Resolving missense variants of uncertain significance in TSC2

Genetic variants in genes in the mammalian target of rapamycin (mTOR) signaling pathway, or mTORopathies, are emerging as a common pathway implicated in epilepsy. Variants of uncertain significance (VUS) in mTOR pathway genes present significant issues in patient care and impact whether or not further genetic testing is pursued. There is a critical need to develop tools to functionally characterize and resolve VUSs in mTOR pathway genes and other epilepsy-associated genes. I recently developed a medium throughput assay for VUS resolution in an autosomal recessive mTORopathy in the gene SZT2. My goal in this study is to adapt this assay to incorporate saturation mutagenesis to greatly increase throughput in order to characterize many of the VUSs in the TSC2 gene. Genetic variants causing TSC2 haploinsufficiency result in aberrant mTOR signaling and are a major cause of tuberous sclerosis, a multisystem disorder characterized by hamartomas, epilepsy, and other CNS manifestations such as autism. In this proposal, I will determine the pathogenicity for approximately 12.5% of all TSC2 VUSs using saturation mutagenesis and a TSC2 knockout landing pad cell line. I will further develop a machine learning algorithm to improve in silico pathogenicity prediction for missense TSC2 variants. The development of this approach is translatable to other mTORopathy genes, and eventually other epilepsy-associated genes and will allow me to generate preliminary data for an NIH R01 grant.