Identification of a leader exon and a core promoter for the rat tuberous sclerosis 2 (Tsc2) gene and structural comparison with the human homolog

Hereditary renal carcinoma in the Eker rat is an excellent example of predisposition to a specific cancer being transmitted as a dominant trait. Recently, we identified a germline mutation of the tuberous sclerosis 2 (Tsc2) gene in the Eker rat. In the present study, we analyzed the upstream region of the Tsc2 gene. A novel leader exon (exon 1a) in a CpG island was found, and core promoter activity was identified in a 242-bp region of this island. Exon 1a and the promoter region were conserved in the human TSC2 gene. In addition, a rat homolog of a gene found upstream of TSC2 in human has been identified, indicating that the genomic organization around Tsc2/TSC2 is conserved between the two species. Characterization of the 5' region of Tsc2 and TSC2 will facilitate studies of the regulation of the gene and its disregulation in tumorigenesis.     

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.     

Biallelic mutations of the Tsc2 gene in chemically induced rat renal cell carcinoma

A number of cancer genes have been identified by the study of hereditary human cancers and shown to be involved in sporadic genesis of the same tumors. We have identified a germline mutation in the rat homologue of the human tuberous sclerosis (TSC2) predisposing gene in the Eker rat model. In this study, we searched for mutations of the Tsc2 gene in chemically induced non-Eker rat renal cell carcinomas (RCs). N-ethyl-N-hydroxyethylnitrosamine (EHEN)- and diethylnitrosamine (DEN)-induced non-Eker rat primary RCs were subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis using specific primers covering all exons of the Tsc2 gene (41 coding exons and 1 non-coding exon). We simultaneously searched for mutations in the Vhl gene, a rat homologue of von Hippel-Lindau disease (VHL) gene, as well as the Tsc2 gene. Mutations in the Vhl gene were not detected in any rat RCs (0/8). In contrast, Tsc2 gene mutations were detected at a high frequency in EHEN-induced RCs (2/3) and DEN-induced RCs (3/5) (total 5/8) (p < 0.05). By a direct cloning approach utilizing PCR analysis in 2 applicable cases, we could demonstrate the presence of intragenic somatic mutations in both alleles of the Tsc2 gene. Our results suggest that Tsc2 gene inactivation plays an important role in EHEN- and DEN-induced RCs as well as in Eker rat RCs.     

Loss of heterozygosity in tuberous sclerosis hamartomas

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.     

"Ideal" length of stay after colectomy: whose ideal?

The tuberous sclerosis 2 (TSC2) gene is thought to function as a growth suppressor in sporadic and TSC-associated hamartomas and tumors. Clusters of dysplastic glial cells are a common feature of cortical tubers and subependymal nodules in tuberous sclerosis patients. In an effort to identify TSC2 gene alterations in sporadic gliomas, we detected a novel polymorphism adjacent to the 3'splice site of intron 4. We evaluated the distribution of this variant allele in a series of 244 patients with glial tumors, including 55 gangliogliomas, 31 pilocytic astrocytomas (WHO grade I), 50 astrocytomas (WHO grades II and III), and 108 glioblastomas (WHO grade IV). The allelic distribution in the general population was estimated by examining 381 healthy blood donors. This rare allele appeared in the control population and in the patients with astrocytic gliomas with a virtually identical frequency (8.14%, and 8.20%, respectively). The frequency of the rare allele in gangliogliomas, however, was significantly higher (15.5%; p = 0.024). The fact that both gangliogliomas and cortical tubers in tuberous sclerosis contain neuronal and astrocytic elements and may resemble each other histologically suggests that the TSC2 gene may be involved in the development of these tumors. The rare allele of the TSC2 gene emerges as a candidate for a predisposing factor for the formation of sporadic gangliogliomas.     

