is found repeatedly in Swedish breast cancer families, whereas the remaining four BRCA2 mutations are observed infrequently in such families.

The mean ages at breast cancer diagnosis of BRCA1 and BRCA2 mutation carriers were 34.8 and 31.7 years, respectively, or 34.0 years for the whole group of BRCA mutation carriers, as compared with a mean age of 36.8 years among the 213 BRCA-negative case subjects (P = .0037). When the material was subdivided into age groups (<31 years, 31–35 years, and 36–40 years), there was a statistically significant trend toward more mutation-positive case subjects among the younger age groups (P = .0027; Table 4Go). Among the 81 women diagnosed below the age of 36 years, nine had a BRCA1 mutation and four had a BRCA2 mutation. Thus, in this group, the frequency of BRCA mutations was 16% (95% CI = 8.8% to 26%) (Table 4Go).


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Table 4. Prevalence of BRCA1 and BRCA2 mutation carriers in different age groups and among index case subjects with bilateral breast cancer*
 
The frequency of BRCA1 and BRCA2 mutations in index case subjects with or without a family history of breast and ovarian cancer is shown in Table 5GoGo. Of the 50 BRCA-tested subjects with at least one first-degree relative or two first- or second-degree relatives with breast or ovarian cancer, 13 (26%; 95% CI = 14% to 40%) were BRCA1 or BRCA2 mutation carriers compared with seven (3.9%; 95% CI = 1.6% to 7.8%) of 180 case subjects without this degree of family history, which is statistically significantly more frequent (P<.001). Of the 71 index case subjects with at least one first-degree relative with breast or ovarian cancer, according to the criteria allowing for male gene transmission, 11 (15%; 95% CI = 8.0% to 26%) had a BRCA1 mutation and three (4.2%; 95% CI = 0.88% to 12%) had a BRCA2 mutation. The remaining six BRCA mutations occurred in women with a weaker (two case subjects with one second-degree relative) or no (four case subjects with no more than a third-degree relative with breast or ovarian cancer) family history. According to the conventional criteria, 13 (65%) of 20 mutation carriers came from families with at least one additional first-degree relative with breast or ovarian cancer. By use of the extended criteria, allowing for gene transmission through a healthy male, 14 (70%) of 20 index individuals presented with this indirect sign of dominant mendelian inheritance. In one case, the family history of a BRCA1 mutation carrier was unknown.


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Table 5, A. Numbers of mutation carriers in relation to family history of breast or ovarian cancer*
 

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Table 5, B. Numbers of mutation carriers in relation to family history of breast or ovarian cancer; male gene transmission accounted for by considering two affected females related through a man to be first-degree relatives*
 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We have analyzed the frequency of a positive family history of breast and/or ovarian cancer as well as the frequency of BRCA1 and BRCA2 germline mutations in a population-based series of 262 case subjects with early-onset breast cancer. The sample was unselected for family history of any cancer and includes all invasive breast cancer cases occurring in women below the age 41 years in south Sweden during 6 consecutive years. Mutation screening was performed in 89% of the subjects and covered the whole coding region of BRCA1 and BRCA2, in contrast to other similar studies in which mutation analysis was restricted to founder mutations (11,13,14,17,21). In this genuine population-based sample, the inclusion rate is higher than that in any other previously published study in this field. Peto et al. (15) based their investigation on two large case–control studies of breast cancer patients diagnosed before age 36 years (n = 755) and diagnosed from ages 36 through 45 years (n = 644), respectively. However, mutation screening was done in only 617 (44%) subjects, the major reason for not obtaining samples being that the patients were dead, were seriously ill, or refused to donate a sample. This selection most probably resulted in a skewed distribution of survival as well as of family history of cancer in the tested and untested sets (15). An Australian study by Southey et al. (22) included 91 randomly selected subjects from a population of about 640 women with invasive breast cancer diagnosed under the age of 40 years, which gives an inclusion rate of 14%. A Finnish study of prevalence of founder mutations (17) is more representative of its target population because 82% of the breast cancer cases were included; however, since only founder mutations were analyzed, a substantial proportion of mutation carriers may have been overlooked. Malone et al. (16,24) have published results from a population-based study in which the rate of BRCA1 and BRCA2 mutations was determined in a sample of cases subjects with breast cancer diagnosed before age 35 years; 203 (66%) subjects from a population of 306 breast cancer patients were analyzed for germline mutations in BRCA1 and BRCA2. The recently published study from the Anglian Breast Cancer Study Group (18) is the largest study published so far. A total of 1435 case subjects (51%) of a population of 2805 eligible case subjects were screened for mutations in BRCA1 and BRCA2 (Table 1Go).

