Gerald Hurowitz, M.D.·Published 12 June 2008·23 min read
Key points
The study of clinical syndromes that include an increased incidence of breast cancer has provided insight into the mechanisms by which genetic mutations result in the development of breast cancer.
DeMichele: Departments of Medicine and Epidemiology, The University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania B.
The occurrence of breast cancer in association with diverse childhood neoplasms in the Li-Fraumeni/SBLA (s oft tissue and bony sarcomas, b rain tumors, l eukemias, and a drenocortical carcinomas) syndrome and the association between breast and ovarian cancer represent some of the most intensively studied examples.
BRCA1 In 1990, chromosome 17q21 was identified as the location of a susceptibility gene for early-onset breast cancer, now termed BRCA1.
The study of clinical syndromes that include an increased incidence of breast cancer has provided insight into the mechanisms by which genetic mutations result in the development of breast cancer. The most frequently identified pedigrees contain site-specific breast cancer (i.e., breast cancer in these families is not found in association with inherited susceptibility to other cancers, such as ovarian)and are thought to represent the effect of a single genetic abnormality. BRCA1 and BRCA2 are examples, and others are likely to be identified in the future. Breast cancer also has been noted to occur in association with other cancers.
The occurrence of breast cancer in association with diverse childhood neoplasms in the Li-Fraumeni/SBLA (s oft tissue and bony sarcomas, b rain tumors, l eukemias, and a drenocortical carcinomas) syndrome and the association between breast and ovarian cancer represent some of the most intensively studied examples. Finally, an increased frequency of breast cancer may occur in patients with hereditary syndromes that include nonmalignant manifestations as well, such as Cowden disease and Muir-Torre syndrome.
BRCA1 In 1990, chromosome 17q21 was identified as the location of a susceptibility gene for early-onset breast cancer, now termed BRCA1. Shortly thereafter, linkage between the genetic marker D17S74 on 17q21 and the appearance of ovarian cancer in several large kindreds was also demonstrated. Initial estimates suggested that BRCA1 mutations were responsible for more than 90% of breast cancer cases in families with apparent autosomal dominant transmission of breast cancer and at least one case of ovarian cancer, and 45% of cases in families with breast cancer only. However, the percentage of site-specific breast cancer cases attributed to BRCA1 mutations rose to almost 70% if the median age of onset of breast cancer in the families was younger than 45 years.
Using direct mutation testing, several groups have attempted to refine the estimates of mutation prevalence and breast cancer penetrance in BRCA1 carriers found in less striking families. In families identified through clinics treating high-risk breast cancer, BRCA1 mutations appear to be responsible for 20% to 30% of breast cancer cases and 10% to 20% of ovarian cancer cases. Estimates of BRCA1 prevalence in unselected breast cancer patients are not well documented, although initial studies suggest that the proportion is in the range of 3% as defined by two different studies.
Population Genetics of BRCA1 and BRCA2 The population genetics of BRCA1 and BRCA2 reflect several basic human evolutionary principles. It is postulated that each gene has undergone multiple mutations, and that these mutations have migrated with the people in which they occur. Certain "founder mutations" are now known to exist in BRCA1 and BRCA2. These are ancient mutations that have occurred in specific ethnic populations many generations in the past. They are thought to persist because the development of disease normally occurs after childbearing age, so that individuals carrying these mutations are able to pass them on to subsequent generations with little impact of the mutated alleles on survival of the species.
Written and revised by
Gerald Hurowitz, M.D.
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Founder mutations so far have been identified in at least nine separate ethnic subpopulations, including those in Iceland, Finland, Hungary, Russia, France, Holland and Belgium, Israel, Sweden and Denmark, and Norway. A comprehensive review of these studies by Szabo and King reveals the similarities and differences in mutation rate, penetrance, and nature of the mutations among various population groups. The proportion of high-risk families with breast or ovarian cancer appears to vary widely by population group; mutations in BRCA1 are most common in Russia (79% of families with breast/ovarian cancer), followed by Israel (47% of families) and Italy (29%).
