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[501] Functional Analysis of the von-Hippel Lindau Gene Product. Kaelin WG Jr. Dana-Farber Cancer Institute, Boston, Massachusetts.
Tumor development in von Hippel-Lindau patients is due to mutation or loss of the remaining wild-type VHL gene (located at chromosome 3p25) in susceptible cells. In keeping with Knudson's hypothesis, this gene is also inactivated in the majority of sporadic renal cell carcinomas and cerebellar hemangioblastomas.
Reintroduction of a wild-type VHL cDNA into VHL-/- renal carcinoma cells (RCC) inhibits their ability to form tumors in nude mice. The VHL gene product (pVHL) bears little resemblance to other proteins in currently available databases. A frequently mutated region of pVHL binds to elongin B and C in vitro and in vivo. Elongin B and C, when bound to elongin A, generate a transcriptional elongation complex called elongin/SIII. pVHL competes with elongin A for binding to elongin B and C, thereby inhibiting elongin/SIII activity.
We have shown that VHL-/- RCC produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain (PDGF-B) under normoxic and hypoxic conditions.
Reintroduction of wild-type pVHL into these cells specifically inhibited the production of these mRNAs under normoxic conditions, thus restoring their previously described hypoxia-inducible profile. Thus, pVHL appears to play a critical role in transduction of signals generated by changes in ambient oxygen tension. Deregulation of VHL-associated gene expression may account for the hypervascular nature of VHL-associated neoplasms.
[502] Pathology of Hemangioblastomas and Endolymphatic Sac Tumors. Richard S. Laboratoire de Neuro-Oncologie, EPHE, Hôpital Necker, Paris, France.
Hemangioblastoma is the emblematic lesion of von Hippel-Lindau (VHL) disease. Its sites of predilection are the neuraxis (60% of 495 French patients) and the retina (50%). This benign neoplastic entity shows a double, vascular and cellular, differentiation. Within a well-defined capillary network are found trabeculae of polyhedric lipid-laden cells ("stromal cells"). A large number of mast cells is also frequently present. The principal differential diagnosis of hemangioblastoma consists of metastasis from a "clear cell" renal carcinoma; of course both are frequently associated in VHL disease.
Despite numerous immunochemical and electron microscopic studies, the histogenic origin of hemangio-blastoma remains enigmatic. The strong vimentin positivity of stromal cells, in conjunction with their lack of reactivity to the majority of other markers, led to the conclusion that stromal cells are poorly differentiated cells of mesenchymal origin. There is now further evidence to suggest fibro-histiocytic differentiation. More recently, a diffuse cytoplasmic expression of ezrin, that is normally restricted to epithelial cells, has been demonstrated. The cellular origin of erythropoietin, responsible for the polycythemia secondary to cerebellar hemangioblastoma, has not been elucidated.
In contrast to hemangioblastoma, endolymphatic sac tumors are very rare, presenting as a locally aggressive papillary tumor of temporal bone. Fewer than 60 cases have been reported, and 15% were associated with VHL. The incidence of this unusual tumor is probably under-estimated; it may be histologically confused with choroid plexus papilloma or metastasis from papillary adeno-carcinomas.
[503] Somatic VHL Mutations in Clear Cell Renal Carcinoma. Brauch H1, Hornauer M1, Rödi H1, Filipowicz M1, Feurer M1, Weirich G1, Pomer S2, Block T3, Störkel S4, Glavic D5. 1,5Laboratory of Molecular Pathology, Technical University Munich, Germany, and University of Ljubjana, Slovenia; 2,3Dept Urology, University Heidelberg and Technical University Munich, 4Dept Pathology, U. Münster, Germany.
