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Identification of Novel Genetic Factors Contributing to Clinical Phenotypes in Large Families with BRCA1 and BRCA2 Mutations
Background: Although functional roles for BRCA1 and BRCA2 in DNA repair and transcriptional regulation have been described, the mechanism by which these genes drive tumor progression is not clear. For example, BRCA1 and BRCA2 are widely expressed in various tissue types, however, the predominant site of tumor development is breast or ovary. One model postulates that the estrogen-rich environment of the female reproductive organs, especially during puberty and pregnancy, allow cells with dysfunctional BRCA1/2 genes to escape apoptosis. The choice of which organs- breast, ovary, breast and ovary- are affected, however, is not clear. In addition, families with inherited BRCA1/2 mutations are characterized by incomplete penetrance, with 15-20% of mutation carriers never developing breast or ovarian cancer.
Objective/Hypothesis: The objective of this research is to perform global profiling on DNA from members of large families with breast cancer and known BRCA1/2 mutations to identify genetic factors that 1) influence the ability of cells to become tumorigenic and 2) the site of primary tumor development. We believe, based on preliminary data from one large family, that genes and genetic relationships exist that promote or confer a protective advantage within the background of BRCA1/2 mutations.
Specific Aims: 1) to collect blood and tumor samples from nine large (>500 persons) families with breast cancer and known BRCA1/2 mutations for use in CGH and LOH analysis, 2) to perform comparative genomic hybridization (CGH) analysis of genomic DNA from family members with breast and/or ovarian tumors, other types of tumors, or no tumors to identify heritable genetic factors that promote or confer a protective advantage from overall tumor development, 3) to perform CGH and loss of heterozygosity (LOH) analysis on breast and/or ovarian tumor specimens that develop unique sets of genomic changes that may determine the site of tumor development, and 4) to use statistical analysis to identify genetic changes that show significant associations with biological characteristics and clinical outcome of individuals with breast and ovarian cancer.
Study Design: Fresh blood samples will be redrawn from family members who have already consented to participate in family breast cancer genetic studies. Genomic DNA will be extracted from each blood sample and used as the test probe in CGH analysis to identify genetic abnormalities that are inherited either throughout each family or within branches of large families with specific tumor phenotypes. Archival tumor specimens will be collected from family members who have had breast and/or ovarian cancer. DNA will be extracted from these specimens, in the case of small tumors, after laser-assisted microdissection. These DNAs will be used in CGH and LOH analysis to identify those changes that characterize the specific phenotypes, including primary tumor site of each tumor.
Potential Outcomes and Benefits of the Research: By performing CGH on DNA from blood samples from large families with breast cancer, we expect to identify genetic factors that work in conjunction with BRCA1/2 to either promote tumor development or to confer a protective advantage in individuals genetically predisposed to breast cancer. In addition, by looking at genetic changes in the tumor, we will identify genetic factors that are not inherited but develop within and contribute to the tumor site, phenotype and biological characteristics of individual tumors. Although not within the scope of this three-year proposed work, we expect that these genetic modifiers, as with BRCA1/2 will also contribute to the development of sporadic breast cancers. These genes will provide novel tools for risk assessment and early diagnoses and will serve as novel molecular targets for drug and gene therapies.
Breast cancer is the most common cancer in women in Western countries and causes more deaths than any other cancer except lung. The incidence of breast cancer in the United States has risen by approximately 1.2% per year since 1930; today, one in eight American women will develop breast cancer during her lifetime. Development of breast cancer has been associated with a number of factors, both environmental and genetic. The identification of two genes linked to breast cancer in families, BRCA1 and BRCA2, provided new tools for the early diagnosis of women who have inherited a BRCA1 or BRCA2 mutation, and are therefore at increased risk of developing breast and ovarian cancer. Fifteen to twenty percent of women who inherit a BRCA1 or BRCA2 mutation, however, never develop breast or ovarian cancer, thus BRCA1/2 mutations are not solely responsible for tumor development. In addition, women with BRCA1 or BRCA2 germ-line mutations may develop breast, ovarian, or breast and ovarian cancer. These differences in the site and overall risk of tumor development are likely influenced by additional genes.
In this study, we propose to look for DNA alterations in family members, affected and unaffected, from nine large families with BRCA1 or BRCA2 mutations and a prevalence of cancer breast. Identification of these families, most of which have pedigree information on over 500 people, is complete, and the type of the BRCA1/2 mutation is known. By comparing genetic changes between individuals with cancer to those without, we can identify genes that influence the ability of BRCA1/2 mutations to promote overall tumor development. By looking at changes that develop within breast tumors but not ovarian tumors (and vice versa), we can also identify genes that determine where a tumor develops. We will use comparative genomic hybridization (CGH) which is a chip-based technology that allows the detection of DNA abnormalities at ~300 chromosomal regions simultaneously, without a priori knowledge of the underlying genetic defect(s) to detect large DNA alterations. CGH results will be complemented by loss of heterozygosity (LOH) analysis, which detects chromosomal loss using defined DNA sequences that are known to vary widely in the population.
Preliminary data has been generated from a subset of patients from one large family with a known BRCA1 mutation. Using CGH, we have detected amplification of a small region of chromosome 2 which contains the MSH2 gene, a gene that is involved in repairing DNA damage and is mutated in a large proportion of individuals with hereditary nonpolyposis colorectal cancer. This amplification was seen in individuals with a number of types of cancer including breast, ovarian, cervix, and rectum. How amplification of this gene might lead to increased risk of tumor development is not clear. Additional studies on DNA samples from this family will allow us to make associations between amplification of this region and tumor development. We also expect to identify additional genetic changes in this family and in the other eight large families with breast cancer that contribute to overall tumor development.
While CGH will be performed on the DNA of members of large families, we will also use CGH and LOH analysis to examine genetic changes found within breast and ovarian tumors of affected family members. The majority of changes within a tumor is specific to that tumor and need not match those changes seen in tumors from other family members or even from separate tumors within the same individual. We believe that these tumor-specific changes will allow us to identify genes that, in conjunction with BRCA1 or BRCA2 mutations, influence where a primary tumor develops. In addition, based on previous work in our laboratory examining chromosomal changes seen in sporadic breast cancers, we believe that we will be able to associate changes at particular chromosomal regions with tumor characteristics, such as the risk of metastasis, recurrence and mortality. These factors, although working against a background of germ-line BRCA1 or BRCA2 mutations, will likely influence tumor development in sporadic cancers, and thus will serve as new diagnostic tools and molecular targets for gene and clinical therapeutics.