Moreover, the population dynamics of the groups are very different; only a small number of different mutations are seen in the Russian and Israeli populations, whereas a large number of unique mutations are found in the Italian population. Thus, the number of families affected does not accurately reflect the number of different mutations present. BRCA2 mutations appear to be more common than BRCA1 mutations only in Iceland, where a single mutation accounts for virtually all of the BRCA2-associated breast and ovarian cancer.
The BRCA1 and BRCA2 mutations among the Ashkenazi Jewish population have been studied most intensively. The two most common mutations in BRCA1 are 185delAG and 5382insC, each accounting for approximately 10% of the total. These two mutations, along with a mutation in BRCA2, 6174delT, have been identified with the highest frequency in the Ashkenazi Jewish population.
The mutations 185delAG and 5382insC have been shown to occur in individuals of Ashkenazi descent with a combined frequency of approximately 1%; the estimated frequency of BRCA1 mutations in an unselected white population is less than 0.1%. This observation has subsequently been expanded to include the Moroccan, Iraqi, and Yemenite Jewish populations. Analysis of germ-line BRCA1 mutations in several cohorts of Jewish women suggests that just over 20% of Jewish women developing breast cancer before age 40 carry the 185delAG mutation. Even more strikingly, estimates suggest that 30% to 60% of all Ashkenazi Jewish women with ovarian cancer carry either a common BRCA1 or BRCA2 germ-line mutation. Other BRCA1 mutations have also been detected in the Jewish population but at much lower levels. {pagebreak}
Other Types of Cancers Associated with Mutations in BRCA1 Risk for cancers other than breast and ovarian cancer also appears to be increased in the presence of an inherited BRCA1 mutation. The first such indication came from the study of a large Icelandic family group with breast and ovarian cancer, which suggested that prostate cancer also is a component of the BRCA1 syndrome. Subsequently, the Breast Cancer Linkage Consortium estimated a relative risk of 3.33 for prostate cancer and a relative risk of 4.11 for colon cancer in males thought to carry BRCA1 germ-line mutations.
However, the excess colon cancer risk in this study reflects the experience of only a few families, which suggests either very low penetrance with regard to colon cancer or a limited number of specific mutations that increase colon cancer risk. No significant excesses were observed for cancers originating from other anatomic sites, and male breast cancer is only rarely associated with BRCA1 germ-line mutations.
Pathobiology of BRCA1-Associated Breast Tumors The relationship between BRCA1 and tumor prognosis is one of the key clinical questions that followed the identification of BRCA1. In the first study to address this question, Lynch et al. analyzed 180 tumors from families with hereditary breast/ovarian or site-specific breast cancer. Ninety-eight of the 180 tumors they studied were classified as the "BRCA1 group." These tumors were thought to have the highest likelihood of arising as a result of BRCA1 mutations but were not directly tested for mutations.
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The BRCA1 group was found to include more aneuploid and more high S-phase tumors; however, surprisingly, disease-free survival was longer for patients whose tumors were of this type than for those whose tumors were thought less likely to have BRCA1 mutations. Subsequent studies have verified that BRCA1-associated tumors are often high grade and tend to be estrogen-receptor and progesterone-receptor negative. Genetic studies of BRCA1-related breast and ovarian tumors further indicate that these tumors are both genetically unstable and highly proliferative.
Whether the aggressive pathologic features described in association with BRCA1-associated breast tumors translate into reduced survival or lack of response to standard treatment strategies is not yet clear, but data are accumulating that these high-grade lesions behave as would be expected. In addition, a study of Ashkenazi Jewish BRCA1 mutation carriers suggested that a BRCA1 mutation is an adverse prognostic factor in its own right. Ongoing large-scale studies are currently under way to address this issue in mutation carriers, in the context of other known prognostic factors.
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BRCA1 Gene Structure and Mutation Spectrum The BRCA1 gene is composed of 24 exons, or coding regions, and is translated into a protein consisting of 1,863 amino acids. This is important from a clinical standpoint in the context of genetic testing, because this very large size makes screening the entire gene for mutations technically demanding and costly. The entire gene covers approximately 100 kb of genomic sequence, and in the case of BRCA1, this region contains a large number of repetitive elements called Alu repeats.
