Key words: BRCA2 - breast cancer – DNA repair – genetic instability – familial cancer – modifier gene – hereditary cancer syndrome – mutation – chromosome aberration – growth control – RAD51 – 9p – transcription – CRAZY 9 – DSB – double stranded break – Li-Fraumeni syndrome – Cowden syndrome – Muir-Torre syndrome – Ataxia telangiectasia – Peutz-Jegher syndrome

Abstract
The identification of the breast cancer susceptibility genes BRCA1 and BRCA2 a few years ago has been greeted with great excitement and has raised hopes that they might illuminate the common mechanisms of this disease. Today we have to recognise that these expectations remain unfulfilled. Mutations in BRCA1 and BRCA2 account only for a relatively small proportion of breast cancers, even within the group of familiar clusters, they seem to be virtually non-existing in sporadic breast cancers. A substantial proportion of familiar breast cancer clusters has failed to provide evidence for an association with mutations in either BRCA1 or BRCA2, thus we have to look forward to the identification of additional breast cancer susceptibility genes. What has been most disappointing is that the mutation status of BRCA1/2 can provide only limited information for cancer risk. Initial assessments had indicated a risk of close to 90% for mutation carriers to develop breast cancer until age 75 – a value that turned out to be restricted to high-risk families in which the BRCA1 and BRCA2 genes had been genomically mapped. In unselected clusters the risk appears much lower, some estimates suggest less than 40%. Both BRCA1 and BRCA2 large encode proteins that appear to have a plethora of functions, with a conspicuous association to DNA repair and DNA recombination, and probably transcription activation. Defects in DNA repair can result in cancer predisposition syndromes and are recognized as being instrumental in cancer progression. Central questions have remained unanswered: What is the function of damaged BRCA1 and BRCA2 genes in breast cancer risk? What is the basis of large variations of risk conferred to the patients by identical mutations? How can the predictive value of mutation surveys be increased?

Introduction
The annual incidence of women world wide currently diagnosed with breast cancer is approximately 1 million [1], with one out of twelve woman diseased in Western-Europe and the United States. The mortality rate is approximately 30%, making breast cancer the highest cause for death among women 50 to 55 years of age [2,3] Women with familial history of breast cancer are at highest risk, additional risk factors include a number of environmental challenges such as ionising radiation [4,5] alcohol consumption, estrogen replacement therapy [6,7], early menarche [8], no children [9] and late menopause [10].

Genetic Risk Factors
Conservative estimates of the proportion of breast cancers developing within familial clusters have ranged between 5 and 10%. The best characterized genetic risk factors are represented by germline mutations in BRCA1 on 17q21 [11] and BRCA2 on 13q12 [12]. Initially, the mutation in one of these genes was generally considered to confer high risk of close to 90% (Figure 1). Subsequent surveys of familial breast cancer clusters unselected for high risk soon corrected this view. A study of Ashkenazi Jews with familial background revealed a relative risk of little more than 55% to develop breast cancer until age 75 (Figure 1), and a subsequent study of a comparative population but without familial background showed approximately 35% relative risk for mutation carriers to develop breast cancer. This variability in the penetrance seriously complicates patient counseling [13].

Epidemiological surveys suggest that perhaps the majority of breast cancer developing in familial clusters is not associated with mutations in BRCA1 or BRCA2. Obviously as yet unidentified risk-genes play a significant role, with recent epidemiological data suggesting the proportion of breast cancers developing in familial clusters as high as 25% of all breast cancers (Figure 2). A fraction of familial breast cancer clusters develops as part of rare hereditary cancer syndromes [14].

Peutz-Jegher syndrome
The Peutz-Jegher syndrome is caused by a mutation of the gene STK11 on chromosome 19p13 [15]. Clinical manifestations include punctuated increases of melanin on the lips. Additionally the incidence of benign ovarian tumors, and of cancer of the breast, pancreas and testes. The average age at diagnosis for breast cancer is at 39 years [16].

Cowden syndrome
Cowden syndrome results from mutation of the gene PTEN on chromosome 10q23.3. Mutation carriers have a risk for breast cancer, additional tumors include follicle-cell carcinoma, thyroid cancer, ovarian cancer as well as polyps and cancers of the gastro-intestinal tract. Breast cancer is seen in 20-30% of female mutation carriers. It appears that PTEN is not responsible for high-risk familial clusters unlinked to BRCA1 or BRCA2 [17,18].

