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    Does BRCA2 Justify Prophylactic Mastectomy? Allele-Specific Methylation Markers of BRCA2 v. AS3 Losses in Breast Cancer

    Scientific Abstract:
    TITLE: Does BRCA2 justify prophylactic mastectomy? Allele-specific methylation markers of BRCA2 v. AS3 losses in breast cancer. BACKGROUND. Germ line mutations in the BRCA1 and 2 genes show strong correlation with familial breast cancer. In contrast, in the non-familial (sporadic) form of the disease (97% of all), somatic mutations in BRCA2 are conspicuously rare and present only a moderate increase in cancer risk. Genomic losses in the BRCA2 area, however, showed significant correlation with cancer and linked to the D13S171 microsatellite marker. Surprisingly, in spite of the genomic losses, BRCA2 expression remained intact in many cancers, indicating that the deletions targeted another gene in the area. The finding predicted a tumor suppressor next to BRCA2 and prompted us to re-investigate the BRCA2 genomic region. Our data established that D13S171 is misannotated in the databases. The correct marker position is separated by three genes and 0.3 million base pairs from BRCA2 and ruled out that D13S171 is directly linked to BRCA2 (Am. J. Hum.Genet., 2001). D13S171, however, links directly and maps physically to another gene, AS3 (or APRIN) (J.St.Bioc.Mol.Biol, 1999). We showed that AS3 shares features with the predicted suppressor, it arrests breast cancer cell proliferation (PNAS, 2000) and involved in terminal differentiation and growth arrest (Endocrinology, 2002). We found that the promoters of both AS3 and BRCA2 had CpG islands, the hallmarks of methylation-based gene silencing (Proc.Am.Assoc Canc.Res. 2002). OBJECTIVE/HYPOTHESIS. The above results challenge the role of BRCA2 in sporadic breast cancer and focus the attention on a new gene, AS3. Our hypothesis is that the BRCA2-based cancer risk estimates have been flawed by the marker misannotation, with potentially tragic consequences in patient decisions on breast cancer prevention strategies. Tens of thousands of women are tested every year for BRCA2 mutations and thousands undergo prophylactic mastectomy, with emotional and physical consequences for life. At the same time, the gene behind the marker, AS3, remains unnoticed. The objective of the proposed studies is, therefore, to clarify the real contributions of BRCA2 and AS3 in sporadic breast cancer. Genetic changes in BRCA2 are insignificant in this group, but mutations are not the only mechanism in functional losses. We propose that the CpG islands we identified present an alternative option, to analyze epigenetic changes. To investigate epigenetic silencing of BRCA2 and AS3 and to establish their individual contributions to breast cancer, we propose the following Aims. SPECIFIC AIM 1. To establish and optimize the methods for DNA methylation studies in the BRCA2 and AS3 promoters and to work out a novel allele-specific and methylation-specific PCR (MSP) method. SPECIFIC AIM 2. To study the methylation status of breast cancer cell lines in vitro and to establish correlations with phenotypes and methylation status. SPECIFIC AIM 3. To study BRCA2 and AS3 silencing in human breast cancer samples. To establish laser capture microdissection to isolate DNA from commercially available breast cancer tissue microarrays and to perform allele-specific MSP. STUDY DESIGN. In Specific Aim 1, commercially available and tissue culture DNA samples will be used to develop and optimize a novel methodology in the detection of allele-specific methylation. We have the complete SNP and CpG maps and sequences of both genes and we will try various primers and techniques. In samples where insufficient haplotype differences limit the application of the method, we are well-versed to perform regular methylation-specific PCRs. In Specific Aim 2, using the optimized MSP on various breast cancer cell lines, the goal is to investigate if BRCA2 and AS3 methylation patterns correlate with the phenotypes. In Specific Aim 3, we will use breast cancer tissue arrays and we will optimize laser capture microdissection and MSP methodologies. The proposed pilot experiments will establish the techniques and generate data for large-scale clinical studies. POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH. Thousands of women undergo prophylactic mastectomy based on their BRCA2 mutation status. Our data challenge the role of BRCA2 in the majority of cases and focus the attention on AS3, a new suppressor candidate gene in breast cancer. We will establish novel prognostic markers to detect epigenetic gene silencing from needle biopsy samples. Our data will help to dissect the roles of the two genes in breast cancer and will establish new preventive strategies based on correct genetic and epigenetic platforms. Ultimately, our goal is to save thousands of women from the adverse consequences of a potentially unnecessary amputative surgery.