Of clinical importance, these repetitive elements facilitate the generation of large deletions and duplications in BRCA1, creating disease-associated mutations that are not detectable by methods in current use in commercial testing laboratories. The prevalence of these mutations in the United States is not known yet, but three such mutations account for 36% of BRCA1 mutations in the Netherlands. These mutations clearly represent a potential source of a significant number of false-negative BRCA1 tests.
Now that BRCA1 has been identified, more than 500 coding region sequence variations have been detected. A listing and description of most known BRCA1 mutations is available on the Breast Cancer Information Core (BIC) website. Surprisingly, almost all described mutations are found in the germ line. Somatic BRCA1 mutations are rare in sporadic breast and ovarian tumors, a finding which suggests that BRCA1 coding region mutations play a limited role in the development of sporadic breast cancer. Whereas only one report of an individual with two BRCA1 mutations exists in the literature, a number of individuals who carry both BRCA1 and BRCA2 mutations have been described.
Preliminary work has begun to identify possible correlations between specific BRCA1 mutations and the types of cancers that subsequently develop. Two studies have suggested that mutations in the 5' half of BRCA1 predispose to both breast and ovarian cancer, whereas mutations closer to the 3' portion of the gene are predominantly associated with site-specific breast cancer.
This correlation has been observed in several European studies but is rarely seen in the BRCA1 population in the United States. It appears to be due largely to the reduced penetrance of ovarian cancer associated with these mutations rather than to an increased incidence of breast cancer arising in association with mutations in the 5' end of the gene. Finally, mutations occurring at either end of BRCA1 may be associated with a more severe phenotype, as defined by high tumor grade, suggesting that these two regions may be important for the control of mammary cell growth. Identification of these specific genotype/phenotype correlations will ultimately aid clinicians in recommending appropriate screening, prevention, and treatment strategies in women with known BRCA1 mutations, but at present these data are too preliminary to be used in counseling women with known mutations.{pagebreak}
Biological Function of BRCA1 To date, numerous studies have linked normal BRCA1 function to the cellular processes involved in transcription, apoptosis (programmed cell death), cell cycling, DNA repair, and developmental biology. The evidence supporting each of these functions for BRCA1 is summarized in the sections that follow.
Convincing evidence now exists that BRCA1 is a nuclear protein, and it is likely to play a role in altering the expression of other genes and in the response to DNA damage. Some controversy arose with the report of the presence of a granin motif in BRCA1, which is characteristic of secreted molecules normally found in the cytoplasm and membrane of cells.90 However, overwhelming evidence now suggests that BRCA1 is not secreted; and that the observations made suggesting that BRCA1 is a secreted molecule may have arisen due to a lack of specificity of the early BRCA1 antibodies.
For the most part, BRCA1 protein does not display homology to any other known motif or gene. The two exceptions are the RING finger domain at the beginning of BRCA1 and the BRCT motif at the end of the gene. The 126 base pair region of homology to a RING finger motif near the amino terminus of the protein binds zinc and is thought to be involved in protein-protein interactions.
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Two novel proteins, BAP1 (BRCA1 activator protein 1) and BARD1 (BRCA1-associated RING domain protein 1), have now been identified that bind the BRCA1 RING finger. BAP1 is thought to enhance BRCA1-mediated cell growth suppression, whereas BARD1 may be involved in response to DNA damage. Further study of the function of both BARD1 and BAP1 should help define the function of BRCA1.
BRCA1 and Transcription Evidence is mounting in support of a role for BRCA1 as a transcriptional coactivator-a protein that facilitates transcription of genes in the presence of direct transcriptional activators. First, BRCA1 has been shown to interact with two key components of the cell's transcription machinery-the RNA polymerase holoenzyme and the transcription factor CREB binding protein.
In addition, two groups have characterized a transcriptional activation domain in the carboxy terminus of the BRCA1 protein. Finally, BRCA1 has been shown to bind p53, a critical component of response to DNA damage that is itself known to be a direct transcriptional activator. The interaction between BRCA1 and p53 has been associated with activation of p21 expression; this leads to a cell cycle pause, which may be necessary for DNA damage repair to take place. Without this pause, which could perhaps not occur if BRCA1 was absent or mutated, mutations in additional genes could accumulate, facilitating malignant transformation.
