2. Academic Validation
  3. An integrated, scaled approach to resolve TSC2 variants of uncertain significance

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

  • bioRxiv. 2026 Jan 18:2026.01.16.699909. doi: 10.64898/2026.01.16.699909.
Carina G Biar 1,  Ziyu R Wang 2,  Nathan D Camp 3,  Daniel L Holmes 2,  Melinda K Wheelock 2,  Sriram Pendyala 2,  Abby V McGee 2,  Pankhuri Gupta 2,  Abbye E McEwen 2 4,  Malvika Tejura 2,  Marcy E Richardson 5,  Jamie D Weyandt 5,  Taylor Coleman 5,  Ross Stewart 6,  Daniel Zeiberg 6,  Allyssa J Vandi 2,  Samantha Dawson 3,  Predrag Radivojac 6,  Lea M Starita 2 4,  Gemma L Carvill 1,  Richard G James 3 7,  Douglas M Fowler 2 4 8,  Jeffrey D Calhoun 1
Affiliations

Affiliations

  • 1 Ken and Ruth Davee Department of Neurology, Northwestern Feinberg School of Medicine, Chicago, Illinois.
  • 2 Department of Genome Sciences, University of Washington, Seattle, WA.
  • 3 Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA.
  • 4 Brotman Baty Institute for Precision Medicine, Seattle, WA, USA.
  • 5 Ambry Genetics, Aliso Viejo, CA.
  • 6 The Institute for Experiential AI, Northeastern University, Boston, MA.
  • 7 Department of Pediatrics and Pharmacology, University of Washington, Seattle, WA.
  • 8 Department of Bioengineering, University of Washington, Seattle, WA, USA.
PMID: 41648336 DOI: 10.64898/2026.01.16.699909
Abstract

Obtaining a precise genetic tuberous sclerosis diagnosis is a challenge as many missense TSC2 variants are variants of uncertain significance (VUS). VUS in TSC2 have been resolved by one-at-a-time functional assays, but these assays cannot scale to the 3,634 TSC2 missense VUS observed so far. To address this challenge, we used massively parallel Sequencing to measure the steady-state abundance of almost 9,000 TSC2 missense variants and developed an mTOR pathway activity assay using genome editing and cell sorting to generate activity scores for 391 missense variants. 1,288 of 8,891 (14.49%) missense variants assayed had altered TSC2 abundance, and 69 of 391 (17.65%) missense variants assayed had altered mTOR pathway activity. Calibration and integration of these data into classification of variants identified in a clinical cohort putatively reclassified 212 of 276 (76.8%) TSC2 missense VUS. These datasets will lead to improved genetic diagnosis of tuberous sclerosis with potential positive impacts on the clinical management of patients and their families.

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