The tumor suppressor gene model predicts that the sporadic counterparts of a hereditary cancer should follow the same inactivation mechanism of the tumor suppressor gene as in the hereditary type. It has been shown that germline mutations in the VHL tumor suppressor gene confer a genetic predisposition to VHL disease, and somatic VHL mutations in concert with 3p loss of heterozygosity represent rate limiting events in the tumorigenesis of renal clear cell carcinoma and sporadic hemangioblastoma (Latif et al., 1993, Gnarra et al., 1994, Foster et al., 1994, Shuin et al., 1994, Whaley et al., 1994, Glavac et al., 1996). The studies on renal cancer included more than 230 renal cell tumors diagnosed according to the WHO standards, and more than 140 tumors diagnosed according to the classification of Thoenes et al. (1986). The latter distinguishes carcinomas with transparent cytoplasms into the common clear cell carcinomas and the rare chromophobe carcinomas. The chromophobe carcinomas are regarded to have a more favorable prognosis, making the distinction of clinical importance.
In order to test if somatic VHL mutations are specifically associated with clear cell carcinomas, or if they also contain features common to other tumor entities, we analyzed 146 kidney tumors (39 cell lines and 107 primary tumors) for somatic VHL mutations. Histo-pathological diagnosis identified 75 clear cell, 1 mixed cell, 1 dedifferentiated, 24 chromophilic, and 12 chromophobic carcinomas, as well as 10 oncocytomas; for 23 carcinomas, detailed histopathology was not available. 52 somatic VHL mutations were identified by exon-specific PCR followed by non radioactive SSCP and sequencing, as well as hypermethylation analysis of CpG islands in exon 1.
There were 24 frameshift, 11 missense, 6 splice site, 3 nonsense, 1 in-frame deletion, and 7 hypermethylation mutations; 45% of the point mutations affected exon 2. Most carcinomas with a somatic VHL mutation also showed loss of the homologous 3p allele. VHL mutations were identified in 40/75 (53%) clear cell carcinomas, one mixed cell, one dedifferentiated, and 7 carcinomas of unknown histopathological type. Three non-clear cell carcinomas showed a somatic VHL mutation: these were 2 chromophilic and 1 chromophobic carcinoma respectively. None of the oncocytomas showed a somatic VHL mutation.
Our data confirm that somatic VHL mutations are important steps in the development of renal cell carcinomas, but not of oncocytomas. Somatic VHL mutations afflict at least 53% of clear cell carcinomas. The rare finding of somatic VHL mutations in non-clear cell carcinomas raises the question whether these carcinomas may be true chromophilic or true chromophobic carcinomas, or if they represent tumors with clear cell content. Somatic VHL mutation analysis, together with cytological and histological typing, may allow more precise statements with regard to prognosis of renal cell carcinomas. This should remain under scrutiny.
[504] Detection of VHL Gene Mutations in Sporadic
Renal Cell Carcinoma and Development of a VHL Database of Germline and
Somatic Mutations. Béroud C1, Joly D1,2, Tarlet D1, Martin
N1, Richard S2, Chauveau D2, Grünfeld JP2, Junien C1. 1U383 INSERM
and 2Service de Néphrologie, Hôpital Necker Enfants Malades,
Paris, France.
Renal cell carcinoma (RCC) is the most frequent tumor of the kidney and accounts for about 3% of all human adult cancer. It is usually sporadic but can occur as a hereditary form in association with von Hippel-Lindau disease (VHL). Since the isolation of the VHL gene in 1993, mutations have been reported in more than 200 VHL families and in nearly 100 sporadic RCC. To understand the mutational events involved in sporadic RCC, we performed mutation analysis in a large series of 170 sporadic RCC with matched normal kidney tissue samples. We used six sets of primers spanning the three VHL gene exons for PCR amplifications followed by SSCP analysis of the PCR products. Somatic VHL gene mutations of samples were found in exon 1 in 17.5% (27:155), in exon 2 in 12% (17:143), and in exon 3 in 16.5% (17:143). The common A or G polymorphism at nt19 was also detected, with frequencies of 0.65 (A) and 0.35 (G). This marker was used to investigate loss of heterozygosity (LOH) in these tumors. Among tumors with mutations, we identified LOH in 83% of informative cases. Sequencing revealed deletions in 59% of cases, insertions in 17%, nonsense mutations in 10%, and substitutions in 14%.