    Lay Abstract:
    TITLE: Does BRCA2 justify prophylactic mastectomy? Allele-specific methylation markers of BRCA2 v. AS3 losses in breast cancer. Two genes have been identified to play major roles in the familial form of breast cancer, BRCA1 and BRCA2. Most of the breast cancer cases, however, do not fall into this category, and in a large number of cases these genes do not appear to be involved. The conspicuous lack of genetic changes (mutations and deletion) in BRCA2 in these cancers and reports of BRCA2 mutations without cancer are even more perplexing. An important genetic marker is used to detect genomic losses in BRCA2, it is called D13S171. Genetic losses in this marker show significant correlation with cancer, but in several cases the expression of BRCA2 was still detected, indicating that the deletions targeted a gene other that BRCA2 in the area. Altogether, the data raised the possibility that a strong cancer gene, other than BRCA2 locates next to BRCA2. The conflicting data prompted us to perform detailed sequence and marker analysis in the area. We found that D13S171 is not a direct marker for BRCA2 losses, it is 0.3 million base pair away from BRCA2. More importantly, the marker was mapped in the middle of another gene we call AS3 (new name APRIN). We found that AS3 expression correlates with growth arrest in vivo, and expression of the gene arrested the proliferation of a breast cancer cell line in vitro. We also found that both the AS3 and BRCA2 promoters share a feature (“CpG island”), a known target for down regulating gene expression, called “methylation silencing". The above results represent a significant challenge to the perceived role of BRCA2 in breast cancer. If BRCA2 turns out to be the wrong gene to screen sporadic breast cancers, it has far-reaching and tragic consequences for the patients. Tens of thousands of women are tested every year for BRCA2 mutations and thousands undergo surgery to remove one or both breasts (called “prophylactic mastectomy”), with grave emotional and physical consequences for life. For the above reasons, it is timely to clarify the contributions and the real roles of BRCA2 and AS3 in breast cancer. Investigating only genetic changes in BRCA2 in the past resulted in limited data in a few cases and may not be the major mechanism in losing the function. We propose that the CpG islands we identified present an alternative option, to analyze changes which are not based on DNA sequence, called “epigenetic” changes. Epigenetic mechanisms in gene silencing are known to play a major role in other cancers. To investigate if epigenetic silencing of BRCA2 and/or AS3 is a major mechanism also in breast cancer and to establish their individual role, we propose the following experiments. We plan to establish and optimize the methods for DNA methylation studies in the BRCA2 and AS3 promoters, using methylation-specific amplification reactions (MSP). We propose to study the methylation status of breast cancer cell lines in vitro and to study BRCA2 and AS3 silencing in human breast cancer samples. We will use commercially available breast cancer tissue samples to isolate DNA and to perform the assays. The proposed studies represent pilot studies only, to establish the techniques and to generate data for large-scale comprehensive clinical investigations. We believe that the potential outcomes and the benefits of the proposed research are several-fold. Thousands of women undergo prophylactic mastectomy based on genetic tests that include BRCA2 analysis. We showed that a body of evidence challenged the role of BRCA2 in the majority of cases. Our data also point to another gene in the area, AS3, a potential candidate breast cancer suppressor gene. We will establish the methods to analyze epigenetic silencing of the two genes in breast cancer. Our results will develop a series of novel markers that will use very little sample (needle biopsy) for early diagnosis and to generate prognostic information. The results will ultimately establish the roles of the two genes in breast cancer and will save thousands of women from the tragic consequences of potentially unnecessary prophylactic mastectomy.