MUTATION AND GENETIC ANALYSIS OF NEUROFIBROMATOSIS

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Neurofibromatosis type 1 (NF1) is among the most prevalent of human genetic diseases, with a frequency of 1/3000 individuals in all ethnic groups. The major features include pigmented dermal regions, neurofibromas (benign tumors) arising from nerve sheaths, bone deformities, and hamartomata of the iris. Most patients are diagnosed in childhood or adolescence, as the symptoms tend to progress with age and hormonal surges. Medicine currently can only offer standard treatment of some individual symptoms - no cure or treatment to retard progression exists. The NF1 gene is an approximately 300 kb locus on chromosome 17 which is disrupted by constitutional mutations in NF1 patients. It is thought to be a tumor suppressor gene, based on phenotype being associated with loss of function. A widely accepted hypothesis suggests that loss or alteration of function from both NF l alleles in a cell (the other loss the result of somatic mutation, a "second hit") results in the phenotype. The long-term goal of this project is to identify NF1 gene mutations in order to answer specific questions regarding the genetics of the disorder, and to examine the two-hit hypothesis. First, it is important to define a set of mutations to categorize NF1 mutations by type and location within the gene, since very little mutational data currently lists. Secondly, the issue of penetrance is not clear-do all individuals with constitutional NF1 mutations express symptoms? Penetrance will be studied by identifying NF1 gene mutations in a series of "new mutation" families, as well as in families in which the transmission is not clearly autosomal dominant (such as multiple affected children from normal parents, or "skipped" generations). DNA and RNA will be analyzed by several molecular genetic techniques designed to detect mutations, including pulsed field electrophoresis, and PCR-based analysis of individual exons and cDNA fragments. This research may also provide a better estimate of the true rate of occurrence of "new mutation." In addition, two recent studies using linked markers suggest that a very high proportion of NF1 mutations arise in the paternal germline, unrelated to age. Parental origin of each new mutation identified in this study will be analyzed using polymorphisms within the coding regions of the NF1 gene itself. Any maternally-arising mutations will be the focus of further study, to investigate whether there are any characteristics that set them apart from paternally-derived mutations. To investigate the "two-hit" hypothesis, mutational analysis will be performed on the DNA from blood and from several physically separate neurofibromas from the each of several patients. Discovery of the same constitutional mutation yet different "second" mutations in each tumor would verify the tumor suppressor hypothesis. The data from these studies will significantly contribute to research into the function of the NF1 gene and the mutation data base, which will be useful for development of DNA diagnosis and further NF1 research.