Molecular Test for Inherited Mutations in Breast Cancer

描述（由申请人提供）： 乳腺癌是最常见的恶性肿瘤，发生在妇女和癌症死亡的第二大原因。虽然大多数乳腺癌是散发性的（非遗传性的），但大约10%是由于BRCA 1和BRCA 2基因的遗传突变。人群研究表明，这些基因的突变导致乳腺癌的终生风险约为80%，卵巢癌的终生风险为40- 65%。虽然医生以前依赖于乳腺癌的高风险家族史来识别携带BRCA 1/2突变的女性，但最近的一项研究表明，50%的携带者没有明显的家族史。通过查明这些携带者将改善公共卫生，因为有越来越多的治疗选择，这可以大大降低风险。然而，基于直接测序的检测BRCA 1/2突变的常规方法对于人群筛查来说太昂贵（约3，000美元）且劳动密集型。 该项目的目标是开发一种具有成本效益的技术来筛查BRCA 1/2基因的突变。将开发和评价两种不同的方法;两种方法均基于PCR扩增BRCA 1/2基因组DNA和mRNA重叠片段肽的体外表达。一种方法利用新开发的基于ELISA的蛋白质截短试验（ELISA-PTT）来检测链截短，这占BRCA 1/2所有突变的90%。与传统的蛋白质截短试验相比，ELISA-PTT无需电泳和放射性。第二种方法同样不需要电泳或放射性标记，利用体外表达蛋白质的质谱分析（MASSIVE-PRO）来扫描所有可能的突变，包括氨基酸取代。该方法的关键是开发具有非常低水平的蛋白水解活性的体外表达系统。我们的初步研究表明，这两种方法都是可行的，可以提供一个非常低的成本和高通量的替代全DNA测序。一个关键的项目科学家是Alex Garvin博士，他是BRCA 1/2蛋白截短测试的专家，也是MASSIVE-PRO的开发者。该技术将与Jessica K. Booker博士说，北卡罗来纳州大学医学院（UNCSM）分子诊断实验室副主任使用了一个来自乳腺癌患者的经验证的基因组DNA和mRNA样本库。在第二阶段，将开发和临床评估筛选BRCA 1/2突变的优化系统。

DESCRIPTION (provided by applicant): Breast cancer is the most common malignancy, which occurs in women and the 2nd leading cause of cancer death. While the majority of breast cancers are sporadic (not inherited), approximately 10 percent are due to inherited mutations in the BRCA1 and BRCA2 genes. Population studies show that mutations in these genes lead to a lifetime risk of approximately 80 percent for breast cancer and 40-65 percent for ovarian cancer. Although physicians have previously relied on a high-risk family history of breast cancer to identify women at risk for carrying mutations in BRCA1/2, a recent study shows that 50 percent of carriers have no significant family history. Public health would be improved by identifying such carriers since there are an increasing number of therapeutic options, which can significantly reduce risk. However, conventional methods of detecting BRCA1/2 mutations based on direct sequencing are too expensive (approximately $3,000) and labor intensive for population screening. The objective of this project is to develop a cost-effective technology to screen for mutations in the BRCA1/2 genes. Two different methods will be developed and evaluated; both based on in vitro expression of peptides from overlapping segments of PCR amplified BRCA1/2 genomic DNA and mRNA. One approach utilizes a newly developed ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations, which constitute 90 percent of all mutations in BRCA1/2. In contrast to conventional protein truncation tests, ELISA-PTT eliminates the need for electrophoresis and radioactivity. A second approach, which again does not require electrophoresis or radiolabels, utilizes mass spectrometric analysis of in vitro expressed proteins (MASSIVE-PRO) to scan for all possible mutations, including amino acid substitutions. A key to this approach is the development of an in vitro expression system, which has very low levels of proteolytic activity. Preliminary studies by us demonstrate that both approaches are feasible and can offer a very low cost and high throughput alternative to full DNA sequencing. A key project scientist is Dr. Alex Garvin, an expert on BRCA1/2 protein truncation tests and developer of MASSIVE-PRO. The technology will be extensively evaluated in collaboration with Dr. Jessica K. Booker, Associate Director of the Molecular Diagnostics Laboratory at the University of North Carolina School Of Medicine (UNCSM) using a repository of validated genomic DNA and mRNA samples from breast cancer patients. During Phase II, an optimized system for screening BRCA1/2 mutations will be developed and clinically evaluated.