Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.     

An analysis of phenotypic variation in the familial cancer syndrome von Hippel-Lindau disease: evidence for modifier effects.

von Hippel-Lindau disease (VHL) is a dominantly inherited familial cancer syndrome predisposing to ocular and CNS hemangioblastomas, renal-cell carcinoma (RCC), and pheochromocytoma. Both interfamilial and intrafamilial variability in expression is well recognized. Interfamilial differences in pheochromocytoma susceptibility have been attributed to allelic heterogeneity such that specific missense germ-line mutations confer a high risk for this complication. However, in most cases, tumor susceptibility does not appear to be influenced by the type of underlying VHL mutation. To probe the causes of phenotypic variation, we examined 183 individuals with germ-line VHL gene mutations, for the presence and number of ocular tumors. The prevalence of ocular angiomatosis did not increase with age, and the distribution of these tumors in gene carriers was significantly different than the expected stochastic distributions. Individuals with ocular hemangioblastomas had a significantly increased incidence of cerebellar hemangioblastoma and RCC (hazard ratios 2.3 and 4.0, respectively). The number of ocular tumors was significantly correlated in individuals of 12 degree relatedness but not in more distantly related individuals. These findings suggest that the development of VHL ocular tumors is determined at an early age and is influenced by genetic and/or environmental modifier effects that act at multiple sites. Functional polymorphisms in the glutathione-S-transferase M1 gene (GSTM1) or the cytochrome P450 2D6 gene (CYP2D6) did not show a significant association with the severity of ocular or renal involvement.     

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe

von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management. Received: 10 November 1995 / Revised: 1 March 1996     

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.     

Molecular study of a new family with hereditary renal cell carcinoma and a translocation t(3;8)(p13;q24.1)

An alternative model has been proposed for the development of clear-cell renal cell carcinoma (RCC) in families where chromosome 3 translocations segregate with the disease. In this model, the existence of a translocation involving chromosome 3 would favour the non-disjunctional loss of the derivative chromosome carrying the 3p segment. Additionally, subsequent somatic mutations in the VLH gene, located in 3p25-26, would inactivate this tumour suppressor gene. In the present work, we describe a new family with two clear-cell RCC affected members and a t(3;8)(p13;q24.1) translocation in two consecutive generations. We observed loss of the derivative chromosome carrying the 3p segment (der(8)) and somatic mutation of the VHL gene in the left-kidney tumoral tissue of the proband. His right-kidney tumour carried a different VHL mutation and loss of heterozygosity (LOH) was not detected. The mother of the proband was also clear-cell RCC-affected but the tumoral tissue analysed did not carry any VHL gene mutations. Another member of the family, a maternal aunt, had a papillary RCC and did not carry this translocation, the LOH on 3p or the VHL somatic mutations. Haplotype analysis of the three affected members revealed that they did not inherit a common region on 3p, confirming the different genetic origin of both tumour types. Finally, the presence of RCC in other non-available members of the family highlights the overall risk for RCC in families with chromosome 3 translocations. In the present work, we have confirmed the proposed mechanism for the development of clear-cell RCC in this family, although we cannot discard the existence of other genes, in addition to VHL, being involved in hereditary RCC.     

Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.     

VHL综合征遗传学研究

VHL综合征(von Hippel-Lindau syndrome,VHL;MIM 193300)是一种常染色体显性遗传的多系统肿瘤综合征,最常见临床表现是视网膜或中枢神经系统(central nervous system,CNS)血管母细胞瘤.CNS血管母细胞瘤和肾细胞癌(renal cell carcinoma,RCC)的并发症是VHL患者最主要的死因.VHL综合征主要因VHL基因(the vonHipple-Lindau gene,VHL)突变所致,细胞周期素D1基因(the cyclin D1 gene,CCND1)突变和蛋白异常也可能参与其发生.目前已建立了多个VHL基因缺陷动物模型.在此就VHL综合征的遗传学研究进展作一概述.     

Expression of the Von Hippel-Lindau tumor suppressor gene,VHL, in human fetal kidney and during mouse embryogenesis.

