Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 tumour suppressor gene. The disease is characterized by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction and dermatological abnormalities. TSC2 encodes tuberin, a putative GTPase activating protein for rap1 and rab5. The TSC1 gene was recently identified and codes for hamartin, a novel protein with no significant homology to tuberin or any other known vertebrate protein. Here, we show that hamartin and tuberin associate physically in vivo and that the interaction is mediated by predicted coiled-coil domains. Our data suggest that hamartin and tuberin function in the same complex rather than in separate pathways.      

Novel TSC2 mutations and decreased expression of tuberin in cultured tumor cells with an insertion mutation

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by hamartomas in many organs. Two genes responsible for TSC, TSC1 and TSC2, were recently identified. TSC1 and TSC2 encode the proteins hamartin and tuberin, respectively, and 337 different mutations have been reported in these genes thus far. Here, we report six novel TSC2 mutations including one missense mutation, two nonsense point mutations, two frameshifts, and an insertion mutation. The insertion mutation is unique because of its location at an exon/intron boundary that results in triplication of a 34-bp sequence. Cultured tumor cells from the patient with this insertion mutation exhibited a decreased level of tuberin as revealed by Western blotting, suggesting that the mRNA of TSC2 is not translated as efficiently or the translated protein exhibits reduced stability. Five novel polymorphisms of TSC2 were also identified. As previously reported, the missense mutations were located in the GTPase activating protein-related domain of TSC2 encoded in exons 34-38. No TSC1 mutations were identified in the present subjects.     

Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.

Critical functions of hamartin and tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302-430) and tuberin (amino acids 1-418) interacted strongly with one another. A region of tuberin encoding a putative coiled-coil (amino acids 346-371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719-998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593-595delACT) or tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and tuberin.     

Analysis of all exons of TSC1 and TSC2 genes for germline mutations in Japanese patients with tuberous sclerosis: report of 10 mutations

Twenty-seven Japanese patients with the tuberous sclerosis complex (TSC), consisting of 23 sporadic and 4 familial cases, were tested for mutations in the TSC1 and TSC2 genes, using single-strand conformational polymorphism analysis and direct sequencing. Four possible pathogenic mutations were found in the TSC1 gene, including three frame shifts and a nonsense mutation in a familial case. All mutations were expected to result in a truncated hamartin gene product. The TSC2 gene analysis identified six possible pathogenic mutations only in the sporadic cases, including two frame shifts, one in-frame deletion, and three missense mutations. Two of the TSC2 mutations were expected to result in a truncated tuberin gene product. These results of the Japanese TSC patients were compatible with the reports from Europe and the United States, i.e., (1) TSC1 mutations are rarer in sporadic cases than in familial cases, (2) substantial numbers of sporadic cases arise from mutations in the TSC2 gene, and (3) mutations of the TSC1 gene may cause premature truncation of hamartin.     

Expression of the TSC2 product tuberin and its target Rap1 in normal human tissues.

The tuberous sclerosis-2 (TSC2) gene is linked to tuberous sclerosis (TSC), a dominantly inherited genetic syndrome in which inactivation of the normal TSC2 allele is associated with the development of mostly benign tumors and focal dysplasias. TSC2 encodes the protein tuberin, which is a widely expressed 180-kd polypeptide that exhibits specific GTPase activating activity toward Rap1 in vitro and co-localizes with Rap1 in cultured cells. In this study, we have performed immunohistochemical analyses, using affinity-purified anti-tuberin antibodies, to study the distribution of tuberin in a panel of normal human organs that are commonly affected by TSC. Cryosections indicated that tuberin is widely expressed at low levels. More intense staining of tuberin, in the cryosections and in paraffin sections, was observed in the small blood vessels of many organs, including the kidney, skin, and adrenal gland. High levels of tuberin were also detected in cortical neurons and cerebellar Purkinje cells. These findings imply that loss-of-function mutations in TSC2 might lead to the development of highly vascularized tumors, subcortical tubers, and focal atrophy of the cerebellar cortex, which are features commonly associated with TSC. Moreover, Rap1 was also found to be highly expressed in many of the same cells that contained high levels of tuberin, suggesting a functional interaction between tuberin and Rap1 in these tissues.     

