The TSC1 and TSC2 gene products interact to form the TSC complex, an important negative regulator of the mechanistic target of rapamycin (mTOR) complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance (VUS) affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC. This article is protected by copyright. All rights reserved.