Estrogen treatment enhances hereditary renal tumor development in Eker rats

Hormonal influences are known to affect the development of renal cell carcinoma in man and laboratory animal models. We tested the hypothesis that estrogen treatment or ovariectomy of rats modulates renal tumor development using tuberous sclerosis 2 (Tsc2) heterozygous mutant (Eker) rats in which a germline mutation predisposes the animals to renal cell tumor development. Two-month-old female wild-type and Eker rats were ovariectomized or sham-operated and treated with placebo or 5 mg 17beta-estradiol in s.c. pellets for 6 or 10 months. Rats were examined at 8 or 12 months of age, at which time the numbers of renal tumors and preneoplastic foci were quantitated and the severity of nephropathy was assessed. In contrast to what may have been expected, prolonged estrogen treatment enhanced the development of hereditary renal cell tumors, with a 2-fold greater number of preneoplastic and neoplastic renal lesions compared with untreated Eker rats. Ovariectomized Eker rats had 33% fewer renal lesions than the unmanipulated control group. No tumors or preneoplastic lesions were present in wild-type rats at either time point. Estrogen treatment increased the severity of nephropathy in both wild-type and Eker rats, whereas ovariectomy was protective against nephropathic changes. Although estrogen is not a rat renal carcinogen, it enhanced the development of hereditary renal cell tumors when administered to Eker rats. Eker rats heterozygous for a mutation in the Tsc2 locus provide a good model in which to study how genetic and hormonal factors contribute to the development of renal cell tumors and to understand the influence genetic susceptibility has on the development of renal cell carcinoma.     

Study of the viscosity of excited membranes using combined dispersion spectroscopy

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous lesions. Although hamartomas can occur in almost any organ, they are most common in the brain, kidney, heart, and skin. Allelic loss or loss of heterozygosity (LOH) in TSC lesions has previously been reported on chromosomes 16p13 and 9q34, the locations of the TSC2 and TSC1 genes, respectively, suggesting that the TSC genes act as tumor-suppressor genes. In our study, 87 lesions from 47 TSC patients were analyzed for LOH in the TSC1 and TSC2 chromosomal regions. Three findings resulted from this analysis. First, we confirmed that the TSC1 critical region is distal to D9S149. Second, we found LOH more frequently on chromosome 16p13 than on 9q34. Of the 28 patients with angiomyolipomas or rhabdomyomas, 16p13 LOH was detected in lesions from 12 (57%) of 21 informative patients, while 9q34 LOH was detected in lesions from only 1 patient (4%). This could indicate that TSC2 tumors are more likely than TSC1 tumors to require surgical resection or that TSC2 is more common than TSC1 in our patient population. It is also possible that small regions of 9q34 LOH were missed. Lastly, LOH was found in 56% of renal angiomyolipomas and cardiac rhabdomyormas but in only 4% of TSC brain lesions. This suggests that brain lesions can result from different pathogenic mechanisms than kidney and heart lesions.     

Distribution of glial fibrillary acidic protein in central nervous system lesions of tuberous sclerosis

The distribution of glial fibrillary acidic protein (GFAP) in the central nervous system (CNS) lesions of tuberous sclerosis (TS) was examined using antiserum against GFAP and the peroxidase antiperoxidase method of Sternberger. In cortical tubers there were islands of gemistocytic astrocytes staining intensely for GFAP and occasional giant cells having some cytoplasmic staining. The majority of the cortical giant cells had no GFAP. The islands were separated by areas devoid of astrocytes with perikaryal staining. A faintly staining fibrous network was found between these islands. The majority of cells in the subependymal nodules stained. The retinal phakoma stained but not as intensely as the subependymal nodules. There was no staining whatsoever in the giant cell subependymal tumors. Absence of GFAP staining in the subependymal giant cell tumors makes their classification as astrocytomas less certain.     

Medication control in hospitals: a practical approach to the problem of drug-drug interactions

We present 19 patients with tuberous sclerosis complex and subependymal giant cell astrocytoma. The mean age at the time of tumor diagnosis was 9.4 years (range, 1.5 to 21 years). Computed cranial tomography (CT) or cranial magnetic resonance imaging (MRI) identified the lesion which was resected in all cases. Seven patients had hydrocephalus and there was an interval increase in the tumor size or a large tumor without hydrocephalus in 12 patients. Surgical criteria included: (1) presence of hydrocephalus; (2) interval increase in tumor size; (3) new focal neurologic deficit attributable to the tumor; and/or (4) symptoms of increased intracranial pressure. Eight patients were identified through a surveillance program involving annual computed cranial tomography. All of these eight patients had their tumor removed prior to the development of symptoms, none had neurologic deficits which persisted after surgery, and none has so far developed recurrent subependymal giant cell astrocytoma. In contrast, of the 11 patients from the non-surveillance group 7 were symptomatic at tumor diagnosis, 1 had a complicated postoperative course, 2 developed recurrent giant cell astrocytoma, and 1 had an extensive lesion that could not be completely excised. Periodic cranial imaging may help to identify subependymal giant cell astrocytomas in tuberous sclerosis patients before they become symptomatic. Earlier diagnosis and treatment could reduce surgical morbidity and the risk of tumor recurrence.     

