Phenotype-Genotype Relationships in Tuberous Sclerosis

Summary 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). Mutations in TSC2 are both more common and produce more severe disease phenotypes. TSC1/2 regulate signal transduction through mTOR, a major signaling hub important for cell growth, cell survival, neuroinflammation and other cellular functions. Neurological symptoms are major contributors to morbidity in TS, with the majority of affected children developing intractable epilepsy. FDA- approved mTOR inhibitors reduce seizures in some individuals with TS and in TS mouse models. 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. Mechanisms underlying this variability 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 of the primary mutations in TSC1/2. It has been proposed that mutations lead to complete loss of function, but this has not been rigorously tested and seems unlikely, considering that mutations are highly diverse; from single point mutants to large deletions spanning entire exons. This R21 application, therefore, has two goals. Firstly, we will test the hypothesis that distinct human TSC2 mutations contribute to the variability in disease severity. Secondly, mutations will be tested using a platform optimized for a personalized medicine approach for infants newly diagnosed with TS. The long-term goal is to be able to provide data on the functional effects of a child’s TSC2 mutation by their first birthday, guiding prognosis and treatment. The rapid screening platform utilizes a cre/lox system in Tsc2fl/fl mouse cells and intact mice, facilitating a lentivirus-based strategy to delete the mouse gene and replace it with the human mutation. Lentiviral vectors can be easily modified to express different human TSC2 variants for patient testing. Initial proof-of-concept studies will test six human mutations in cell culture and in intact mice, focusing on neuronal morphology, epilepsy severity and single nuclei RNA-Seq analyses as a first step to understand differences in mutant-induced molecular changes. Mutations affecting different protein functional domains and covering a range of clinical severities were selected. The project will benefit from synergistic collaborations between CCHMC’s TS clinic and basic science labs. This research may be a first step towards the development of rapid screening platforms useful for diagnosis and drug discovery in TS, making a personalized medicine approach for TS feasible. Project Number: 1R21NS141450-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Christina Gross (+1 co-PI) | Institution: CINCINNATI CHILDRENS HOSP MED CTR, CINCINNATI, OH | Award Amount: $445,500 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CN-U (82)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11311523