A reliable cell-based assay for testing unclassified TSC2 gene variants.
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.
Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands.
Ricardo
R
Coevoets
Sermin
S
Arican
Marianne
M
Hoogeveen-Westerveld
Erik
E
Simons
Ans
A
van den Ouweland
Dicky
D
Halley
Mark
M
Nellist
D001681:Biological Assay; D002478:Cells, Cultured; D004252:DNA Mutational Analysis / Q000379:methods; D014644:Genetic Variation; D006801:Humans; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D058570:TOR Serine-Threonine Kinases; D014402:Tuberous Sclerosis / Q000235:genetics; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics*
C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases