Phenotype-Genotype Relationships in Tuberous Sclerosis
Principle Investigators: Steve Danzer, PhD, Division of Anesthesia, Christina (Nina) Gross, PhD, Division of Neurology, Darcy Krueger, MD, PhD, Division of Neurology
Tuberous sclerosis (TS) is a rare genetic disorder associated with benign tumors and neurological symptoms including autism and epilepsy. TS is caused by mutations in the genes coding for tuberous sclerosis complex proteins 1 and 2 (TSC1/2). TSC1/2 regulate signal transduction through mTOR, a major signaling hub important for cell growth and survival, neuroinflammation, and other cellular functions. MTOR regulated pathways are associated with autism and epilepsy, and multiple FDA-approved mTOR inhibitors improve neurological symptoms in some individuals with TS and in TS mouse models. The single available disease mechanism-targeted treatment, rapamycin, is only effective in a subset of patients. However, not all TS patients respond clinically to mTOR inhibitors and treatment can have problematic side effects. Moreover, the severity of TS is highly variable with disease manifestations ranging from minimally symptomatic to life-threatening complications. Mechanisms for this disease diversity are poorly understood and have been identified as one of five priority focus areas in the NIH strategic plan for TS research. Considerable effort is being made to understand the impact of secondary somatic mutations, genetic modifiers, and environmental factors, but surprisingly, little is known about the biological consequences (and thus contribution to disease severity) of the primary mutations in TSC1/2. It has been proposed that mutations lead to complete loss of function, but this has not been tested thoroughly and seems unlikely, considering that mutations are highly variable – from single point mutants to large deletions spanning entire exons.
We hypothesize that distinct human TSC2 mutations differentially dysregulate neuronal growth, gene expression, and disease severity. We are testing this hypothesis in a TS mouse model, in which we will lentivirally replace one Tsc2 allele with human TSC2 variants using a cre/lox system. The specific variants of TSC2 to be used to test our hypothesis are expected to lead to changes in pathways regulated by TSC2 important for neuronal function. We have chosen the mutations based on the published literature, the extent of neurological abnormalities, and clinical disease burden present in actual TS patients at Cincinnati Children’s. We are pursuing two aims, both of which will use in vitro neuron culture systems and intact mice. In aim 1, we are using confocal imaging and EEG analyses to test the effects of the human variants on mTOR signaling, neuronal morphology, and neurophysiology. In aim 2, we are using ribosome tagging to isolate the transcriptome of neurons expressing the human variants and identify the biological pathways altered by the specific mutations. An in silico screen will identify drug candidates correcting these defects. We predict that TSC2 variants will produce a range of phenotypes and that the extent of dysregulated gene expression will correlate with phenotype severity. We are following a rigorous approach, including randomization, blinding, and consideration of animal sex. We anticipate that this research will be a first step towards the development of rapid screening platforms useful for diagnosis and drug discovery for individuals with TS.