BACKGROUND: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome that has a dominant inherited pattern which predisposes affected individuals to a variety of tumours. The most frequent tumors are hemangioblastomas of the central nervous system and retina, renal cell carcinoma (RCC), and pheochromocytoma. The recent identification and characterization of the VHL gene on human chromosome 3p and mutational analyses confirms the VHL gene functions as a classical tumor suppressor. Not only are mutations in this gene responsible for the VHL syndrome, but mutations are also very frequent in sporadic RCC. MATERIALS AND METHODS: VHL expression in human kidney and during embryogenesis, was analyzed by in situ mRNA hybridization with 35S-labeled antisense VHL probes, derived from human and mouse cDNAs, on cryosections of human fetal kidney and paraffin sections of murine embryos. RESULTS: In human fetal kidney, there was enhanced expression of VHL within the epithelial lining of the proximal tubules. During embryogenesis, VHL expression was ubiquitous in all three germ cell layers and their derivatives. Expression occurred in the cerebral cortex, midbrain, cerebellum, retina, spinal cord, and postganglionic cell bodies. All organs of the thoracic and abdominal cavities expressed VHL, but enhanced expression was most apparent in the epithelial components of the lung, kidney, and eye. CONCLUSIONS: In human fetal kidney, the enhanced epithelial expression of the VHL gene is consistent with the role of this gene in RCC. There is widespread expression of the VHL gene during embryogenesis, but this is pronounced in areas associated with VHL phenotypes. These findings provide a histological framework for investigating the physiological role of the VHL gene and as basis for further mutational analysis.     

Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC)

Spontaneous renal cell carcinoma (RCC) occurs with a high frequency in Eker rats carrying a germline alteration of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene. To determine the frequency with which the wild-type allele of the Tsc-2 gene is lost in RCC and the ability of DHPLC to detect loss of heterozygosity (LOH) at this gene locus, fresh-frozen and paraffin-embedded formalin-fixed tumors from heterozygous Eker rats (Tsc-2(Ek/+)) were examined for LOH at the Tsc-2 locus. LOH was determined by quantitation of peak areas of PCR products specific for the mutant and wild-type Tsc-2 alleles. For normal DNA isolated from heterozygous animals, the allele ratio (AR) of mutant to wild-type PCR products was empirically determined to be 1.5+/-0.3 (n=30) and LOH was defined as >2 standard deviations away from this mean, i.e. any AR >2.1. Analysis of 15 spontaneous frozen RCC samples showed LOH in 10/15 samples (66%). Carcinogen-induced tumors exhibited an even higher frequency of LOH, with 6/6 paraffin-embedded, formalin-fixed tumors exhibiting LOH. 100% concordance was observed between the results obtained by DHPLC and traditional methodologies. Therefore, LOH appears to occur with a high frequency in both spontaneous and carcinogen-induced RCC in this animal model and DHPLC is a sensitive and high throughput methodology for detecting this type of genetic alteration.     

Von Hippel-Lindau disease (VHL): a need for a murine model with retinal hemangioblastoma

Von Hippel-Lindau (VHL) disease is a highly penetrant autosomal dominant systemic malignancy that gives rise to cystic and highly vascularized tumors in a constellation of organs. Patients with VHL disease commonly present with hemangioblastomas in the central nervous system and the eye while other manifestations include pheochromocytoma, clear cell renal cell carcinoma, endolymphatic sac tumors of the middle ear, pancreatic cystadenomas, epididymal and broad ligament cystadenomas. Animal models inactivating the VHL gene product in various organ tissues have been constructed over the past 15 years to parse its HIF-associated mechanisms and its link to tumorigenesis. These models, despite advancing our understanding the molecular role of VHL, are by and large unable to recapitulate the more common features of human VHL disease. Up to date, no model exists that develop retinal hemangioblastomas, the most common clinical manifestation. The purpose of this review is: (1) to discuss the need for an ocular VHL model, (2) to review the animal models that recapitulate clinical VHL disease and (3) to propose potential mechanisms of tumorigenesis for the development of ocular VHL.     

Software and database for the analysis of mutations in the VHL gene.

VHL is a tumor suppressor gene localized on chromosome 3p25-26. Mutations of the VHL gene were described at first in the heritable von Hippel-Lindau disease and in the sporadic Renal Cell Carcinoma (RCC). More recently, VHL has also been shown to harbor mutations in mesothelioma and small cell lung carcinoma. To date more than 500 mutations have been identified. These mutations are mainly private with only one hot spot at codon 167 associated with pheochromocytoma. The germline mutations are essentially missense while somatic mutations include deletions, insertions and nonsense. To standardize the collection of these informations, facilitate the mutational analysis of the VHL gene and promote the genotype-phenotype analysis, a software package along with a computerized database have been created. The current database and the analysis software are accessible via the internet and world wide web interface at the URL:http://www.umd.necker.fr     

The von Hippel-Lindau Tumor Suppressor Gene Inhibits Hepatocyte Growth Factor/Scatter Factor-Induced Invasion and Branching Morphogenesis in Renal Carcinoma Cells

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.     