In our study, information about family history of breast and ovarian cancers was known in 75% of the case subjects who dropped out. An absence of family history seemed to be more prevalent among the dropouts (66%) than among the subjects who were screened for mutations in BRCA1 and BRCA2 (50%). Even if the difference is not statistically significant (data not shown), the finding illustrates the risk for selection bias toward a positive family history among subjects included in a mutation-screening study and, consequently, the risk of overestimation of the penetrance of breast and ovarian cancers if such estimations are made from the material. Similarly, all subjects with bilateral breast cancer were included in the study, again indicating that there might be an underrepresentation of cases with hereditary factors among subjects not included in a population-based study. The only way to overcome this possible source of bias is a high inclusion rate among subjects screened for mutations.

A striking observation in this study was the high prevalence of a positive family history of breast or ovarian cancer, suggesting a strong contribution of genetic factors in early-onset breast cancer. This observation was even more pronounced if the possibility of male gene transmission was considered, using a definition of first-degree relative that "upgraded" second-degree relatives to first-degree relatives, provided that they were both affected with breast or ovarian cancer and that they were related to each other through an unaffected male. Almost one third (30%) of the index case subjects had at least one first-degree relative with breast or ovarian cancer, and 13% presented with a family history of at least three cases of breast or ovarian cancer among first-degree relatives in the family (including the index case subject). We believe that the use of the more conservative definition leads to an underestimation of the contribution of hereditary factors in a sample such as this and that this possibility must be accounted for so as not to overlook case subjects with obviously hereditary cancer. Rates of positive family history of different cancer types in different studies are difficult to compare, since criteria for a positive family history vary. For example, Newman et al. reported, in a large segregation analysis, that more than 12% of affected index case subjects under the age of 55 years had an affected mother or sister with breast cancer (2) and that 49% of the case subjects in a population-based estimate of the contribution of BRCA1 among U.S. case subjects with breast cancer who were 20–74 years old had any relative with breast or ovarian cancer (10). Peto et al. (15) reported a positive family history of breast cancer in merely 6.7% of breast cancer patients under the age of 36 years, a percentage that is based on the presence of breast cancer before age 60 years in the mother or a sister and that most likely represents an underestimation due to selection bias in the material. In the large study from the Anglian Breast Cancer Study Group (23) including subjects under age 55 years, 14% of the subjects had at least one first-degree relative with breast cancer. Malone et al. (24) reported that 44% of their patient sample came from families with a relative with breast cancer. Because reliable and independently controllable data on family history of breast and ovarian cancers were available for the great majority of index case subjects, we believe that the data presented in this article are close to the true rates in the population.

