Neurofibromatosis type I (NF1) is one of the most common inheritable autosomal dominant disorders. Although it is clear that mutation on NF1 is the major initial culprit responsible for the disease, mutation of NF1 alone cannot explain the diverse clinical symptoms. The affected individuals even among members of the same family display a high degree of phenotypic variation. No phenotype-genotype (NF1 mutation) correlation has been observed. These observations strongly suggest involvement of other genes, here termed modifier genes, in the NF1 disease development. Our recent preliminary studies imply that two proteins involved in expression regulation of the NF1 gene at the pre-mRNA splicing level function as potential modifiers of the disease. In this proposal, we will determine if and how these pre-mRNA splicing regulators play an important role as modifier genes of the NF1 disease.

The NF1 gene can generate multiple protein products through a mechanism called alternative splicing, a process that generates more than one mature RNA transcripts from one precursor RNA by selectively joining the exons. One major alternative splicing event occurs at exon 23a, which is located in the middle of the protein known for its tumor suppressor function of the NF1 gene product neurofibromin. The neurofibromin that does not contain the amino acids of exon 23a is a strong tumor suppressor, while the neurofibromin that contains the 21 amino acids encoded in exon 23a is a much weaker tumor suppressor. Thus, the tumor suppressor activity of neurofibromin can be controlled by alternative splicing regulators that affect inclusion of exon 23a. We hypothesize that such factors play an important role in modifying the function of NF1 protein product and will test this idea in this proposal.

There are two major objectives in this proposal. We will first investigate how alternative splicing modulates the function of neurofibromin. Particularly, we will generate and test how mutations on and around exon 23a affect inclusion of this exon. Furthermore, our recent studies identified two alternative splicing factors that affect exon 23a inclusion. We will determine how these two factors influence the tumor suppressor activity by up- and down-regulating these factors in mammalian cell cultures. The second goal of these studies is to determine the biological consequence of altered ratio of the two neurofibromin products (with or without exon 23a). We will create a unique system that will allow us to readily modify the NF1 alleles. We will use this system to generate mouse embryonic stem (ES) cell lines that express the two NF1 products at different ratios. Subsequently, we will use a recently developed differentiation strategy to derive neural stem cells from these ES cells to examine the cell growth and differentiation potential of these cells.

The results of these studies will define the function of alternative splicing regulators in NF1 disease development. Splicing regulator as NF1 modifier genes is an extremely under studied yet very important area. Establishment of these modifier genes will help explain the diverse clinical variability and provide significant insight into the etiology of the NF1 disease. Potentially, they will provide novel basis for disease diagnosis and therapy.