Tuberous sclerosis complex (TSC) is a known cause of epilepsy, mental retardation, and autism. Structural abnormalities known as tubers and subependymal giant cell astrocytomas (SEGAs) in the brain of TSC patients disrupt the normal anatomic connections in the brain and cause seizures and cognitive dysfunction. We have learned that tubers and SEGAs form by complete inactivation of the TSC1 and TC2 genes due to a two-hit mutational mechanism in which both copies of the TSC genes are affected by mutations. We also have learned that loss of TSC gene function leads to activation of the mTOR cell pathway cascade that governs cell size. mTOR activation leads to abnormally enlarged cell types. Our plan is to study human TSC brain samples to define the effects of loss of TSC1 or TSC2 function in the developing brain. Our goal is to provide a genotype-phenotype analysis that includes both germline and somatic second hit mutations. We will define germline events and the types of second hit mutations in tubers and SEGAs as a strategy to form a comprehensive view of the mutational mechanisms that lead to formation of these lesions. We will establish a database of germline and second hit mutations and correlate these events with specific cellular features of the lesion such as numbers of giant cells and astrocytes, size of the tuber or SEGA, and number of proliferative cells to define the actual cellular effects of the specific second hit mutation superimposed on the existing germline event. These studies will provide the first attempt to analyze genotype-phenotype correlations of both germline and second hit mutations. In a second set of experiments, we will study post-mortem TSC brain tissue to define cells in the cerebral cortex, thalamus, basal ganglia, and cerebellum that exhibit mTOR cascade activation as a strategy to determine to what extent subtle structural abnormalities in TSC brain (aside from tubers and SEGAs) may contribute to epilepsy or neurocognitive abnormalities.

The studies will provide direct mutational evidence for how tubers and SEGAs are formed and potentially how specific mutations can be associated with specific cellular and clinical features of TSC. These can benefit a broad variety of TSC patients including those with infantile spasms, intractable epilepsy, autism, and cognitive disabilities. We propose that in the very near future complete genotyping of resected tissue specimen including germline and second hit events can be performed and may yield pivotal insights into clinical outcome measures in TSC patients such as response to surgery, response to medications, and likelihood of normal cognitive development. Perhaps most exciting is that by defining the mutational spectrum of tubers and SEGAs, we can begin to understand the developmental epochs in which these lesions form so that preventive therapy can be designed.