Molecular epidemiology has been used to analyze the mutational spectrum of the various mutations and to reveal a direct causal effect between carcinogen exposure and a specific cancer. Taking advantage of our experience in designing databases with the 4th Dimension (4D) package from ACI for three genes: p53 (>5,000 mutations), APC (>700 mutations), and FBN1 (75 mutations), we developed a VHL gene mutation database including >400 mutations.
A preliminary study showed that germinal and somatic mutations differ both in type of event (76% substitutions in germinal mutations vs. 70% frame-shifts in somatic mutations), and in the distribution of these mutations along the VHL gene. Considering the substitutions: transitions were found in 71.4% of germinal mutations, and trans-versions in 66.7% of somatic mutations, giving support to the hypothesis that a toxic factor is involved in sporadic RCC. For VHL patients, the mutational event is associated with the occurrence of RCC (frameshifts vs. substitutions). It is crucial to collect new molecular and clinical data, to define new critical regions of the VHL protein, to identify a toxic factor involved in sporadic RCC and to evaluate the risk for RCC to develop each specific germinal mutation.
[505] Mutational Analysis of the von Hippel Lindau
Tumor Suppressor Gene in 106 Sporadic Renal Cell Carcinomas: Involvement
of the Clear Cell Type as Well as the Chromophilic Type. Decker
H J, Neuhaus C, Brauch H, Weidt E, Storkel S, Brenner W, Huber C. Dept
Hematology, Urology, U. Mainz, and Molecular Pathology Laboratory, U.
Munich, Germany.
We have collected 125 sporadic renal cell carcinomas (RCC) over a period of 3 years, and performed cytogenetic and molecular analysis on these tumors and on corresponding normal kidney tissues. The tumors were classified in accordance to the Mainz Classification system. We applied PCR/SSCP with primers for all exons of the VHL gene. Besides a high percentage of clear cell carcinomas, 33% of chromophilic tumors also showed mutations in the von Hippel Lindau (VHL) tumor suppressor gene. Using cytogenetic, fluorescent in situ hybridization and minisatellite analysis, we detected homozygous loss in virtually all RCC with VHL gene mutations, underscoring its tumor suppressing nature.
Our findings also indicate a close genetic relation between the two forms of RCC: the clear cell and the chromophilic type, both of which are derived from the proximal tubule of the nephron.
[506] Frequent Somatic Mutations and Loss of Heterozygosity of the VHL Tumor Suppressor Gene in Sporadic Central Nervous System Hemangioblastomas. Kanno H1, Ito S1, Yamamoto I1, Kondo K2, Yoshida M2, Yao M2, Shuin T3. Dept 1Neurosurgery, 2Urology, Yokohama City University School of Medicine, Dept 3Urology, Kochi Medical University, Japan.
Hemangioblastoma is often associated with von Hippel-Lindau (VHL) disease. It is believed that inactivation of both alleles of the VHL gene on chromosome 3p25 is essential for tumorigenic processes in hemangioblastomas associated with VHL disease. We analyzed 20 cases of sporadic central nervous system (CNS) hemangioblastomas for somatic mutations of the VHL gene with single-strand conformation polymorphism (SSCP) analysis of tumor DNA, and for loss of heterozygosity (LOH) on chromosome 3p using 9 microsatellite probes.
We detected abnormal SSCP patterns in 10 cases (50%). Of the 10 possibly mutated cases, we successfully characterized 5 cases by direct sequencing. Somatic mutations in these 5 cases were 3 missense mutations and 2 deletions. Although 4 of 10 cases with abnormal SSCP patterns showed a recurrence of tumor, 10 cases with normal SSCP did not. LOH at loci on chromosome 3p was found in 10 of 15 cases (67%).
Our results suggest that the VHL tumor suppressor gene is involved in the development of sporadic CNS hemangioblastomas.
[507] Basic Mechanisms of Transcript Elongation by RNA Polymerases. Chamberlin JM. Dept Biochemistry and Molecular Biology, U. California, Berkeley, CA 94720.
Transcription of genes in cells involves the copying of one of the two strands of DNA, through Watson-Crick base pairing (A=T(U) and G=C) to produce an RNA product. This is the first step in gene expression, and is closely regulated in all cells. Transcription is carried out by an enzyme, RNA polymerase, together with a variety of transcription factors. The transcription process can be represented as a cycle with four steps:
1) The binding of RNA polymerase to a promotor region in DNA to form an active promotor complex.
2) The initiation of an RNA chain, and escape from the promotor.
3) RNA chain elongation, in which the enzyme moves along the DNA synthesizing RNA without dissociation.
4) RNA chain termination and release, in which the enzyme encounters a termination signal, releases its RNA chain and dissociates to reenter the cycle at step 1.
Although it has long been known that the first step of transcription can be regulated, it has only recently become clear that each of the four steps is an important regulatory target. We have studied the elongation and termination steps of transcription, to try to understand their basic mechanisms, and to understand how regulation can be effected at these steps. In the past few years, it has become clear that elongation is a complex process, in which the enzyme moves along DNA, passing through a number of blocks to elongation as it proceeds from the promotor to the terminator. These blocks can cause the RNA poly-merase to pause, a state from which it can easily continue elongation, to arrest, a state from which the enzyme normally cannot continue without the action of a transcription factor, or to terminate, ending elongation. Release of RNA polymerase from the arrest state often involves cleavage of up to 15nt from the growing end of the nascent RNA, allowing the enzyme to move backwards, and freeing it to reenter elongation.
A model for the basic mechanism of elongation that we proposed recently involves the discontinuous movement of RNA polymerase along DNA in an "inchworm" like manner. This movement is controlled by two sets of binding sites on the enzyme: two DNA binding sites that grip the DNA duplex near the leading and trailing edges, and two RNA binding sites that hold the nascent RNA firmly to the polymerase. This model provides simple explanations for the processes of transcript arrest, transcript cleavage and termination release, as well as for the action of transcription factors that facilitate or suppress elongation.
[508] Phosphorylation of the von Hippel Lindau Disease Tumor Suppressor Protein by Casein Kinase II. Stackhouse TM1, Kishida T2, ,Chase D3, Ferris DK1, Kuzmin I2, Geil L1, Orcutt ML2, Sakashita N4, Takeya M4, Renbaum P2, Zbar B2. 1Intramural Research Support Program, SAIC Frederick, 2The National Cancer Institute, 3The National Institute on Aging/Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA, 4Dept Pathology, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan.
Von Hippel-Lindau disease (VHL) is an inherited multiple system neoplastic disorder. Recent evidence suggest the VHL protein may play a role in transcription elongation. The VHL protein displays cell density dependent trafficking between the cytoplasm and the nucleus. We tested whether the VHL protein is a phospho-protein by studying VHL proteins precipitated from metabolically-labeled renal carcinoma cells. We also developed an in vitro assay to measure and characterize binding of a cellular kinase by the VHL protein.
Using a panel of VHL deletion mutants, we demonstrated that kinase binding and phosphorylation sites were located within amino acids 1-60. Within this region of the VHL protein there are three serines with consensus sequence recognized by casein kinase II (CKII). Quercetin, a competitive inhibitor of CKII, and a 10 amino acid CKII substrate peptide, antagonized phosphorylation of the VHL protein by the cellular kinase.
Phosphopeptide maps of the VHL protein labeled in vivo, in vitro in the kinase binding assay, and with purified CKII, were identical. Phosphoamino acid analysis demon-strated that only serines were labeled with phosphate.
We conclude that serines 33/38/43 of the VHL protein are targets for phosphorylation and that the kinase responsible for this phosphorylation is CKII.
[509] Characterization of the VHL Tumor Suppressor Gene and VHL Knock-Out Mice. Kley N, Gavin B, Gao J, Whaley J, Naglich J, Seizinger BR. Dept Molecular Genetics, Oncology, Bristol-Meyers Squibb Pharmaceutical Research Institute, Princeton, NJ.