Distinct effects of single amino-acid changes to tuberin on the function of the tuberin-hamartin complex

Tuberous sclerosis is an autosomal dominant human disorder caused by inactivating mutations to either the TSC1 or TSC2 tumour suppressor gene. Hamartin and tuberin, the TSC1 and TSC2 gene products, interact and the tuberin-hamartin complex inhibits cell growth by antagonising signal transduction to downstream effectors of the mammalian target of rapamycin (mTOR) through the small GTPase rheb. Previously, we showed that pathogenic tuberin amino-acid substitutions disrupt the tuberin-hamartin complex. Here, we investigate how these mutations affect the role of tuberin in the control of signal transduction through mTOR. Our data indicate that specific amino-acid substitutions have distinct effects on tuberin function.     

Genetic analysis and prenatal diagnosis for ten families affected with tuberous sclerosis complexCSCD

Objective: To provide prenatal diagnosis for families affected with tuberous sclerosis complex and explore the correlation between phenotype and genotype. Methods: For probands from 10 families, all exons and splicing regions of the TSC1 and TSC2 genes were analyzed with high-throughput DNA sequencing. Suspected mutations were verified by Sanger sequencing. Results: All probands were found to have mutations, which included 1 case with TSC1 mutation and 9 cases with TSC2 mutations (missense mutations in 6, nonsense mutations in 2, and frameshifting mutation in 1 case). Prenatal diagnosis was provided for 9 cases, and 1 fetus was found to carry a mutation. Genetic analysis has identified a novel pathogenic mutation (TSC2 c. 2415-2416 ins GT). Conclusion: Identification of pathological mutations for tuberous sclerosis complex can facilitate genetic counseling and prenatal diagnosis for the affected families. © 2018 West China University of Medical Sciences. All rights reserved.     

Cloning and evaluation of RALGDS as a candidate for the tuberous sclerosis gene TSC1

RALGDS is a 115 kDa protein which was identified by its ability to enhance guanine nucleotide exchange for the ras family member ral . It also binds to activated ras and rap1 , and appears to function as part of a signalling complex in downstream events following rap1 activation. Here we report the identification of full-length cDNA clones for human RALGDS, isolated from a brain cDNA library. The predicted protein has strong sequence homology to rat and murine isoforms of RALGDS in the N- and C-terminal regions, but an internal region (aa 250–380) shows relatively high divergence with only 42% identical amino acid residues. The human RALGDS gene is contained within a 30 kb region of 9q34, approximately 200 kb proximal to the ABO gene, within the current critical region for the tuberous sclerosis gene TSC1. Partial genomic structure was determined; it consists of at least 11 exons. Based upon analysis of Southern blots from 110 TSC patients, genomic DNA SSCP analysis, and RT-PCR analysis which demonstrated RNA expression of both alleles in patients from 9q34-linked TSC families using intragenic polymorphisms, we conclude that RALGDS is not likely to be TSC1.     

Co-Localization of TSC1 and TSC2 Gene Products in Tubers of Patients with Tuberous Sclerosis

Two genes, mutations in which result in the phenotype of tuberous sclerosis (TSC), have recently been cloned. TSC2 on chromosome 16p 13.3 encodes the protein tuberin, which appears to have growth regulating properties. TSC1 on chromosome 9q34 encodes hamartin which, as yet, has no specified cellular functions. Polyclonal antibodies were raised to synthetic peptides representing portions of tuberin and hamartin and used in immunoblots and immunohistochemical studies to localize the proteins in surgically resected neocortical tubers from four TSC patients. On Western blots of autopsy brain specimens, K-562 cell, and NT2 lysates, each antibody labelled a single band at the expected molecular weight. In immunohistochemical protocols on paraffin embedded tissue, antibodies to both tuberin and hamartin prominently labelled atypical and dysmorphic neuroglial cells that are a defining feature of TSC tubers. Some abnormal cells within cortical tuber sections were labelled with both tuberin and hamartin antisera. Our results suggest that tuberin and hamartin are both robustly expressed in similar populations of neuroglial cells of TSC tubers, even in the presence of TSC1 or TSC2 germline mutations. The roles of these gene products in normal and abnormal cortical development, tuber pathogenesis and the generation of seizures remain to be defined.     

