Advanced Molecular Diagnostic Test for Neurofibromatosis

描述(申请人提供)：神经纤维瘤病(NF)是一种常染色体显性遗传病，由NF1和NF2基因突变引起。目前，超过10万美国人患有1型神经元病(NF1)，这是遗传病患病率最高的疾病之一(1/3,500名新生儿)。NF1是由NF1基因突变引起的，NF1基因编码肿瘤抑制蛋白神经纤维蛋白。大多数突变(&gt；80%)是链截断，通常会导致蛋白质失活。检测这种突变的首选方法是蛋白质截断测试(PTT)。然而，传统的PTT对于常规的临床应用有许多缺点。出于这个原因，目前的诊断是基于既定的临床症状。这项提议的目的是开发一种具有成本效益的技术来筛查NF1基因的突变。这将通过更早和更可靠地识别患有NF1的人而对公共卫生产生重大好处，并与NINDS的使命保持一致。两种不同的方法将被开发和评估，这两种方法都是基于从PCR扩增的NF1基因组DMA和mRNA的重叠片段中体外表达的多肽。一种方法利用新开发的基于ELISA的蛋白质截断测试(ELISA-PTT)来检测主要由移码突变引起的链截断，该突变占NF1所有突变的80%以上。与传统的蛋白质截断试验不同，ELISAPTT不需要电泳法和放射性测定法。第二种方法，基于体外表达蛋白质的质谱分析(MASS-PRO)，能够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具有非常低水平的蛋白分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，可以提供一种完全DMA测序的非常低成本和高吞吐量的替代方案。这项技术将与阿拉巴马大学基因组学主任Ludwin Messiaen博士合作，使用来自NF1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NF1突变的优化系统并进行临床评估。

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.