NF1 Neuronal Genotype-Phenotype Relationships
Abstract
Neurofibromatosis type 1 (NF1) is a highly variable and unpredictable genetic disease. Symptoms include tumors of the nervous system, called neurofibromas. These are benign, but often numerous and highly disfiguring, but can eventually form malignant tumors, which are often fatal. However, while the role of the NF1 gene in tumor formation has received much attention, the non-tumor symptoms of NF1 also greatly impact the lives of those with the disease. These symptoms include skeletal and vascular abnormalities, pigmentation defects, reduced overall growth, and cognitive deficits. The latter is seen in 50%-70% of children with NF1. Cognitive and behavioral problems associated with NF1 are a major impairment to children s functioning and affect learning, encompassing attention problems, memory problems, spatial perception difficulties, and selective problems in reading or mathematics. No effective therapy for any NF1 symptom yet exists. The NF1 gene, which is mutated in the disease, produces a large protein, called neurofibromin. The normal role of neurofibromin is to serve as a regulator of an important group of proteins called Ras, which are involved in communication within cells. As a consequence of mutations in NF1, Ras becomes abnormally activated. The diverse symptoms of NF1 are likely due to Ras controlling many important signaling pathways, to differing degrees, in all types of cells in the body. Although Ras presents an obvious therapeutic possibility for NF1, there are multiple problems with using this approach. Chronically blocking Ras is an inappropriate strategy for treating the many serious but non-life-threatening symptoms of NF1, especially in children. Development of targeted therapies hinges on improving our understanding of the precise signaling pathways downstream of Ras responsible for NF1 symptoms. Patient mutations causing NF1 have been identified throughout NF1 gene -- not just within the segment known to control Ras. We hypothesize that investigating these mutations may give a clue to the diverse clinical symptoms and the variable nature of NF1. We propose using a dual approach to investigate the molecular and cellular consequences of NF1 mutations on altered neuronal function that underlie the cognitive impairment in children with NF1. A fruit fly (Drosophila) model of NF1 will be used to rapidly assess the function of NF1 patient mutations occurring outside of the Ras-regulating regions. This approach relies on the fact that the majority of mutations in human NF1 are found in regions in the fly NF1 gene that are conserved. Flies mutant for NF1 have a number of defects that we can use to test human mutations. These include altered cell signaling, abnormalities in the junctions (synaspes) between fly neurons, and impaired ability to learn and remember. By rapidly testing NF1 mutations for altered function, we can rapidly select the most informative ones for the second part of our proposed study. Guided by the results of the fly experiments, we will use the latest gene editing technology, called CRISPR, to generate selected mutations in human induced pluripotent stem cells (iPSCs). These iPSCs will be differentiated into neurons, and we will examine neuronal morphology, proliferation, signaling, and electrophysiological properties to look for correlates with specific mutations in different regions of NF1. Together, these unbiased approaches will investigate the wide variety of disease-causing NF1 mutations, correlating them to altered neuronal function, which ultimately contributes to cognitive deficits in NF1 patients. We expect that these experiments, combining rapid functional testing in fruit flies, with validation in human neurons will allow us to establish genotype-phenotype relationships for a number of patient-derived NF1 mutations, which is not feasible to do in other experimental systems. Using CRISPR and iPSC technology, we will reduce the effects of genetic bac
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 31, 2017
- Source ID
- W81XWH1610220
Entities
People
- James Walker
Organizations
- Massachusetts General Hospital
- United States Army