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BRCA1 and Development Wild-type BRCA1 appears to play a critical role in both mouse and human development. The first studies of the role of BRCA1 in development were performed by in situ hybridization of mouse embryos and indicated that BRCA1 was widely expressed in many developing tissues. These investigations also demonstrated that BRCA1 was highly expressed in the mouse mammary gland during development and pregnancy.
Proof that BRCA1 is necessary for normal mouse development was provided by the derivation of four BRCA1 "knockout" mouse strains, each of which died in early embryonic development. The mechanisms by which BRCA1 exerts an effect on mouse development and its role in human development remain unclear; however, this pathway provides another means of defining BRCA1 function.{pagebreak}
BRCA1 and Cell Cycling BRCA1 has been associated with the orderly and efficient progression of the cell through the cell cycle, as originally suggested by studies of breast cancer cell lines demonstrating that BRCA1 and cyclin A RNA levels increased in parallel in response to hormone treatment. BRCA1 also has been shown to be phosphorylated in a cell cycle–dependent manner. Thus, BRCA1 may play a role in G1/S-phase checkpoint control or may be up-regulated during the cell cycle in response to cellular messages.
BRCA1 and Response to DNA Damage Finally, BRCA1 appears to play an important role in response to DNA damage. BRCA1 complexes with Rad51 and BRCA2, both of which have been associated with the cellular response to double-strand DNA breaks. Rad51 is the human homologue to an Escherichia coli recombination and repair protein, RecA. Additional work in this area suggests a role for BRCA1 not just in response to damaged DNA, but also in the control of normal recombination and thus in genome integrity.
In further evaluating the role of BRCA1 and Rad51 in response to DNA damage, foci containing both proteins were visualized in the cell nucleus and shown to disperse in response to the presence of hydroxyurea, ultraviolet radiation, mitomycin C, and gamma radiation-all agents that induce double-strand breaks in DNA. After dispersal, BRCA1, BARD1, and Rad51 all accumulated in structures known to play a role in repairing damaged DNA.
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Further attempts to identify clinically relevant damaging agents and repair pathways involving BRCA1 showed that doxorubicin and ultraviolet radiation down-regulate BRCA1 expression in a mutant p53 ovarian cancer cell line, suggesting that BRCA1-associated DNA repair may be dependent on p53 function.
Resistance to cis-diamine dichloroplatinum (CDDP) also has been associated with induction of BRCA1 expression, and antisense BRCA1 expression has been associated with CDDP sensitivity. Finally, BRCA1 nullizygous mouse embryonic stem cells have been shown to be hypersensitive to ionizing radiation and hydrogen peroxide and are unable to perform preferential transcription–coupled repair of oxidative DNA damage.115 Taken together, these data suggest that BRCA1 and BRCA2 associate with Rad51 and play a role in response to DNA damage, and they suggest a role for BRCA1 as a key guardian against malignant transformation. Why breast and ovarian cancer are the primary manifestations of BRCA1 mutations, and not widespread malignancy, remains an intriguing mystery.
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BRCA2 Initial progress toward the identification of a second breast cancer susceptibility gene came from a linkage analysis of 22 families with multiple cases of early-onset female breast cancer and at least one case of male breast cancer. Linkage between male breast cancer and polymorphic genetic markers on band 13q12-13 identified the BRCA2 locus. In 1995, the partial sequence of BRCA2117 and six germ-line mutations that truncated the putative BRCA2 protein were published, and shortly thereafter, the complete DNA coding sequence and exonic structure of BRCA2 was published by another collaborative group.
Frequency of BRCA2-Associated Breast Cancer No studies have been performed on BRCA2 in the general population because of the difficulty in screening the entire coding sequence for mutations. However, one study of selected breast cancer patients identified BRCA2 mutations in only 2.7% of women with breast cancer younger than 32 years. This finding suggests that BRCA2 mutations may contribute to fewer cases of early-onset breast cancer than do BRCA1 mutations. This observation is supported by studies identifying BRCA2 mutations in 10% to 20% of families at high risk for breast and ovarian cancer.