Diagnosis and management of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal-dominant neurocutaneous disorder with a high spontaneous mutation rate. Understanding of this disorder has greatly increased in recent years. Two chromosomal loci can produce the TSC phenotype: 9q34 and 16p13. These appear to code for proteins that have a tumor suppressor function. TSC results in hamartomas that affect various organ systems, most commonly brain, skin, heart, and kidney. Previously thought to consist of intractable seizures, facial angiofibromas, and dementia, increasing numbers of persons with less severe involvement have been identified. Diagnostic criteria, various types of lesions, and medical management are reviewed.     

Generation of metastatic variants of Eker renal carcinoma cell lines for experimental investigation of renal cancer metastasis

We and others have demonstrated that a mutation in Tsc2 is the rate-limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S-Lk9d-SLM cell lines from a non-metastatic RC cell line (Lk9dL) by co-implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis-promoting events in renal carcinogenesis.     

Malignant tumors of the kidney, brain, and soft tissues in children and young adults with the tuberous sclerosis complex

BACKGROUND: The tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the brain, heart, skin, and kidney. Malignant tumors also can occur in patients with tuberous sclerosis, particularly in the kidney, although they occur less frequently than benign tumors. The types of malignancy that occur in TSC have not been characterized fully. METHODS: Clinical and pathologic features of 8 malignant tumors from 6 TSC patients ranging in age from 22 months to 21 years are reviewed. Six tumors were renal, one was from the inguinal region, and one was from the brain. The tumors were analyzed for loss of heterozygosity (LOH) in the chromosomal regions of the TSC1, TSC2, and VHL genes. RESULTS: Three patients (ages 7, 8, and 20 years) had renal cell carcinomas (RCCs). Two of these patients had multifocal RCCs. All three patients with RCC also had prominent multifocal dysplasia of renal cyst epithelium. Two patients (ages 20 and 21 years) had malignant angiomyolipomas (1 renal and 1 inguinal). One patient (age 22 months) had a Grade 4 giant cell astrocytoma (glioblastoma multiforme). LOH in the region of the TSC2 gene was found, either in the malignant tumor or in benign tumors, in all five patients whose DNA could be analyzed. CONCLUSIONS: Children with TSC, as well as adults with the disease, are at risk for developing malignant tumors. Two types of renal malignancy occur in TSC: RCC, which appears to arise from dysplastic renal cyst epithelial cells, and malignant angiomyolipoma. Tumors cytologically similar to malignant angiomyolipomas also may occur at extrarenal sites. LOH analyses suggest that the majority of patients with TSC who develop malignant tumors have germline TSC2, rather than TSC1, gene mutations.     

Loss of tuberin from cerebral tissues with tuberous sclerosis and astrocytoma

We studied the expression of tuberin, the TSC2 gene product, in cerebra with or without tuberous sclerosis. Tuberin was abundant in the gray matter of normal cerebra, but was undetectable in the subependymal astrocytomas from 3 patients with tuberous sclerosis. In 1 patient, cortical tubers and normally appearing cortical tissue also showed a marked loss of tuberin. These results indicate the critical role of tuberin in the neuropathology of tuberous sclerosis.     

Subependymal tumor with metaplastic bone formation: a case report

A 26-year-old woman was admitted to our hospital because of headache. CT scan and MRI showed a right subependymal nodule and a left ventricular tumor, neither of which had any enhancement nor were they stained in angiography. Although no skin abnormality was detected, the patient was suspected of tuberous sclerosis. The diagnosis was made because of the subependymal nodule on CT scan and MRI. On June 29, 1995, total removal of a left ventricular tumor was performed by a transcortical approach. Histological sections of this tumor consisted of astrocytic and meningothelial components, containing metaplastic bone formation. Histological diagnosis was dysplastic subependymal tumor. Postoperative course was uneventful. Regrowth of the tumor has not been observed as of now. This case was suspected to involve factors of tuberous sclerosis from a subependymal nodule. However, the ventricular tumor was not diagnosed as a subependymal giant cell astrocytoma.     

