A reliable cell-based assay for testing unclassified TSC2 gene variants

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Tuberous sclerosis complex (TSC) is a genetic disorder characterized by seizures, intellectual disability, and hamartoma formation.
• Mutations in TSC1 or TSC2 genes disrupt the TSC1-TSC2 protein complex, affecting mammalian target of rapamycin (mTOR) signaling.
• Understanding the functional impact of TSC2 variants is crucial for diagnosing and managing TSC.

Purpose of the Study:

• To develop and validate a semiautomated in-cell western (ICW) assay for assessing TSC1-TSC2 complex function.
• To investigate the effects of amino acid changes on TSC1-TSC2-dependent mTOR inhibition.
• To characterize novel TSC2 variants identified in individuals with TSC.

Main Methods:

• Development of a straightforward, semiautomated in-cell western (ICW) assay.
• Application of the ICW assay to evaluate the functional impact of 20 different TSC2 variants.
• Assessment of TSC1-TSC2 complex activity and its inhibition of mTOR signaling.

Main Results:

• Successfully developed and implemented a rapid and reproducible ICW assay.
• Characterized 20 TSC2 variants, determining their impact on TSC1-TSC2 complex function.
• Identified 12 of the tested TSC2 variants as pathogenic, demonstrating significant disruption of mTOR inhibition.

Conclusions:

• The developed ICW assay is a valuable tool for the rapid and reproducible characterization of TSC2 variants.
• This assay facilitates the assessment of novel TSC2 mutations' effects on TSC1-TSC2 complex activity.
• The findings contribute to a better understanding of TSC pathogenesis and variant classification.