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    Research Grants Awarded

    A Novel Genomic Approach To Finding New Genes For Inherited Breast Cancer:Rare Structural Variants In High-Risk Families

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    (1) RATIONALE. As genes for inherited breast cancer are increasingly well characterized, it is clear that among their mutations are genomic duplications or deletions ranging in size from a few kb to more than 100 kb. Among known tumor suppressor genes - including BRCA1, BRCA2, and CHEK2 - between 3% and 20% of all inherited mutations are genomic deletions or duplications. These so-called genomic rearrangements are equivalent to structural variants (SVs), albeit rare ones. Like other structural variants, those in tumor suppressor genes can be detected in human genomic DNA with high-density CGH microarray technology. (2) HYPOTHESIS. We suggest that novel genes for inherited breast cancer can be identified via a subset of their mutations detectable with high density CGH microarrays. Our goal is to find novel breast cancer genes by identifying rare structural variants in constitutional DNA of breast cancer patients from high-risk families, then validate the genes by identifying other mutations in unrelated breast cancer patients. If our hypothesis is correct, we will identify several novel candidate genes for inherited breast cancer, each harbouring multiple mutations (some small and some large) in our cohort of more than 1000 breast cancer families. (3) RESEARCH AIMS AND DESIGN. Our project involves five aims. Aim 1. We will use NimbleGen CGH microarrays, specifically the 2.1-million feature HD2 platform, to screen constitutional genomic DNA from a discovery series of 300 breast cancer patients. These 300 patients are probands of families with four or more cases of breast cancer, but with no mutations in BRCA1, BRCA2, or any other known breast cancer gene. (We have screened them all.) We will identify all structural variants (SVs) in the DNA of each proband. Aim 2. We will filter the SVs detected in each patient to identify those that are rare; that is, specific to one or more probands and not found in the general population. We will exclude all common SVs using databases developed this year that indicate the exact locales of known SVs genome-wide, based on several thousand controls evaluated with various microarray platforms. Aim 3. We will verify each putative rare SV by quantitative PCR. We will sequence each validated rare SV that impacts a gene(s) to identify its exact breakpoints. We will determine if a gene or genes are deleted, duplicated, or disrupted by the SV. In the past six months, we have carried out this analysis for more than 200 individuals in our lab and have developed an efficient pipeline for breakpoint and event characterization. Aim 4. We will evaluate each SV in the family of the proband in whom it was discovered. We will determine if the SV is co-inherited with breast cancer in the family; that is, whether other breast cancer patients in the family inherited the proband?s SV. This step is possible because our participants in each family include not only probands, but also virtually all surviving relatives with breast cancer and the adult children of women who have died of the illness. Aim 5. We will define as candidates those genes that are deleted, duplicated, or disrupted by a rare SV co-inherited with breast cancer in a family. We will sequence these candidate genes in our validation series of 1000 probands. The goal of this step is to search for point mutations and small frameshifts in the same gene(s) that were disrupted by the SV in one family. The identification of multiple mutations of functional consequence is a hallmark of all known genes for inherited breast cancer, and the strongest proof of existence of a new breast cancer gene. With our present cohort of families, and with high-density microarrays currently in use in our lab, we could have detected BRCA1 and BRCA2 and CHEK2 with this approach, knowing nothing of these genes a priori. It is irresistible to apply the approach to search for new genes for inherited breast cancer. (4) UNIQUE CONTRIBUTION TO THE BREAST CANCER PROBLEM. The application of genome-wide screening for structural mutations to the problem of inherited breast cancer is novel. Genomic screening for SVs has been successfully applied to the characterization of somatic mutations in cancers and to identification of inherited mutations for neurodevelopmental traits. It is now possible to apply the approach to identification of germline mutations in cancer, because technology has become available that is sufficiently sensitive to pick up duplications and deletions of sizes typically seen among inherited genomic rearrangements. Our laboratory is among the first to have this technology in hand. To apply this approach to inherited breast cancer requires the involvement of a large number of families at high-risk of breast cancer. Our participating families are ideally suited to this effort and have provided genealogies, clinical information, DNA, RNA, and blood for us to generate immortalized lymphoblast cell lines. We remain in contact with virtually all families, now often over three generations. All probands have been fully sequenced and screened for genomic rearrangements in BRCA1, BRCA2, and other breast cancer genes. For this project, we will work with the approximately 1000 probands and their families who do NOT carry a mutation in any known breast cancer gene. We believe the integration of this new technology with the genetic material from our large cohort of participating families is the best opportunity in decades to identify novel genes for inherited breast cancer.

