Molecular Test for Inherited Mutations in Breast Cancer

乳腺癌遗传突变的分子检测

基本信息

批准号：
6834918
负责人：
SADANAND GITE
金额：
$ 18.7万
依托单位：
AMBERGEN, INC
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-01 至 2006-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6834918
关键词：
bioassay biotechnology brca gene breast neoplasm /cancer diagnosis breast neoplasms diagnosis design /evaluation enzyme linked immunosorbent assay gene mutation genetic disorder diagnosis high throughput technology

项目摘要

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.

描述（由申请人提供）：乳腺癌是最常见的恶性肿瘤，发生在女性和癌症死亡的第二大原因。尽管大多数乳腺癌是零星的（未遗传），但大约10％是由于BRCA1和BRCA2基因中的遗传突变引起的。人口研究表明，这些基因的突变导致乳腺癌的终生风险约为80％，而卵巢癌的突变风险为40-65％。尽管医师以前曾依靠高风险的乳腺癌家族史来识别有BRCA1/2中携带突变的妇女，但最近的一项研究表明，有50％的携带者没有重要的家族史。由于越来越多的治疗选择可以大大降低风险，因此可以通过确定此类载体来改善公共卫生。但是，基于直接测序的BRCA1/2突变的常规方法太昂贵（大约3,000美元），并且用于人口筛查的劳动量很高。该项目的目的是开发一种具有成本效益的技术，以筛选BRCA1/2基因中的突变。将开发和评估两种不同的方法；两者都基于PCR扩增BRCA1/2基因组DNA和mRNA的重叠段的肽的体外表达。一种方法利用新开发的基于ELISA的蛋白质截断测试（ELISA-PTT）来检测链截断，链接构成BRCA1/2中所有突变的90％。与常规的蛋白质截断测试相反，ELISA-PTT消除了对电泳和放射性的需求。第二种方法再次不需要电泳或放射性标记，它利用体外表达蛋白（大量Pro）的质谱分析来扫描所有可能的突变，包括氨基酸取代。这种方法的关键是开发体外表达系统，该系统具有非常低的蛋白水解活性。我们的初步研究表明，这两种方法都是可行的，可以提供非常低的成本和高吞吐量的替代替代完整的DNA测序。一个关键的项目科学家是Alex Garvin博士，他是BRCA1/2蛋白质截断测试的专家，也是Massive-Pro的开发者。该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Jessica K. Booker博士进行广泛评估，并使用乳腺癌患者的经过验证的基因组DNA和mRNA样本的存储库。在II期期间，将开发出一种筛查BRCA1/2突变的优化系统，并通过临床评估。