BRCA2 : A genetic risk factor
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?
Epidemiology of breast cancer
The annual incidence of women world wide currently diagnosed with breast cancer is approximately 1 million , 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. Women with familial history of breast cancer are at highest risk, additional risk factors include a number of environmental challenges such as ionising radiation alcohol consumption, estrogen replacement therapy, early menarche, no children and late menopause.
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 and BRCA2 on 13q12. 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 Ashenazi 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.
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, like Peutz-Jegher syndrome, Cowden syndrome, Muir-Torre syndrome, Li-Fraumeni syndrome, and Ataxia telangiectasia (AT) (reviewed in Schwab et al., 2002).
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 preferencially 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”. 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. In our laboratory, we are pursuing BRCA2 protein functions with the perspective of identifying its possible caretaker role.
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.
DNA repair – A caretaker function for genomic integrity
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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. Homozygous Brca2-/- mice usually die at the end of embryonal development or shortly after birth, although there is a strain-specific difference. This adverse effect seems like a paradoxon in view of the tumor development seen in human breast cancers. Brca2-/-. Surviving Brca2-/- 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. Homozygous Brca2-/- are more sensitive to ionising radiation than their wild type counter parts or Brca2+/- mice, indicating loss of DNA repair. In line with this, Brca2-/- mice show much higher rate of DNA double strand breaks than Brca2+/- mice. The deficiency in DNA repair results in gross chromosomal changes involving translocations and deletions, a chromosomal phenotype missing in heterozygotes.
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. However, 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 our recent observation that has revealed constitutional hyperinstability of a restricted region of the genome, 9p23-24, in independently ascertained BRCA2 families . This instability has been visualized by classical chromosomal fluorescence in situ hybridization (FISH) and involves in most instanes duplications and inversions (Figure 4). In a fraction of cells translocations and amplifications are detectable. 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 and not yet understood so far. The same chromosome is perfectly stable in a wild-type genetic environment of BRCA2 wildtype relatives.
This original observation obviously entails a number of questions that have found their way into ongoing experimental approaches: What is the molecular basis for the CRAZY 9 recombinations in 9p23-24? What mechanism dictates the apparent restriction to this genomic region? What might be the contribution of the BRCA2 protein to this regional instability? And if BRCA2 protein 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.
Perspectives
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.