TSC基因的生物学作用

TSC基因TSC1和TSC2分别编码蛋白hamartin和tuberin,最初在结节性硬化症中发现其存在突变,作为肿瘤抑制因子,除参与mTOR信号途径调节细胞的生长和增殖外,还参与细胞黏附,细胞内吞等过程的调节.     

Genetic analysis and prenatal diagnosis for ten families affected with tuberous sclerosis complexCSCD

Objective: To provide prenatal diagnosis for families affected with tuberous sclerosis complex and explore the correlation between phenotype and genotype. Methods: For probands from 10 families, all exons and splicing regions of the TSC1 and TSC2 genes were analyzed with high-throughput DNA sequencing. Suspected mutations were verified by Sanger sequencing. Results: All probands were found to have mutations, which included 1 case with TSC1 mutation and 9 cases with TSC2 mutations (missense mutations in 6, nonsense mutations in 2, and frameshifting mutation in 1 case). Prenatal diagnosis was provided for 9 cases, and 1 fetus was found to carry a mutation. Genetic analysis has identified a novel pathogenic mutation (TSC2 c. 2415-2416 ins GT). Conclusion: Identification of pathological mutations for tuberous sclerosis complex can facilitate genetic counseling and prenatal diagnosis for the affected families. © 2018 West China University of Medical Sciences. All rights reserved.     

The TSC1 gene product, hamartin, negatively regulates cell proliferation

Tuberous sclerosis is an autosomal dominant hereditary disease caused by mutations in either the TSC1 or the TSC2 tumor suppressor gene. The TSC1 gene on chromosome 9q34 encodes a 130 kDa protein named hamartin, and the TSC2 gene on chromosome 16p13.3 codes for tuberin, a 200 kDa protein. Here we show that expression of hamartin, assayed by immunoblot analyses, is high in G(0)-arrested cells and hamartin is expressed throughout the entire ongoing cell cycle. An interaction of hamartin and tuberin can be detected in every phase of the cell cycle. Ectopic expression of high levels of hamartin attenuates cellular proliferation. We provide evidence that this effect could depend on a coiled-coil region earlier proposed to be involved in binding of hamartin to tuberin. Further investigations revealed that hamartin affects cell proliferation via deregulation of G(1) phase. Our data have a clear impact on understanding the role of hamartin during development of this disease.     

Mutation screening of the entire coding regions of the TSC1 and the TSC2 gene with the protein truncation test (PTT) identifies frequent splicing defects.

Mutation analyses in tuberous sclerosis (TSC) have reported a wide variety of disease-causing aberrations in the two known predisposing genes, TSC1 and TSC2 on chromosomes 9q34 and 16p13, comprising mainly small mutations distributed over the entire genes. So far, all known TSC1 mutations as well as the majority of TSC2 mutations truncate the proteins hamartin and tuberin, respectively. We describe for the first time an RNA-based screening of the entire coding regions of both TSC genes for truncating mutations applying the protein truncation test (PTT). Simultaneous investigation of both TSC genes in a group of 48 unassigned TSC patients, which were previously tested to exclude large intragenic TSC2 rearrangements, revealed aberrant migrating polypeptides resulting from truncating mutations in nine TSC1 cases and in 16 TSC2 cases while three TSC2 cases showed enlarged proteins. TSC1 mutations include two nonsense mutations, four insertions, and three splice mutations. Nineteen mutations identified in TSC2 were composed of four different nonsense mutations in five patients, one deletion, one insertion, and seven different splicing aberrations due to at least eight different mutations found in 12 patients. Additional predicted truncating mutations according to PTT without possible identification of the causative alteration allowed assignment to TSC1 in one and TSC2 in seven cases. Twelve patients without abnormalities in the PTT are assumed to harbor missense mutations, probably in TSC2. The high proportion of TSC2 splicing aberrations strengthens the importance of intronic disease-causing mutations and the application of RNA-based screening methods to confirm their consequences.     

