¹Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands

Correspondence: Dr M Nellist, Department of Clinical Genetics, Erasmus Medical Centre, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. Tel: +31 10 7043357; Fax: +31 10 7049489; E-mail: m.nellist@erasmusmc.nl

Received 15 April 2008; Revised 9 July 2008; Accepted 4 September 2008; Published online 15 October 2008.

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.