Muir-Torre syndrome
Muir-Torre syndrome is caused by mutations in the mismatch repair genes MSH2 (2p22-21) and MLH1 (3p21.3). The clinical feature include cutaneous manifestations of hereditary nonpolypous colorectal cancer (HNPCC). This autosomal dominant syndrome is characterized by a combination of sebaceous gland and malignant visceral tumors [19,16]. Approximately 25% females that carry a mutation in one of these two genes developing breast cancer at an average age of 68 years.

Li-Fraumeni syndrome
Li-Fraumeni syndrome is an autosomal dominant disease that results from mutations of the gene TP53 on 17p13.1. The clinical phenotypes are composed of a number of tumors including sarcomas of the bone and soft tissues, brain tumors and breast cancer at unusually early ages [20]. In case female mutation carriers have not already developed tumors at young age, approximately 50% will develop breast cancer until age 50 years. The life time risk for breast cancer is close to 100%. Analysis of unselected breast cancer patients revealed a frequency of only 1% with TP53 germ line mutation [21,22].

Ataxia telangiectasia
Ataxia telangiectasia (AT) is a recessively inherited disease characterised predominantly by cerebral ataxia and by defects of the immune system. The responsible gene ATM maps to 11q22.3. Homozygous mutation carriers develop Non-Hodgkin Lymphoma at an incidence of almost 100%; additionally, cancers of the ovary and of the breast are frequent [23]. The role of the ATM gene as a risk factor in heterozygous mutation carriers has been discussed controversially. Although relatives of Ataxia telangiectasia patients do have an increase risk for breast cancer [24-26], breast cancer patients below age 40 do not have an increased ATM-mutation frequency [27-30]. This would suggest that mutated ATM either has only low penetrance or is a modifier of other predisposing factors.

Modifier Genes
One of the most interesting aspects of risk conferred by BRCA1 or BRCA2 is that the cancer phenotype resulting from an identical mutation can vary when different familial clusters are looked at (Figure 3). The same mutation can entail low risk or high risk, in case of BRCA2 mutation in one cluster preferentially males can be affected and in another one females; finally, the clinical phenotype in one cluster can be restricted to breast cancer while in another additional cancer types are seen. It is obvious that the principle risk conferred by BRCA1 and BRCA2 can be further modified. Although, the search for such modifier genes is in full swing, their identification will be difficult as they can be expected to be low penetrance genes that in general are difficult to identify.

Functions of the BRCA2 Protein
Cancer-associated genes have been classified into two groups, the so called “gatekeepers” and the “caretaker” [31]. The gatekeepers are represented by the “classical” oncogenes and “tumour suppressorgenes” whose proteins directly are related to cellular growth control. The caretakers were not directly implicated in growth control and alone are not sufficient to a cancer cell, their functional impairment or inactivation results in loss of genomic integrity by deficient DNA repair such that mutations in other genes can occur more frequently. BRCA2 illuminates the point that a single protein can have both caretaker functions by its involvement in DNA repair and gatekeeper function by its role as a transcription factor regulating the expression of other genes [32].

BRCA2 is thought to represent a tumor suppressorgene, which on the cellular level behaves recessively. Tumor cells usually have lost the other, normal allele, in most instances by loss of heterozygosity [33,34].

DNA repair – A caretaker function in genomic integrity
The BRCA2 gene is translated into an 11.5 Kbp mRNA that encodes a 384 KDa protein of 3418 amino acids. The vast majority of mutations are nonsense mutations, which result in premature termination of translation with the consequence of the synthesis of a shorter, C-terminally truncated protein (Figure 4).

Most information about BRCA2 function comes from studies of the murine protein. The nucleotide sequence of the murine BRCA2 gene is 74% similar to the human sequence. On the amino acid level there is 59% identity and 72% similarity [35].

Homozygous BRCA2-/- mice usually die at the end of embryonal development or shortly after birth, although there is a strain-specific difference [36]. This adverse effect seems like a paradoxon in view of the tumor development seen in human breast cancers. Surviving BRCA2-/- mice are smaller than their wild type counterparts, and males show defects in spermatogenesis. Homozygous BRCA2-/- mice are at risk for developing malignant lymphomas, heterozygous BRCA2+/- mice are not tumour prone [36,37]

Homozygous BRCA2-/- are more sensitive to ionising radiation than their wild type counter parts or BRCA2+/- mice [38]), in support of the conjecture that the BRCA2 protein has a role in DNA repair. In line with this, BRCA2-/- mice show much higher rate of DNA double strand breaks than BRCA2+/- mice [36]. The deficiency in DNA repair results in gross chromosomal changes involving translocations and deletions [39], a chromosomal phenotype missing in heterozygotes [38,39].

The BRCA2 protein has an essential role in maintaining chromosome stability through ist participation in recombinational processes, in association with additional well known DNA-repair proteins [40]. The involvement of the human BRCA2 protein in the repair of double stranded breaks (DSB) of DNA is indicated by its cellular co-localisation with RAD51, which is the homolog of the bacterial RecA protein [41,42,35]. The binding sites of RAD51 to BRCA2 have been mapped [41] (Figure 4). BRCA2-/- and Rad51-/- mice exhibit similar hypersensitivity towards ionizing radiation, as if both proteins have similar functions in DNA repair [38,43]. It should also be noted that ScRad51 protein of Saccharomyces cerevisiae and other members of the Rad52 epistasis group participate in DNA repair of DSB, both in mitosis and in meiosis [44,45; for reviews of DSB repair see 46-48]. The observation of a physical association of BRCA2 with BRCA1 [49], as shown by immunoprecipitation, has allowed to lay out a tentative scenario of the interaction of BRCA1 and BRCA2 proteins in the repair double stranded DNA damage. Previous studies had demonstrated hyperphosphorylation of BRCA1 as the result of DNA damage [50,51]. The BRCA1 phosphorylation is mediated by a protein kinase called ATM (mutated in ataxia telangiectasia), which also controls the phosphorylation of other proteins, such as CHK2, NBS1) involved in double strand breaks as well [52-57; reviewed in 58]. During S-phase of the cell cycle the hyperphosphorylated BRCA1 co-localises with BRCA2, RAD51 and BARD1 proteins in nuclear foci [41]. ATM resides in a nuclear complex with BRCA1 and phosphorylates BRCA1 after exposure of cells to gamma-radiation. Phosphorylated BRCA 1 activates homologous recombination in cooperation with BRCA2, RAD51 and other repair proteins. The exact contribution of BRCA2 is unclear as yet. The biochemical connection between BRCA1, BRCA2 and ATM defines a cellular DNA repair pathway that should be deficient in a significant number of breast cancers. Yet, BRCA1 or BRCA2 mutation carriers do have one damaged allele in all of their somatic cells. Why then are they at risk predominantly for breast cancer? Also, in view of the mechanistic link of BRCA1 and BRCA2 with ATM in DSB repair, why then are mutations in ATM predominantly associated with lymphoid malignancies?

Central questions of the role of mutated BRCA2 in breast cancer development remain unanswered. While BRCA2-/- mice have provided clues about the basic roles of the BRCA2 protein in genomic integrity, heterozygote BRCA2+/- mice have failed to provide evidence for any phenotype related to the mutation of one allele. It should be clear, however, that it is the heterozygous BRCA2+/- genotype that confers the cancer risk in humans. This testifies to the fact that mouse models vary in the degree at which they faithfully mimic genetic mechanisms of human disease. Obviously many species-specific factors dictate the susceptibility, the phenotype and the growth pattern of tumors. Could there be some degree of haplo-insufficiency in human heterozygotes? An answer to this question could come from a recent observation that has revealed constitutional hyperinstability of a restricted region of the genome, 9p23-24, in independently ascertained BRCA2 families [59,60]. This instability has been visualized by classical chromosomal fluorescence in situ hybridization (FISH) and involves in most instanes duplications and inversions (Figure 5A). In a fraction of cells translocations and amplifications [61] are detectable (Figure 5B).

These recombinations must involve double strand breaks, and the principle mechanisms are somehow reminiscent of V(D)J recombination events. The apparent lack of instability in other genomic areas is provocative yet not understood so far. The same chromosome is perfectly stable in a wild-type genetic environment of BRCA2 wildtype relatives [60]. This original observation obviously entails a number of questions: What is the molecular basis for the recombinations in 9p23-24? What mechanism dictates the apparent restriction to this genomic region? What might be the contribution of BRCA2 to this regional instability? And if BRCA2 is involved, is it loss of function by disabled repair or gain of function by deregulated recombination, possibly in association with RAD51 and other proteins as well? What is the identity of the genetic material damaged by the recombinations? And finally, what might be the effect on breast cancer risk and phenotype? All these questions are amenable to experimental approaches, the answers should soon be at hand and will possible shed light on the role of BRCA2 in 9p23-24 genomic instability.