In contrast to BRCA1 mutations, in association with which almost all breast cancer appears in women, BRCA2 mutations are associated with a 6% lifetime risk of male breast cancer. Although in absolute terms this represents significantly less cancer risk to men than to women, the relative risk represents a similar 100-fold increase over the general population risk.
The first study to examine this issue after BRCA2 was identified estimated that BRCA2 mutations account for 14% of all male breast cancer. However, these estimates were generated from a cohort heavily weighted toward male breast cancer cases with a significant family history of female breast cancer; thus, 15% was thought to be an overestimate of attributable risk of male breast cancer for an unselected population. The only population-based study of the frequency of BRCA2 mutations in men with breast cancer who were unselected for family history supports this hypothesis. This study demonstrated a 4% prevalence of BRCA2 mutations in men identified from a cancer registry. However, this was a small study, and 30% of the subjects reported a family history of female breast or ovarian cancer, potentially biasing the results of this study as well.
The lack of more conclusive data reflects the fact that familial male breast cancer is very difficult to study because of the rarity of breast cancer in men and the even smaller number of cases that occur in first-degree or second-degree male relatives. In the past 100 years, only 16 families with two or more cases of male breast cancer have been reported in the literature. Analysis of these families suggests that familial male breast cancer is likely to be as heterogeneous as familial female breast cancer. In addition, more than one-half of these families with male breast cancer include first-degree relatives with other types of cancers, such as oropharyngeal, ovarian, and female breast cancer. Nine of the families for which data were available reported cases of female breast cancer.
In these families, average age at diagnosis of breast cancer in female relatives was 46 years; this is considerably younger than the average age at diagnosis in the general population (57 years) and is consistent with the findings in studies of familial breast cancer in females. Thus, the data from families with combined male and female breast cancer argue strongly for the existence of at least one additional male breast cancer susceptibility gene. One potential candidate, a germ-line mutation in the androgen receptor gene, has been identified in two families to date, supporting the belief that this is a very rare cause of inherited male breast cancer. More work is needed to further clarify the role of BRCA2 and other genes in susceptibility to male breast cancer; this currently remains an area of intensive study. {pagebreak}
Other Types of Cancer Associated with Mutations in BRCA2 BRCA2 mutations are associated with elevated risks for the development of a variety of other cancers. Higher than expected rates of prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, basal cell carcinoma, fallopian tube tumors, and bladder carcinoma have been reported in association with BRCA2 mutations. However, the full spectrum of cancers associated with BRCA2 remains poorly defined.
One approach to defining the risk of other cancers associated with BRCA1 and BRCA2 mutations is the study of women with multiple primary cancers. In the initial report of such a study, 63 women with multiple primary cancers (of which one was breast cancer) were analyzed for BRCA1 and BRCA2 mutations. Seven (17.9%) of the 39 women with breast cancer and a nonovarian second primary cancer had either a BRCA1 or BRCA2 mutation. Second cancers included two cases of unspecified skin cancers; a single case each of uterine leiomyosarcoma, brain cancer, bladder cancer, leukemia;and both thyroid and colon cancer in one proband. Overall, 29 (46%)of 63 families screened had a mutation in either the BRCA1 or BRCA2 gene. These data suggest that cancer susceptibility in general is increased in carriers of these mutations.
Pathobiology of BRCA2-Associated Breast Cancer One study found that, unlike BRCA1-associated tumors, BRCA2-associated tumors are similar to sporadic tumors in the number of mitoses, degree of pleomorphism, and proportion classified as medullary. In this study, the proportion of tubular carcinomas was significantly lower in the BRCA2-associated group than the sporadic group, whereas rates of invasive ductal carcinoma, invasive lobular carcinoma, and ductal carcinoma in situ were no different in the BRCA2-associated and the sporadic groups.
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However, other researchers who compared 85 cases presumed to be BRCA2-related (familial, BRCA1 negative) with 90 BRCA1-confirmed cases and 187 sporadic cases found that the BRCA2 group had a significantly greater proportion of tubular cancers. Little is known yet about the natural history of breast cancer due to BRCA2 mutations; prospective longitudinal studies are under way to attempt to address this question.