Mutational analysis of TSC1 and TSC2 genes in Japanese patients with tuberous sclerosis complex

We have surveyed the mutations of TSC1 and TSC2 from 38 (25 sporadic, 11 familial, and 2 unknown) Japanese patients with tuberous sclerosis complex. In 23 of 38 subjects, we detected 18 new mutations in addition to 4 mutations that had been previously reported. We also found 3 new polymorphisms. The mutations were not clustered on a particular exon in either of the genes. Seven TSC1 mutations found in 3 familial and 4 sporadic cases were on the exons (3 missense, 2 nonsense point mutations, a 1-base insertion, and a 2-bp deletion). Fifteen TSC2 mutations were found in 5 familial cases, 10 sporadic cases, and 1 unknown case. The 12 mutations were on the exons (8 missense, 1 nonsense point mutations, a 1-bp insertion, a 5-bp deletion, and a 4-bp replacement) and 3 point mutations were on the exon–intron junctions. Although the patients with TSC2 mutations tend to exhibit relatively severe mental retardation in comparison to those with TSC1 mutations, a genotype–phenotype correlation could not yet be established. The widespread distribution of TSC1/TSC2 mutations hinders the development of a simple diagnostic test, and the identification of individual mutations does not provide the prediction of prognosis. Received: April 5, 1999 / Accepted: June 12, 1999     

A mutation screen of the TSC1 gene reveals 26 protein truncating mutations and 1 splice site mutation in a panel of 79 tuberous sclerosis patients

The entire coding region of the TSC1 gene has been screened for mutations in 79 unrelated patients with tuberous sclerosis. Causative mutations have been found in 27 of these patients and five other variations in the gene have been identified. 26 of the mutations are predicted to cause premature truncation of the protein product of the gene and one mutation is in a splice site. The mutation screen has revealed that TSC1 mutations are rarer in sporadic tuberous sclerosis patients than in familial cases. We have also found that the only previously described case of non-penetrance can no longer be described as such, and that a single ungual fibroma is not necessarily diagnostic of tuberous sclerosis, important findings for the genetic counselling of tuberous sclerosis patients.     

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.     

The expression of hamartin, the product of the TSC1 gene, in normal human tissues and in TSC1- and TSC2-linked angiomyolipomas.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous tumors in multiple organs, including subependymal giant cell astrocytomas, cardiac rhabdomyomas, and renal angiomyolipomas. Mutations in two genes are associated with TSC: TSC1, which was cloned in 1997, and TSC2, which was cloned in 1993. We report here the expression of hamartin, the product of the TSC1 gene, in normal human tissues and in renal angiomyolipomas from TSC1- and TSC2-linked patients. By Western blot analysis, hamartin is strongly expressed in brain, kidney, and heart, all of which are frequently affected in TSC. By immunohistochemical analysis, the expression pattern of hamartin in normal human tissues was almost identical to that of tuberin, the product of the TSC2 gene. This is consistent with the recent finding that tuberin and hamartin interact and with the clinical similarity between TSC1- and TSC2-linked disease. Strong hamartin expression was seen in cortical neurons, renal tubular epithelial cells, pancreatic islet cells, bronchial epithelial cells, and pulmonary macrophages. Hamartin was also expressed in endocrine tissues, including islet cells of the pancreas, follicular cells of the thyroid, and the zona reticularis of the adrenal cortex. In eight angiomyolipomas from a TSC1-linked patient, no hamartin expression was detected, whereas tuberin, the product of the TSC2 gene, was expressed. In 19 angiomyolipomas from a TSC2-linked patient, in whose angiomyolipomas loss of tuberin expression had previously been shown, hamartin expression was present. These data suggest that tuberin and hamartin immunoreactivity can distinguish tumors with underlying TSC1 mutations from those with TSC2 mutations. This differentiation might have diagnostic implications.     

Protein truncation test for screening hamartin gene mutations and report of new disease-causing mutations.

Considering the prevalence of truncating mutations in the tuberous sclerosis (TSC) hamartin gene (TSC1), we devised a protein truncation test (PTT) to analyze the full length coding sequence of TSC1. Studying 12 sporadic cases and three familial forms by a combination of PTT and single-strand conformation polymorphism analysis (SSCA), we found 5/15 mutations while PTT alone detected 4/15 truncating mutations, two of which escaped SSCA analysis. SSCA alone picked up one missense mutation and two mutations also detected by PTT. Interestingly, a TSC1 mutation was identified in all three familial forms (3/3) while the rate of mutation detection was lower in sporadic cases (2/12). In conclusion, PTT proves to be a useful technique for the rapid detection of disease-causing mutations in the TSC1 gene.