Transcriptional regulation – A gatekeeper function controlling cell cycle progression
In contrast to the BRCA1 protein the role of BRCA2 in transcriptional regulation remains largely unexplored. The first indications for BRCA2 being a transcription factor came from the observation that exon 3 (aa23-105) has an amino-acid homology to the transcription factor JUN [62]. This region activates transcription in yeast and mammalian cells. The same domain is subjected to phosporylation in vivo, which is thought to regulate BRCA2 activity. Further indication for a transcriptional activity comes from the observation that BRCA2 could be co-immunoprecipitated with the transcription co-activator P/CAF [p300/cBP-associated factor; 63], protein that has histone-acetylase activity [64]. Because many other known transcription factors also have histone-acetylase activity (GCN5, CBP/p300, TAFII250, SRC-1) it is assumed that chromatin changes by histone-acetylation are a factor in transcriptional regulation. More recent studies also suggest a role of BRCA2 in cell cycle progression [65]. The exact role of BRCA2 in transcription regulation has remained elusive. In particular, it is completely unclear as to how the transcriptional activation by BRCA2 might figure into breast cancer development.

Conclusion
Only a handful of years has gone by since the discovery of the BRCA2 gene – during this time period we had to learn that what initially did appear simple has become complex and difficult to unravel. The initial expectations of BRCA2 mutation screening as a tool for reliable prediction of breast cancer risk have faded away, due to the unpredictable penetrance of the individual mutation. The analysis of the BRCA2 protein has already uncovered two major functions, one in DNA repair, the other in transcriptional regulation. Given the precedence of the tumor suppressor TP53, which so far has been resistant to reveal which of its many functions has a particular role in cancer development, it can be expected that a protein of the size of BRCA2 will be an even more difficult challenge. On the positive side, the study of BRCA2 has rapidly expanded our insights into one element of the complex mechanisms that govern genomic integrity. This is one more example that the motivation to study one of the most common malignancies, breast cancer, can lead to profound knowledge about basic cellular mechanisms. When patiently pursued the further accrual of information about BRCA2 functions is likely to lead to the identification of novel therapeutic tools and to a better assessment of cancer risk in mutation carriers.

Acknowledgments
Work in the authors laboratory is supported by grants from the Deutsche Krebshilfe, the Bundesministerium für Forschung und Technologie, by the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ) and Israeli’s Ministry of Science (MOS), and by core support through the DKFZ.

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Figure legends

Figure 1
Breast cancer risk of women with BRCA mutation until age 75. Estimates for high risk families revealed approximately 87% penetrance [22]. A lifetime risk of 56% was estimated in a population of Ashkenazi volunteers not selcted for high risk [66]. A penetrance of 36% was estimated in an Ashkenazi population without family history [67,68, modified].

Figure 2
Sporadic and inherited forms of breast cancer. The majority of breast cancer cases develops sporadically, an estimated 20% to 25% of cases develops within familial clusters. Identified genetic risk factory include germline mutation in BRCA1 and BRCA2, a fraction of familiar breast cancers develops as part of rare hereditary cancer syndromes. A third BRCA-gene, tentatively referred to as BRCAX, is postulated. The genetic risk factors in the majority of familial breast cancer clusters remain to be determined [from 68, modified].

Figure 3
Epidemiological indicators for the existence of modifier genes. Identical mutations in BRCA2 can result in different cancer phenotypes in different families.

Figure 4
Structure of the BRCA2 protein. Mutations in most instances result in the synthesis of C-terminally truncated proteins lacking the nuclear localization signal and the C-terminal RAD51 interaction site; NLS, nuclear localization signal [details in 60].

Figure 5
CRAZY 9: Hyperinstability in 9p23-24 in normal cells of family members with BRCA2 mutation.
A. Inversions and duplications in two family members. Instability appears restricted to 9p23-24. The different cells of the same individual differ with respect to distal 9p, often both homologues are affected and differ from each other [in part from 60, modified].
B. Occasionally, translocations and low level amplifications of 9p23-24 are seen.

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