    Lay Abstract:
    STUDY HYPOTHESIS. The goal of this project is to discover new genes for inherited breast cancer. We propose to identify in DNA of familial breast cancer patients a special class of mutations that are detectable using genomic screening tools that became available this year. This goal is based on a three-part hypothesis. (1) We suggest that mutations in as-yet-unknown genes are responsible for inherited breast cancer in patients with severe family history who do not carry mutations in BRCA1 or BRCA2 or other known breast cancer genes. (2) We suggest that each of these as-yet-unknown breast cancer genes carries many different cancer-associated mutations. This has proven true for all breast cancer genes discovered so far. (3) We suggest that some of these diverse mutations will be detectable by newly developed technologies. These distinctive mutations are known as structural variants (SVs) and have been identified, by us and by others, in known breast cancer genes. We suggest that they exist in as-yet-unknown breast cancer genes as well. Based on this hypothesis, we propose to use newly developed molecular screening technologies to search for big mutations in the normal genomic DNA of breast cancer patients whose severe family histories remain unexplained. That is, we will find new genes by finding their most flagrant mutations. After finding each such mutation in DNA from one of our participants, we will test whether other women with breast cancer in the same family inherited the same mutation. A mutation co-inherited with breast cancer in a family is an excellent clue that the gene harbouring it predisposes to breast cancer. We will test this possibility by sequencing normal genomic DNA from hundreds of other familial breast cancer patients for other (presumably smaller) mutations in the same gene. Multiple different, deleterious, cancer-associated mutations in a gene demonstrate its role in inherited breast cancer. HOW THE PROJECT UNIQUELY ADVANCES OUR UNDERSTANDING OF BREAST CANCER AND LEADS TO REDUCTIONS IN INCIDENCE AND MORTALITY WITHIN THE NEXT DECADE. The discoveries of BRCA1 and BRCA2 have changed breast cancer prevention and treatment in ways I could not have imagined when I began the search 33 years ago for BRCA1. It is now possible for women to learn if they carry cancer-predisposing mutations in BRCA1 and BRCA2, and if so, to take steps to prevent breast and ovarian cancer. The integration of genetic testing for mutations in BRCA1 and BRCA2 is now an important part of clinical practice. Consensus clinical guidelines have been developed for medical follow-up of women who learn that they carry mutations in these genes. The integration of genetics into oncology has led to prevention of thousands of breast and ovarian cancers and saved the lives of many women. In a complementary way, genetic knowledge can bring tremendous relief to women whose mothers or sisters carried mutations and died of breast cancer, but who themselves did not inherit the mutation. It is now clear that there are at least ten genes for inherited breast cancer. BRCA1 and BRCA2 are the best known, conferring greater than 10-fold increased risks of early-onset breast cancer and of ovarian cancer, and responsible for 5% of all breast cancer in the US and 10% of breast cancer in the US diagnosed before age 45. Nearly all other inherited mutations occur in genes that confer 2- to 4-fold increased risks of breast cancer; that is lifetime risks of 20% to 50%. These genes include CHEK2, PALB2, ATM, BRIP1/FANCJ, RAD50, and NBS1. The formulation of clinical recommendations for care of women with mutations in these genes is presently under intense study. These recommendations are likely to include increased surveillance, including tools such as MRI that are not offered universally. Both our statistical modeling studies and our direct observation of families severely affected with breast cancer suggest that many genes for breast cancer predisposition remain to be found. This is particularly true for families with multiple cases of breast cancer in women but no ovarian cancer or male breast cancer. We estimate that 60% of such families remain unresolved. One of our greatest frustrations is to discover that a family severely affected with breast cancer carries no mutation in BRCA1, BRCA2, or any of the known breast cancer genes. In our studies of extended families at high risk of breast cancer, and their more than 5000 relatives, we have confronted this frustration many hundreds of times. This proposal is the result. THE IMPORTANCE OF THE RESEARCH TO PATIENTS WITH BREAST CANCER. Identifying genes that explain breast cancer in still unresolved families has both immediate and long-term value. The results will be of immediate use for women in the affected families. In addition, the identification of genes responsible for inherited breast cancer reveals pathways critical to breast cancer development generally. The components of those pathways are natural targets for therapeutic intervention.