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

Abstract

Tuberous sclerosis complex (TSC) is characterised by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene or the TSC2 gene. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). We have developed a straightforward, semiautomated in-cell western (ICW) assay to investigate the effects of amino acid changes on the TSC1 -TSC2-dependent inhibition of mTOR activity. Using this assay, we have characterised 20 TSC2 variants identified in individuals with TSC or suspected of having the disease. In 12 cases, we concluded that the identified variant was pathogenic. The ICW is a rapid, reproducible assay, which can be applied to the characterisation of the effects of novel TSC2 variants on the activity of the TSC1-TSC2 complex.

Determining the pathogenicity of patient-derived TSC2 mutations by functional characterization and clinical evidence

“…Recently, a semiautomated cell-based assay has been described to test the effects of unclassified TSC2 variants on protein function by looking at the ability of the mutants to repress S6K1 phosphorylation. 11 Our findings of apparently separable effects of the R505Q mutation on S6K1 and 4E-BP1 phosphorylation suggest that caution may be required in the interpretation of single functional assays.…”

“…11 Confidence in interpreting results is increased when the conclusions from all lines of available evidence concur. However, the data obtained from functional analysis of the R505Q mutation highlight that functional data should be interpreted with caution until functional deficits and phenotypes have been better correlated in larger series.…”

“…A reliable functional assay could greatly facilitate the interpretation of variants of uncertain clinical significance. Few TSC1 or TSC2 mutations have been characterized functionally [9][10][11] and it is still impossible to predict phenotypic effects from the location and type of mutation.…”

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex

“…To assess the effects of the variants on both the formation and activity of the TSC1-TSC2 complex we decided to use immunoblotting followed by infrared scanner-based detection. In our experience, double-label microscopy and GAP assays were too labor intensive and/or unreliable for routine use and, although the in-cell Western was a simple and reliable assay [Coevoets et al, 2009], it required large amounts of (expensive) antibodies and did not provide information on the TSC1-TSC2 interaction.…”

“…Previously, we used immunoblotting, double-label immunofluorescent microscopy, in-cell Western analysis, and GAP assays to study the effects of 47 TSC2 missense and in-frame insertions/deletions and 26 TSC1 missense and inframe insertions/deletions on TSC1-TSC2 activity [Coevoets et al, 2009;Jansen et al, 2006Jansen et al, , 2008Mozaffari et al, 2009;Nellist et al, 2005Nellist et al, , 2008Nellist et al, , 2009. Pathogenic missense changes in the N-terminal region of TSC1 (amino acids 50-224) reduced TSC1 stability [Mozaffari et al, 2009;Nellist et al, 2009], while pathogenic TSC2 missense changes had distinct effects on the TSC1-TSC2 complex, depending on the region of TSC2 that was affected.…”