Skin lesions in children with tuberous sclerosis complex: their prevalence, natural course, and diagnostic significance

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by widespread cutaneous and visceral hamartomas. METHODS: The prevalence of cutaneous lesions in 106 children with TSC (47 boys and 59 girls) aged 1 month-18 years was evaluated from 1984 to 1995. Assessing the diagnostic usefulness of each National Tuberous Sclerosis Association skin criterion was an aim of this study. RESULTS: Hypopigmented macules were the most frequent finding, seen in 103 of 106 children (97.2%). In 66 children they were evident at birth, and in 20 others their presentation was delayed until the first months of age. Facial angiofibromas were seen next most often (79 of 103, 74.5%), followed by a shagreen patch in 51 of 103 (48.1%), "cafe-au-lait" macules in 30 of 103 (28.3%), molluscum fibrosum pendulum (24 of 103, 22.6%), a forehead fibrous plaque (20 of 103, 18.9%), periungual fibromas (16 of 103, 15.1%) and "confetti-like" macules (3 of 103, 2.8%). The hypomelanotic macules were seen within the first 2 years of life in 95 children, as were café-au-lait spots in 24, facial angiofibromas in eight, shagreen patches in six, and forehead fibrous plaques in six, whereas molluscum pendulum and periungual fibromas were not evident. Seizures were seen in 102 of 106 children (98%), with 80 (75%) occurring during the first year of life. CONCLUSIONS: Hypomelanotic macules were the overwhelmingly most common early finding in TSC. Infants with seizures or other possible stigmata of TSC should be carefully evaluated for these hypomelanotic macules, as well as for other associated findings.     

Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation

Rats carrying the Eker tumor susceptibility mutation are genetically predisposed to renal cell carcinoma. Rats heterozygous for the Eker mutation (Eker carriers) develop multiple bilateral renal cell carcinomas by the age of 1 year. Using an in vitro rat kidney epithelial (RKE) transformation assay developed in our laboratory, proximal tubule cells derived from known Eker rat carriers (+/ek) and non-carriers (+/+) were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to determine if cells derived from Eker carriers were more susceptible to in vitro transformation than cells derived from non-carrier animals. The percent transformation frequency following MNNG treatment was 7.5-fold higher in cells derived from carrier animals when compared to cells from non-carrier animals. This increased susceptibility to transformation due to inheritance of the Eker mutation is consistent with a predisposition resulting from inactivation of a tumor suppressor gene. The increased susceptibility of kidney epithelial cells carrying the Eker mutation may prove useful in the further development of the RKE transformation assay as a sensitive tool to identify potential renal carcinogens. In addition, because transformation frequency in the RKE assay measures a very early step in multistage transformation, these results also suggest that alterations (by Loss of Heterozygosity or mutation) at the Eker tumor susceptibility locus are an early event in the development of renal tumors in the rat.     

Evolution of cardiac rhabdomyoma in tuberous sclerosis complex

The objective of the study was to define the longitudinal evolution of cardiac rhabdomyomas (CR) in patients with tuberous sclerosis complex (TSC). A cohort of patients with TSC who had undergone videotaped echocardiographic (ECHO) examination during the 10-year interval (1984-1994) were retrospectively studied by reviewing and quantifying the CR appearance and associated cardiac abnormalities in sequentially obtained ECHO examinations. Sixteen patients with TSC (8 males) underwent a total of 35 recorded studies. Ten of the 16 (62.5%) had CR identified at initial study; none were found in the atria. Localization was the ventricular walls as compared with the ventricular septum by a ratio of 2:1. The number of CRs sequentially studied declined as follows; initial study: 23 lesions in 10 patients; second study: 16 lesions in 8 patients; third study: 12 lesions in 5 patients; and fourth study: 4 lesions in 2 patients. Total CR size index declined at each study as follows: initial index of 2,684; second index of 1,746 (-35% from initial); third index 1,141 (-57% from initial); and fourth index 705 (-74% from initial). Complete spontaneous regression of CR was seen by age 6 years with prolonged gradual resolution thereafter. Two patients had bicuspid aortic valves and two had conduction defects. Patients with TSC who have CR can be expected to experience a decline in both the number and size of CR over time; early complete regression on ECHO occurs before age 6 years.