Screening of 234 patients with early-onset breast cancer resulted in the identification of 16 case subjects with BRCA1 mutations and five case subjects with BRCA2 mutations (Table 4Go). Because the inclusion rate of the present population-based study is higher than that in any other similar study published so far and because the whole coding regions of BRCA1 and BRCA2 were covered in the mutation screening and missense mutations with a high likelihood to be disease associated were considered, our estimates of the relative contribution of BRCA1 and BRCA2 can be anticipated to be close to the true figures. Previously published data from the same geographic area about the contribution of BRCA1 and BRCA2 among South Swedish breast and ovarian cancer families (8) reported a similar distribution between BRCA1 and BRCA2 mutations. Two BRCA1 mutations (1201del11 and 1806C->T) were detected twice and represent known founder mutations in the Swedish population. Two other commonly occurring mutations (2594delC and 3172ins5) were each recorded once in our study. Hence, founder mutations account for six of 16 case subjects with BRCA1 mutations. BRCA2 4486delG represents a previously described Swedish founder mutation (see Table 3Go) (8). Thus, seven (33%) of the 21 BRCA-mutated subjects described in our study are attributable to previously identified founder mutations in the Swedish population. Consequently, by simply screening for founder mutations, two thirds of all mutations would have been missed. The BRCA1 founder mutations and another seven BRCA1 mutations are predicted to result in protein truncation. The five remaining BRCA1 mutations are of missense type. C61G disrupts the N-terminal RING finger domain and is a well-characterized central European founder mutation, whereas R1699Q, A1708E, and G1738E are located in the BRCT (i.e., BRCA1 C-terminal) domain to which a transactivation function has been ascribed (25). On the basis of their location at conserved residues in functionally important domains, their absence in control individuals, their cosegregation with disease in families, or their nonfunctional appearance in yeast or mammalian transactivation assays, we considered these missense mutations to be deleterious (26). The five BRCA2 mutations included four frameshift and one splice-site mutation that are all predicted to result in protein truncation.

Since no mutation detection method can be expected to have 100% sensitivity, the presented rates of mutation carriers should be considered to be minimal estimations of their true frequency in the population. Among the mutations detected, the majority of cases were nonsense and frameshift mutations giving rise to truncated gene products, which is partly a consequence of the methods used for mutation screening. The large exon 11 in BRCA1 and the large exons 10–11 in BRCA2 were screened with the use of PTT, which only detects this type of mutation. The remaining exons were screened with SSCP or with dHPLC, techniques that are presumed to have incomplete mutation detection sensitivity, in spite of thorough optimization (8,20). Their exact sensitivity is difficult to predict; however, dHPLC is likely to be more sensitive than SSCP. In contrast to other similar studies, missense mutations with a high probability of being disease associated were included in the analysis of our study. This approach augments the reported rate of mutations in the study population. If missense mutations were not included, the BRCA1 mutation carrier rate in the population would be 11 (4.7%; 95% CI = 2.4% to 8.3%) of 234, corresponding to 16 (6.8%; 95% CI = 4.0% to 11%) of 234 for all BRCA mutations. However, with the current knowledge of the functional consequences and cosegregation with breast and/or ovarian cancer in families with these altered alleles, we would consider it wrong not to see them as mutations and not to include them in the analyses. The large gene rearrangements that are commonly observed in the Alu-rich BRCA1 gene are overlooked by the PCR-based techniques used in our study. We cannot exclude the presence of as yet undiscovered BRCA genomic rearrangements in the Swedish population, and we may underestimate the potential contribution that founder effects for such a mutation would have. However, screening of more than 500 Scandinavian breast and breast–ovarian cancer families for previously described large rearrangements has resulted in identification of only two such alterations in single families, one being a 5-kilobase (kb) deletion covering BRCA2 exon 3 (27) and the other being a 0.5-kb deletion at BRCA1 exon 22, known as a Dutch founder mutation (Borg Å: unpublished results). These observations indicate that at least these known large rearrangements are infrequent in the Swedish population.

Evidently, BRCA1 accounts for a considerably larger proportion of early-onset breast cancer in southern Sweden than does BRCA2 (6.8% versus 2.1%). In their study of British case subjects with breast cancer diagnosed before the age of 36 years, Peto et al. (15) observed a mutation frequency of 3.5% and 2.4% for BRCA1 and BRCA2, respectively. The corresponding percentages for this age group in our study are 11% and 4.9%. The relatively high proportion of BRCA1-mutated subjects in our study can be partly explained by the inclusion of presumably disease-causing missense mutations in the analysis, but hardly by a strong contribution of the BRCA1 founder mutations. Instead, the difference is presumably caused by population-based differences in mutation frequency. Alternatively, a survival bias in the British study can partly explain the relatively low prevalence of BRCA mutations, in this case most likely due to the relatively long delay between diagnosis and inclusion in the study. The distribution of BRCA1 and BRCA2 in the British population is confirmed in the previously published study from the Anglian Breast Cancer Study Group (18) (Table1Go).