BRCA2 Gene Structure and Mutation Spectrum The coding region of BRCA2 is 11.2 kb in length and is comprised of 26 exons. This is approximately twice as large as BRCA1, itself one of the largest genes isolated to date. This size adds to the difficulties associated with mutation analysis in both the clinical and research settings. Unlike BRCA1, the genomic region of BRCA2 is not rich in repetitive elements, and genomic deletions as the underlying basis of disease-associated germ-line BRCA2 mutations have not been described.
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Like BRCA1, BRCA2 is expressed in most tissues at very low levels, with higher expression in testis and thymus. BRCA2 cDNA has no significant homology to any previously described gene, and the protein contains no previously defined functional domains. However, eight copies of a 30- to 80-amino-acid repeat (BRC repeat) are encoded by exon 11. The functional significance of the conserved repeats remains controversial, but at least some of the repeats form the binding site for the interaction between BRCA2 and Rad51.
More than 250 BRCA2 mutations have been identified to date. A tabulated list of these mutations can be found on the BIC website. Interestingly, several simiarities with BRCA1 are apparent. First, BRCA2 mutations span the entire coding region of the gene, adding little information on important functional regions and making mutation screening difficult in this very large gene. No mutation hot spots have been detected. Second, most mutations reported to date are truncating mutations, again adding little in the way of clues for defining functional regions. Finally, as with BRCA1, few mutations have been identified in the BRCA2 gene in sporadic breast or ovarian cancers, suggesting that mutations in coding regions in BRCA2 do not play a role in sporadic breast cancer pathogenesis.
Several founder mutations have been identified in the BRCA2 gene in specific populations. As discussed previously, the 6174delT mutation has been found in the Ashkenazi Jewish population at a prevalence of 1.2%, with more than 2.5% of the entire Ashkenazi Jewish population now thought to carry one of three specific BRCA1 and BRCA2 mutations. No cases of patients carrying two mutated copies of BRCA2 have been reported, which suggests that this combination may be incompatible with life. However, several groups have reported patients found to have one BRCA1 mutation and one BRCA2 mutation (unpublished data, Breast Cancer Linkage Consortium meeting, 1997).{pagebreak}
Studies of the BRCA2 gene in the Icelandic population have determined that a founder effect also exists in that population; the majority of high-risk breast and ovarian cancer patients share a common haplotype flanking BRCA2, which is associated with 999del5 mutation.55 Research also has shown that 8.5% of breast cancer patients, 7.9% of ovarian cancer patients, and 2.7% of prostate cancer patients under 65 years of age in Iceland carry the 999del5 mutation. Finally, founder mutations have been identified in Finland. These include the 9346-2A>G mutation and the 999del5 mutation, which suggests that individuals from Finland might have introduced this mutation into Iceland. Analysis of haplotypes associated with nine recurrent mutations in BRCA2 suggested that all are ancestral, as no evidence was found of multiple origins of common BRCA2 mutations.
Biological Function of BRCA2 The biological function of the BRCA2 gene is not yet well understood, but like BRCA1, it is thought to play a role in DNA damage response, possibly within some of the same pathways. Importantly, BRCA1 and BRCA2 are now known to be associated with one another, a finding that confirms their function in the same cellular pathways. The regions of the gene that appear to be very similar in human BRCA2 and murine Brca2 have been studied by several groups. Interestingly, both Brca1 and Brca2 are expressed in the mouse mammary gland concurrently, with the steady-state levels of their respective messenger RNAs both up-regulated during pregnancy. Like BRCA1, BRCA2 has been localized to the cell nucleus.
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BRCA2 and Transcription An N-terminal transactivation domain, consisting of repression and activation components, has been identified in BRCA2. Unlike BRCA1, however, BRCA2 has not been shown to interact with the RNA polymerase II holoenzyme, although studies have demonstrated that BRCA2 interacts with both BRCA1 and RAD51. BRCA2 does bind directly to RAD51, which in turn has been shown to bind to both p53 and the RNA polymerase II holoenzyme. These observations suggest a possible role for BRCA2 in regulation of transcription, although no direct BRCA2-DNA interactions have yet been reported.
