Gene Structure and Somatic Mutation Analysis of Neurofibromatosis Type 1

摘要

This research focuses on the characterization of the human neurofibromatosis 1(NF1) locus. Initially, we determined intron-exon boundaries of exons 1 through 27b. In the process of characterizing genomic clones that harbored NF1 exons we identified and partially characterized NF1-homologous loci distributed throughout the genome. With this information we designed oligonucleotides as primers in the polymerase chain reaction (PCR) to specifically amplify from the NF1 locus. NF1 primer sets were adapted to screen DNA for NF1 mutations by an exon-specific PCR approach. Application of this technique enables us to identify both germ-line and somatic NF1 mutations. We identified a somatic mutation in the normal NF1 allele in a dermal neurofibroma from an NF1 individual who is hemizygous at the NF1 locus. This is the first demonstration of complete inactivation of the gene in a neurofibroma, and it suggests that NF1 can act as a tumor suppressor in cells that proliferate as benign tumors in NF1. We also developed primer sets and methodology for PCR genotyping of the NF1 locus, and we identified and adapted NF1 genomic clones for fluorescence in situ hybridization (FISH) analysis. Finally, the NF1 promoter has been subcloned and approximately 4.4 kilobases upstream of the transcription start site has been sequenced.