An integrated, scaled approach to resolve TSC2 variants of uncertain significance

Obtaining a precise genetic tuberous sclerosis complex diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance. Variants of uncertain significance in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3634 TSC2 missense variants of uncertain significance observed so far. To address this challenge, we use massively parallel sequencing to measure the steady-state abundance of almost 9000 TSC2 missense variants and develop an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. We observe that 1256 of 8864 (14.17%) missense variants assayed have altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed have altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassifies 212 of 276 (76.8%) TSC2 missense variants of uncertain significance. These datasets will lead to improved genetic diagnosis of tuberous sclerosis complex with potential positive impacts on the clinical management of patients and their families.