BRCA2 and Development BRCA2 hybridization using mouse embryonic tissue showed widespread expression in many developing tissues, suggesting that BRCA2 may play a role in tissue development. Sharan et al. produced the first Brca2 nullizygous mice.
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The null allele resulted in embryonic lethality at day 8.5 of embryogenesis, the same time that Brca2 expression levels became detectable in the normal animals. Heterozygous mice were healthy and fertile. Suzuki et al. also demonstrated that embryos lacking functional Brca2 die before day 9.5 of development. The death of the Brca2 nullizygous mice during early postimplantation suggests a role for Brca2 in cellular proliferation and development by Sharan and Suzuki.
The mouse models described carried Brca2 mutations that truncated the protein in the region encoded by exon 10 or early exon 11, eliminating the BRC repeats. However, viable mice with two copies of truncated Brca2 have been generated by two groups. In these animals, truncation of Brca2 occurred in regions more toward the 3' portion of the gene than in previous models. The allele described by Connor et al. contained seven of the eight BRC repeats. The Friedman et al. mice retained three of the four BRC repeats, were sickly and growth retarded, but survived to adulthood. Some developed thymic lymphomas. Interestingly, these mice also had a marked defect in chromosomal segregation;cultured fibroblasts demonstrated generation of bizarre chromosomal forms in only three to four passages. These data provide support for the hypothesis that BRCA2 is important in recombination, as suggested by high expression levels in the thymus and testes. {pagebreak}
BRCA2 and Cell Cycling Shortly after the identification of BRCA2, the gene was shown to be regulated in a cell cycle–dependent manner and to be associated with cellular proliferation. Levels of BRCA2 are low in the G1 phase of the cell cycle but increase as the cell approaches the G1-S boundary. The highest levels of BRCA2 are detected in S phase, as with BRCA1. The cell cycle kinetics of BRCA2 appear to be very similar to the kinetics of BRCA1; this suggests that these proteins may be coordinately regulated, consistent with the finding that they interact with each other.
BRCA2 and Response to DNA Damage Several experiments have supported the hypothesis that BRCA2 plays a role in double-strand break repair. First, Morimatsu et al. successfully identified RAD51 as a binding partner of mouse Brca2. Subsequently, Hasty and other researchers demonstrated that Rad51 binds to human BRCA2 (residues 3196-3232) Rad51 is known to be critical for mitotic and meiotic recombination and double-strand DNA break repair. These amino acids are 95% conserved between mouse and human BRCA2, suggesting that the domain is important for BRCA2 function.
Further evidence supporting a role for BRCA2 in DNA damage repair has been provided by studies of Brca2-mutant embryos, which display hypersensitivity to ionizing radiation. After mutant embryos were exposed to gamma radiation, the inner cell mass outgrowth of these embryos was completely ablated, an effect not seen in heterozygous or normal embryos. Mouse embryo fibroblasts from viable Brca2 homozygous mutant animals with mutations toward the 3' portion of the BRC repeats also are hypersensitive to agents that create double-strand DNA breaks.
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Studies in a pancreatic cancer cell line (CAPAN-1) with both copies of BRCA2 deleted have shown that the lack of these repeats leads to hypersensitivity to methyl methanesulfonate treatment. This sensitivity persists with the introduction of BRCA2 containing mutated BRC repeats but is reversed by the introduction of wild-type BRCA2. In addition, CAPAN-1 cells were demonstrated to be highly sensitive to drugs inducing double-strand DNA breaks, such as etoposide, and to ionizing radiation.
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Tumors in mice derived from CAPAN-1 cell implantation also demonstrated hypersensitivity to radiation and mitoxantrone; this finding suggests that irradiation or treatment with DNA-damaging agents may be a useful method for elimination of BRCA2-null cells-a suggestion with clear potential clinical significance for women with BRCA2-related breast cancer.
Angela DeMichele and Barbara L. Weber
A. DeMichele: Departments of Medicine and Epidemiology, The University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania B. L. Weber: Departments of Medicine and Genetics, Breast Cancer Program, Population Science and Cancer Control, The University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania
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