Large deletion at the TSC1 locus in a family with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized 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 on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. Here we describe a deletion encompassing the TSC1 gene and two neighboring transcripts on chromosome 9q34 in six affected individuals from a family with TSC. To our knowledge, this is the first report of such a large deletion at the TSC1 locus and indicates that screening for similar mutations at the TSC1 locus is warranted in individuals with TSC.

Differential localization of hamartin and tuberin and increased S6 phosphorylation in a tuber.

In a tuberous sclerosis patient with a mutation in the TSC1 tumor suppressor gene, no second-hit mutation was found in a resected cortical tuber. Tuber giant cells showed predominantly nuclear hamartin, cytosolic tuberin, and hyperphosphorylation of S6. Differential accumulation of hamartin and tuberin in separate cellular compartments of giant cells may prevent formation of the hamartin-tuberin complex, resulting in increased S6 phosphorylation. These data provide an alternative mechanism for tuberogenesis.

Regulation of tuberous sclerosis complex (TSC) function by 14-3-3 proteins.

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by seizures, mental disability, renal dysfunction and dermatological abnormalities. The disease is caused by inactivation of either hamartin or tuberin, the products of the TSC1 and TSC2 tumour-suppressor genes. Hamartin and tuberin form a complex and antagonise phosphoinositide 3-kinase/protein kinase B/target of rapamycin signal transduction by inhibiting p70 S6 kinase, an activator of translation, and activating 4E-binding protein 1, an inhibitor of translation initiation. Phosphorylation-dependent binding between tuberin and members of the 14-3-3 protein family indicates how the tuberin-hamartin complex may interact with upstream and downstream effectors, and suggests how phosphorylation-dependent regulation of the complex may be controlled.

TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin-hamartin complex.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Inactivating mutations to either of the TSC1 and TSC2 tumour suppressor genes are responsible for the disease. TSC1 and TSC2 encode two large novel proteins called hamartin and tuberin, respectively. Hamartin and tuberin interact directly with each other and it has been reported that tuberin may act as a chaperone, preventing hamartin self-aggregation and maintaining the tuberin-hamartin complex in a soluble form. In this study, the ability of tuberin to act as a chaperone for hamartin was used to investigate the tuberin-hamartin interaction in more detail. A domain within tuberin necessary for the chaperone function was identified, and the effects of TSC2 missense mutations on the tuberin-hamartin interaction were investigated to allow specific residues within the central domain of tuberin that are important for the interaction with hamartin to be pin-pointed. In addition, the results confirm that phosphorylation may play an important role in the formation of the tuberin-hamartin complex. Although mutations that prevent tuberin tyrosine phosphorylation also inhibit tuberin-hamartin binding and the chaperone function, our results indicate that only hamartin is phosphorylated in the tuberin-hamartin complex.

Analysis of TSC2 stop codon variants found in tuberous sclerosis patients.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations to the TSC1 and TSC2 tumour suppressor genes. We detected two sequence changes involving the TSC2 stop codon and investigated the effects of these changes on the expression of tuberin, the TSC2 gene product, and on the binding between tuberin and the TSC1 gene product, hamartin. While elongation of the tuberin open reading frame by 17 amino acids did not interfere with tuberin-hamartin binding, a longer extension prevented this interaction. Our data illustrate how functional protein assays can assist in the verification and characterisation of disease-causing mutations.

Immunological detection of polycystin-1 in human kidney.

Polycystin-1 is the protein product of the PKD1 gene. Mutations in this gene are responsible for most cases of polycystic kidney disease, but little is known about how these mutations lead to the development of cysts. Indeed, even less is known about the normal role of polycystin-1 in the kidney. The cellular localization of polycystin-1 has been the subject of intense investigation by many groups, including ours. In this report we describe our results and compare our data with those of others. We generated 14 different polyclonal antisera against fragments of the predicted 462-kDa polycystin-1 molecule to enable us to investigate the expression of polycystin-1 in cells and tissues by immunocytochemistry, western blotting, and immunoprecipitation. Our antibodies readily recognized a 134-kDa polycystin-1 fragment overexpressed in COS cells and stained the epithelial cells of fetal, adult, and cystic kidney sections with the same pattern as reported by others. However, further investigations revealed that this pattern was not specific for polycystin-1. We could not unequivocally detect polycystin-1 in vivo, either by immunoblotting or immunocytochemistry. Therefore our studies do not support the reported pattern of polycystin-1 expression in the kidney.

The Han:SPRD rat is not a genetic model of human autosomal dominant polycystic kidney disease type 1.

Human autosomal dominant polycystic kidney disease (ADPKD) is a high incidence disorder leading to renal failure in many patients. The majority of cases results from a mutation in the PKD1 gene. The only well documented animal model of ADPKD is the Han:SPRD-Pkd strain. Its genetic basis is unknown as yet. In the current study we determined whether the disease in these rats is genetically linked to the rat homologue of the PKD1 gene. We used the protamine gene as a polymorphic marker (Prm1) of the PKD1 region. Matings of Han:SPRD-Pkd with BB rats and backcross of the offspring with BB yielded animals informative for linkage analysis. This analysis revealed random segregation of the defect and the Prm1 marker, indicating that the model is not caused by a mutation in the PKD1 gene. We conclude that the Han:SPRD-Pkd rat strain is not a genetic model of PKD1.

Angiotensin II induced expression of transcription factors precedes increase in transforming growth factor-beta 1 mRNA in neonatal cardiac fibroblasts.

Angiotensin II (ANG II), a potent vasoconstricting peptide, may act as a growth factor for cardiac muscle cells and induce hypertrophy. We examined the molecular phenotype of neonatal rat cardiac fibroblasts in relation to ANG II by studying the expression pattern of three transcription factors (Egr-1, c-fos and c-jun) and the transforming growth factor-beta 1 (TGF-beta 1). ANG II did not affect cell proliferation and growth of serum deprived neonatal cardiac fibroblasts as predicted from their DNA and protein contents. The expression of Egr-1 and c-fos was induced as early as 15 min that reached maximal levels at 45 min and declined thereafter, whereas c-jun was induced at 45 min and remained elevated up to 2 hrs of ANG II addition. ANG II up-regulated the expression of TGF-beta 1, which became apparent after 1 hr of incubation and reached a plateau between 16-48 hrs. Our results indicate that ANG II transiently stimulates the expression of transcription factors, which may up-regulate TGF-beta 1, that in turn could contribute to the process of myocardial extra-cellular matrix remodeling in hypertrophy.