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    A Tumor-Based Analysis of Uncharacterized DNA Variants in BRCA1/2 Focusing on Undertested Populations

    Scientific Abstract:
    While genetic testing for BRCA1 and BRCA2 has now become part of standard clinical practice, testing often detects uncharacterized variants (UV) in DNA sequence that are of unknown clinical significance. As clinical research has provided better information regarding management of patients with clearly deleterious mutations, the threshold for genetic testing has become more liberalized and the extent of testing greater. Unfortunately, for undertested and underserved ethnic populations, the paucity of knowledge regarding normal polymorphic sequence variation makes the likelihood of finding a UV particularly high. For example, patients of Western European descent have UV results in 12% of tests, but for patients of African descent testing gives UV results in 40% of patients. UV results can compound the complexity of already challenging genetic counseling sessions. While several approaches have been taken to clarify UV results, such as extended testing within families carrying these variants and assays testing BRCA1 function, to date these approaches have been unsatisfactory. Further research clarifying the significance of these genetic variants is therefore of particular importance for undertested/underserved minority populations. Since BRCA1 and 2 are tumor suppressor genes, the loss of function of the wild-type allele is generally an obligate step in tumorigenesis. Therefore, we propose taking a tumor-based approach to the analysis of UV’s. The primary aim of the study will be clarifying the significance of variants by comparing the UV with the loss of normal BRCA1/2 in the associated tumor. Three groups of BRCA1/2 tested breast cancer patients will be identified: negative test result; clearly deleterious mutation; and UV. The paraffin embedded tumor blocks from these patients will be collected and assayed for loss of normal BRCA 1 or 2 function using three methods: standard loss of heterozygosity assays; promoter methylation assays; and fluorescence in situ hybridization (FISH). The negative and positive mutation groups will serve as controls for the results of the assays. While no one UV will be present in numbers adequate for individual statistical analysis, evidence of normal allele loss of BRCA1 or 2 in conjunction with the UV will provide corroborating evidence for a role in carcinogenesis. As secondary aims, the tumor-based assay results will be combined with categorization of the UVs to refine the results and look for more subtle effects. Each UV will be classified for the probability of significance using several approaches. First, a Bayesian statistical analysis of the pedigree will be performed to determine the likelihood that the UV is deleterious. Second, coding sequence changes will be carefully analyzed through evolution for evidence of highly conserved residues that may be altered in the variants. As new information regarding the crystal structure and function of BRCA1 and 2 is obtained, new classification schemes will also be employed. The correlation between the tumor-based assays and these classifications systems will allow greater accuracy in defining the functional significance of the UVs, potentially to a clinically useful degree. The rates of normal allele loss will also be compared between the three groups by subclassifying the UVs using the above approaches. With this approach, evidence for low or moderated penetrance effects for these changes may be demonstrated. While these efforts will not be limited to specific underserved or ethnic groups, the information obtained will be most valuable to those groups as BRCA1/2 testing is extended further into ethnically diverse and underserved populations. To assure representation of these groups, we will team with a community-based organization that provides financial assistance for genetic testing to underserved patients. This collaboration as well as other ongoing outreach efforts will help us focus on the identification and accrual of patients from underserved and undertested populations.

    Lay Abstract: