RESUMO
The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.
Assuntos
Variação Genética , Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Células Cultivadas , Humanos , Modelos Genéticos , Esclerose Tuberosa/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. 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). Recently it has been shown that missense mutations to the TSC1 gene can cause TSC. METHODS: We have used in vitro biochemical assays to investigate the effects on TSC1 function of TSC1 missense variants submitted to the Leiden Open Variation Database. RESULTS: We identified specific substitutions between amino acids 50 and 190 in the N-terminal region of TSC1 that result in reduced steady state levels of the protein and lead to increased mTOR signalling. CONCLUSION: Our results suggest that amino acid residues within the N-terminal region of TSC1 are important for TSC1 function and for maintaining the activity of the TSC1-TSC2 complex.
Assuntos
Mutação de Sentido Incorreto , Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Substituição de Aminoácidos , Linhagem Celular , Humanos , Microscopia de Fluorescência , Proteínas Quinases/genética , Transdução de Sinais , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Ras homolog enriched in brain (Rheb) couples growth factor signaling to activation of the target of rapamycin complex 1 (TORC1). To study its role in mammals, we generated a Rheb knockout mouse. In contrast to mTOR or regulatory-associated protein of mTOR (Raptor) mutants, the inner cell mass of Rheb(-/-) embryos differentiated normally. Nevertheless, Rheb(-/-) embryos died around midgestation, most likely due to impaired development of the cardiovascular system. Rheb(-/-) embryonic fibroblasts showed decreased TORC1 activity, were smaller, and showed impaired proliferation. Rheb heterozygosity extended the life span of tuberous sclerosis complex 1-deficient (Tsc1(-/-)) embryos, indicating that there is a genetic interaction between the Tsc1 and Rheb genes in mouse.