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，发生在女性身上，是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%的乳腺癌是由于BRCA1和BRCA2基因的遗传突变造成的。人群研究表明，这些基因的突变导致乳腺癌的终身风险约为80%，卵巢癌的风险约为40%-65%。尽管医生此前曾依靠乳腺癌高危家族史来识别携带BRCA1/2突变的风险女性，但最近的一项研究表明，50%的携带者没有明显的家族史。通过识别这些携带者将改善公共健康，因为有越来越多的治疗选择，这可以显著降低风险。然而，基于直接测序的传统方法检测BRCA1/2突变太昂贵(约3000美元)，而且对人群筛查来说是劳动密集型的。 该项目的目标是开发一种具有成本效益的技术来筛选BRCA1/2基因的突变。两种不同的方法将被开发和评估；这两种方法都是基于从聚合酶链式反应扩增的BRCA1/2基因组DNA和mRNA的重叠片段中体外表达多肽。一种方法是利用一种新开发的基于ELISA法的蛋白质截断试验(ELISAPTT)来检测BRCA1/2中90%的突变。与传统的蛋白质截断试验相比，ELISAPTT不需要电泳法和放射性。第二种方法同样不需要电泳或放射性标记，它利用体外表达蛋白质的质谱分析(MASS-PRO)来扫描所有可能的突变，包括氨基酸替换。这种方法的一个关键是开发一种体外表达系统，该系统具有非常低的蛋白分解活性。我们的初步研究表明，这两种方法都是可行的，可以提供一种非常低成本和高通量的替代全DNA测序的方法。一位关键的项目科学家是亚历克斯·加文博士，他是BRCA1/2蛋白质截断测试的专家，也是MASSIC-PRO的开发者。这项技术将与北卡罗来纳大学医学院(UNCSM)分子诊断实验室副主任杰西卡·K·布克博士合作，使用来自乳腺癌患者的经过验证的基因组DNA和信使核糖核酸样本储存库进行广泛评估。在第二阶段，将开发一种筛选BRCA1/2突变的优化系统并进行临床评估。

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.