Advanced Molecular Diagnostic Test for Neurofibromatosis

神经纤维瘤病的高级分子诊断测试

• 批准号: 6999394
• 负责人: SADANAND GITE
• 金额: $ 15万
• 依托单位: AMBERGEN, INC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999394
• 关键词: biotechnology; cell line; diagnosis design /evaluation; diagnostic tests; enzyme linked immunosorbent assay; gene mutation; genetic disorder diagnosis; genetic screening; high throughput technology; human genetic material tag; human tissue; mass spectrometry; messenger RNA; neurofibromatosis; neurofibromatosis type 1 protein /gene

DESCRIPTION (provided by applicant): Neurofibromatosis (NF) is an autosomal dominant disorder caused by mutations in the NF1 and NF2 genes. Currently, more than 100,000 Americans suffer from NF type 1 (NF1) which has one of the highest prevalence rates for a genetic disease (1/3,500 births). NF1 is caused by mutations in the NF1 gene which codes for the tumor suppressor protein neurofibromin. Most mutations (>80%) are chain truncating and generally result in inactive proteins. The preferred method to detect such mutations is the Protein Truncation Test (PTT). However, conventional PTT has many disadvantages for routine clinical use. For this reason, current diagnosis is based on established clinical symptoms. The objective of this proposal is to develop a cost-effective technology to screen for mutations in the NF1 gene. This will provide significant benefit to public health by identifying earlier and more reliably persons suffering from NF1 and aligns with the mission of the NINDS. Two different methods will be developed and evaluated which are both based on in vitro expression of peptides from overlapping segments of PCR amplified NF1 genomic DMA and mRNA. One approach utilizes a newly developed ELISA-based protein truncation test (ELISA-PTT) to detect chain-truncations arising mainly from frame-shift mutations, which constitute greater than 80% of all mutations in NF1. In contrast to conventional protein truncation tests, ELISA-PTT eliminates the need for electrophoresis and radioactivity. A second approach, based on mass spectrometric analysis of in vitro expressed proteins (MASSIVE-PRO) is able 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 DMA sequencing. The technology will be extensively evaluated in collaboration with Dr. Ludwine Messiaen, Director of Genomics at the University of Alabama using a repository of validated genomic DNA and mRNA samples from NF1 patients. During Phase II, an optimized system for screening NF1 mutations will be developed and clinically evaluated.

描述（由申请人提供）：神经纤维瘤病（NF）是由NF1和NF2基因突变引起的常染色体显性疾病。目前，超过100,000名美国人患有NF 1型（NF1），该遗传疾病的患病率最高（1/3,500个出生）。 NF1是由NF1基因中的突变引起的，该突变编码为肿瘤抑制蛋白神经纤维蛋白。大多数突变（> 80％）是链截断，通常导致非活性蛋白质。检测这种突变的首选方法是蛋白质截断测试（PTT）。但是，常规PTT有许多常规临床用途的缺点。因此，当前的诊断是基于既定的临床症状。该提案的目的是开发一种具有成本效益的技术来筛选NF1基因中的突变。通过确定患有NF1的人并与Ninds的使命保持一致，这将为公共卫生带来重大利益。将开发和评估两种不同的方法，这些方法均基于PCR扩增的NF1基因组DMA和mRNA的重叠段的体外表达。一种方法利用新开发的基于ELISA的蛋白质截断测试（ELISA-PTT）来检测主要由框架变速突变引起的链截断，该突变大于NF1中所有突变的80％。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。第二种方法基于体外表达蛋白（大量Pro）的质谱分析，能够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品，用于完整的DMA测序。该技术将与阿拉巴马大学基因组学总监Ludwine Messiaen博士合作，使用NF1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，将开发出一种用于筛选NF1突变的系统，并通过临床评估。