Germline-mutations of the von Hippel Lindau tumor suppressor gene (VHL) predispose individuals to various tumors, including hemangioblastomas and renal cell carcinomas. Furthermore, biallelic somatic loss/ inactivation of the VHL gene is a very frequent event in sporadic renal cell carcinomas and cerebellar hemangio-blastomas. Here we present a study showing that introduction of an inducible transgene encoding wild type VHL protein (pVHL) into pVHL-null sporadic renal carcinoma cells is sufficient to inhibit their tumorigenic potential. Importantly, inhibition of growth is observed at levels of exogenous pVHL protein expression that are comparable to those of endogenous pVHL levels detected in normal kidney cells, and does not depend on the presence of the putative acidic pentamer repeat region predicted by the sequence of the cDNA ORF between the two 5' putative start codons. Potential mechanisms of tumor suppression by this ~18 kd pVHL will be discussed.
In an effort to understand further the function of the VHL gene at the organismal level and to develop animal model systems to study VHL-related cancers, we generated a null allele of the mouse VHL gene by homologous recombination in embryonic stem cells. Chimeras and, subsequently, F1 heterozygotes were generated. To date, we have not observed any apparent phenotypic effects in animals heterozygous for the targeted alleles by 9 months of age. No liveborn homozygotes were obtained by F1 heterozygote intercrossings, indicating that the lack of a functional VHL protein results in embryonic lethality. More detailed analyses of embryos at 10.5 dpc and 9.5 dpc indicate that a requirement for a function VHL protein becomes critical around 9/9.5 dpc of development. Alternative approaches that are currently in progress to generate VHL-related animal cancer models will be discussed.
[A1] Genotype-Phenotype Correlations in von Hippel-Lindau Disease. Maher ER1, Webster AR1,2, Richards FM1, Green JS3, Crossey PA1, Moore AT2. Dept 1Pathology, 2Ophthalmology, Addenbrooke's Hospital, Cambridge U., Great Britain; 3Division of Community Medicine, Memorial University of Newfoundland, Canada.
Elucidation of genotype-phenotype relationships may provide clues to structure-function aspects of a gene product. VHL disease characteristically shows variable expression, and interfamilial differences in predisposition to phaeochromocytoma are the best characterized example of phenotypic heterogeneity. A genotype-phenotype correlation has been defined such that most phaeochromocytoma-positive kindreds have missense mutations, while large deletions or protein truncating mutations are most frequent in phaeochromocytoma-negative families [1-3].
In our series of 101 VHL families with defined germline mutations, large deletions and mutations predicted to cause a truncated protein were associated with a lower risk of phaeochromocytoma (9% at age 50 years) than missense mutations (59% at age 50 years) and missense mutations at codon 167 were associated with a particularly high risk of phaeochromocytoma (82% at age 50 years). However, cumulative probabilities of renal cell carcinoma (RCC) did not differ between the two groups (deletion/ truncation mutations 60% and missense mutations 64% at age 50 years respectively). Also there was no association between the type of mutation and age-related risks for retinal and cerebellar haemangioblastomas.
Specific missense mutations may also cause VHL disease with a high risk of phaeochromocytoma and low risk of RCC (e.g. Tyr98His) [4] and familial phaeo-chromocytoma with no features of VHL disease [5,6]. These findings suggest that missense mutations predis-posing to phaeochromocytoma do not have a generalized dominant negative effect, but rather that the VHL protein has tissue-specific effects.
To investigate the relationship between retinal angiomatosis and VHL gene mutations, 180 gene-carriers were examined in detail. No evidence of a correlation between mutation type and number of retinal haemangio-blastomas was detected, but statistical analysis of intra-familial variations provided clear evidence of modifier effects. Thus the expression of both retinal angiomatosis and other VHL tumours may be influenced by one or more distinct genetic loci.
1. Crossey PA, Richards FM, Foster K, et al: Identification of intragenic mutations in the von Hippel-Lindau tumour suppressor gene and correlation with phenotype. Hum Molec Genet 1994; 3:1303-1308
2. Chen F, Kishida T, Yao M, et al: Germ line mutations in the von Hippel-Lindau disease tumor suppressor gene: correlations with phenotype. Human Mutation 1995; 5:66-75.
