Advanced Molecular Diagnostic Test for Neurofibromatosis

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.