Apart from young age and a positive family history of breast cancer, bilateral disease is a hallmark of inherited breast cancer and a criterion for clinical BRCA1 and BRCA2 mutation screening. Contralateral breast cancers were seen at a high rate among the index case subjects of our study. During the follow-up, 18 (6.9%) of the 262 subjects had developed a second primary tumor. Two thirds of these case subjects with bilateral disease either were mutation carriers or had at least one first-degree relative with breast cancer, indicating a close relationship between bilateral disease and inherited risk factors. A positive family history of breast or ovarian cancer and BRCA mutations were also more common among the case subjects with bilateral disease than among those with unilateral disease. Two thirds of the case subjects with bilateral disease were under age 36 years at first diagnosis, and 16 of the 18 women developed both tumors before the age of 41 years. Only one case subject with bilateral disease did not display any of these features [first-degree relative(s) with breast cancer, BRCA1 or BRCA2 mutation, or age under 36 years] typical for hereditary breast cancer.

Whereas BRCA1 has been closely connected to an increased risk of both breast and ovarian cancers (28), the situation is more complex for BRCA2, and the estimated lifetime risk of ovarian cancer in BRCA2 mutation carriers is lower than that in BRCA1 mutation carriers (29,30). Moreover, it has been suggested that the risk for ovarian cancer is higher for truncating mutations in the ovarian cancer cluster region, which constitutes a large part of BRCA2 exon 11, than for other BRCA2 germline mutations (31,32). We have previously suggested (33) that the increased risk of ovarian cancer seen among BRCA2 mutation carriers is due to a selection bias and largely conferred by the preferential screening of index case subjects with both breast and ovarian cancers. In our present study, none of the five BRCA2 mutation carriers had any first- or second-degree relatives with ovarian cancer, while four of them had a strong family history of breast cancer. This finding may support the view that the ovarian cancer risk in BRCA2 germline mutation carriers is not a universal feature of the gene.

Considering the high proportion of case subjects with a positive family history of breast cancer in early-onset breast cancer cases and the relatively low frequency of BRCA1 and BRCA2 mutations, one would envision that other genetic factors are at play. Indeed, a recent study on twins (34) suggests that as many as 27% of all breast cancers are caused by inherited factors. In our study, the breast cancers in 12 (39%) of the 31 index case subjects with a strong family history of breast or ovarian cancer and in 11 (39%) of the 28 case subjects with a strong family history of only breast cancer were due to BRCA1 and BRCA2 mutations. Some remaining families could carry mutations in other susceptibility genes, such as TP53, CHK2, and p16 known to be associated with early-onset breast cancer (3537). It is also suspected that genes that are as yet uncharacterized can explain certain proportions of the early-onset breast cancer cases (38), although some may play a more important role in women with slightly later (at ages 40–60 years) disease onset.


    NOTES
 
Supported by grants from the Swedish Cancer Society; by the Mrs. Bertha Kamprad Foundation; by the Gunnar, Arvid, and Elisabeth Nilsson Foundation; by the John & Augusta Persson Foundation; by the Hospital of Lund Foundations; by the FM Bergqvist Foundation; by King Gustav V's Jubilee Foundation; and by the Nordic Cancer Union.

We thank the following individuals from the Department of Oncology, Lund University Hospital, Sweden: Josefine Börnfors, Ulla Johansson, Gunilla Sellberg, Karin Haraldsson, Therese Sandberg, Philip Oppenheim, Fredrik Holmqvist, and Anna Sällfors for their participation in the mutation screening; Anna Bladström for her statistical advice; Kerstin Nilsson for blood sampling; and Ingrid Mårtensson for the family history investigations.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received December 27, 2000; revised May 30, 2001; accepted June 20, 2001.


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