3. Zbar B, Kishida T, Chen F, et al: Germline mutations in the von Hippel-Lindau disease (VHL) gene in families from North America, Europe and Japan. Human Mutation [in press]
4. Brauch H, Kishida T, Glavao D, Chen F, Pausch F, Hoffler H, Latif F, Leman MI, Zbar B, Neumann HPH: von Hippel Lindau disease with pheochromocytoma in the Black Forest region of Germany: evidence for a founder effect. Hum Genet 1996;95:551-556
5. Crossey PA, Eng C, Ginalska-Melinowska M, et al: Molecular genetic diagnosis of von Hippel-Lindau disease in familial phaeochromocytoma. J Med Genet 1995; 32:885-886
6. Neumann HPH, Eng C, Mulligan L, et al: Consequences of direct genetic testing for germline mutations in the clinical management of families with multiple endocrine neoplasia type 2. JAMA 1995; 274:1149-1151
[A2] Mutation Screening for von Hippel Lindau Disease [VHL] in a Large Kindred. 1,2Hsia YE, 1Moulds JK, 1Hunt JA, 2Yuen J, 1Phillips R, 3Kishida T, 3Zbar B. 1U. Hawai'i, 2Kapi'olani Medical Center, Honolulu, Hawai'i; National Cancer Center, N.I.H., Bethesda, Maryland.
Most familial mutations of the VHL cancer suppres-sor gene have resulted in either protein truncations or missense amino acid substitutions. In the largest family known to be affected with VHL [Med 68:1-29, 1989], and in a second possibly related family, a T to C transition was found at nucleotide [nt] 686, causing a Leu to Pro substitution.
Amplification of this region of the gene by polymerase chain reaction with specially designed primers, created an AciI restriction site specific for the nt 686 transition, allowing simple detection of the mutant allele. When mutation detection was offered to members of this family, the response was very positive. So far, 53 members of the two families have been tested. Of the 14 known to be affected, including one non-symptomatic obligate hetero-zygote, all had this mutation; of 32 with an affected parent, and 7 with an affected grandparent; 9 were heterozygous for the mutant allele; none was homozygous.
Every detected heterozygote had a parent known or shown to be heterozygous.
A majorty of the tested were found to be non-heterozygous because of 11 over age 31 none was heterozygous; 9 were children of asymptomatic, presumably unaffected parents.
Heterozygote screening issues for this adult-onset dominant pre-cancerous condition differs greatly in principle from those for non-treatable disorders such as Huntington disease, because aggressive surveillance and timely intervention for early tumors can preserve health and be life-saving.
The family members found to be affected could be offered close presymptomatic surveillance for early detection of tumors; those found to be unaffected could be assured that they did not need surveillance for early detection of tumors; the non-heterozygous members tested had 31 offspring, who could also be assured they were not at risk for VHL.
Results of Special PCR Mutation Analysis
PCR : Abnormal Normal Total
Known Symptomatic* 14 0 14
Child of Affected 9 32 41
Grand-Child of Affected 7 1 8
Total Tested 30 33 63
PCR: Abnormal = 98, 68, & 30 kb bands, Normal = 98 bp band only
*Includes one non-symptomatic obligate heterozygote
[A3] Deafness due to bilateral endolymphatic sac tumours in a case of von Hippel-Lindau syndrome. Kempermann G, Neumann HPH, Scheremet R, Volk B, Mann W, Gilsbach J, Laszig R: Albert-Ludwig University, Freiburg, Germany.
In a patient with von Hippel-Lindau syndrome, a tumour in the right cerebellopontine angle caused deafness. The tumour was removed and classified as a medullary thyroid metastasis, but on thyroidectomy, no tumour was found. A similar tumor was removed 8 years later from the left petrous bone.
Histology, immunocytochemical findings, and clinical context, reclassified it as a rare endolymphatic sac tumour.
This case supports earlier data that these tumours could be a manifestation of von Hippel-Lindau syndrome.
999 Mutation Detection for VHL in a Large Kindred.Hsia YE
999 Genotype-Phenotype Correlations in von Hippel-Lindau Disease.Maher Eamonn
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