Absence of somatic mosaicism in 17 families with hemophilia B: an analysis with a sensitivity 10- to 1000-fold greater than that of sequencing gels

Most estimates of germ-line mosaicism have been derived from families in which there has been transmission of a mutated allele to two or more children by an unaffected individual. Previously, analyses for somatic mosaicism detected five such individuals by PCR-based sequencing and haplotype analysis at a sensitivity of approximately 1 mutant per 10 wild-type alleles. To determine whether mutations that occur later in embryogenesis also give rise to somatic mosaicism, we analyzed leukocyte DNA from 17 individuals in whom a mutation in the factor IX gene was known to have originated. Methods capable of detecting 1 mutant allele in 100–10 000 were utilized, and no further examples of somatic mosaicism were detected. If confirmed by future studies, the paucity of somatic mosaicism with mutant:wild-type allele frequencies ranging from 1:10 to 1:1000 (relative to the 11% of somatic mosaicism detected with mutant:wild-type allele frequencies of 1:1 to 1:10) may reflect a higher mutation rate and/or germ-line lineage allocation very early in embryogenesis. Received: 14 July 1995 / Revised: 1 April 1996     

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.     

Prevention of renal carcinoma: the nutri-genetic approach

The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors, with somatic and germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and with tobacco smoking, obesity, long term exposure to some nutrients, pollutants, and industrial solvents such as trichloroethylene. Intra and interfamilial variability of expression of germline mutations in the VHL gene and variable susceptibility to carcinogens in the sporadic forms strongly suggest the involvement of conditional modifier genes. In order to identify sub groups of individuals at increased risk because of susceptibility genotypes, we have collected a series of 460 patients who developed an RCC and 79 families with the von Hippel Lindau disease. To collect clinical and mutational data for correlation analysis we have developed a unique tool the Universal Mutation Database. Comparison of the spectrum of germline and somatic mutations in the VHL gene showed that: 1) in sporadic RCC mutations lead more often to truncated proteins (83%), while the remaining mutations (17%), include 3/4 of transversions and 1/4 of transitions. This high proportion of transversions supports the involvement of carcinogens the impact of which is conditioned by the genetic variability of xenobiotic metabolizing enzymes; 2) whereas in familial cases missense mutations are more common; this difference allowed us to define a prognostic factor for the occurrence of RCC in a VHL context. In order to look for genotypes conferring a higher risk we genotyped the RCC patients for 8 different genes (50 genotypes). A significant relationship was observed for several combinations of alleles including CYP1A1 ("variant"), NAT2 and NAT1 (slow) and GSTM1 (null allele). Associations between specific mutational profiles and at risk genotypes at different tumoral stages should allow us to: 1) define more precisely the nature of specific patterns of mutations in relation with the deficiency or overexpression of such or such enzymes in presence of particular carcinogens; 2) demonstrate that certain combinations of genotypes confer a particular risk to develop a specific type of tumor in VHL patients. Thus tracking of potentially carcinogenic substances, through their footprints and through identification of conditionally detrimental genotypes of genes participating in their detoxification should permit a better prevention through an appropriate nutrition adapted to each individual.     

Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma

Hereditary paraganglioma syndrome has recently been shown to be caused by germline heterozygous mutations in three (SDHB, SDHC, and SDHD) of the four genes that encode mitochondrial succinate dehydrogenase. Extraparaganglial component neoplasias have never been previously documented. In a population-based registry of symptomatic presentations of phaeochromocytoma/paraganglioma comprising 352 registrants, among whom 16 unrelated registrants were SDHB mutation positive, one family with germline SDHB mutation c.847-50delTCTC had two members with renal cell carcinoma (RCC), of solid histology, at ages 24 and 26 years. Both also had paraganglioma. A registry of early-onset RCCs revealed a family comprising a son with clear-cell RCC and his mother with a cardiac tumor, both with the germline SDHB R27X mutation. The cardiac tumor proved to be a paraganglioma. All RCCs showed loss of the remaining wild-type allele. Our observations suggest that germline SDHB mutations can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance.     

Folliculin Contributes to VHL Tumor Suppressing Activity in Renal Cancer through Regulation of Autophagy

Von Hippel-Lindau tumor suppressor (VHL) is lost in the majority of clear cell renal cell carcinomas (ccRCC). Folliculin (FLCN) is a tumor suppressor whose function is lost in Birt-Hogg-Dubé syndrome (BHD), a disorder characterized by renal cancer of multiple histological types including clear cell carcinoma, cutaneous fibrofolliculoma, and pneumothorax. Here we explored whether there is connection between VHL and FLCN in clear cell renal carcinoma cell lines and tumors. We demonstrate that VHL regulates expression of FLCN at the mRNA and protein levels in RCC cell lines, and that FLCN protein expression is decreased in human ccRCC tumors with VHL loss, as compared with matched normal kidney tissue. Knockdown of FLCN results in increased formation of tumors by RCC cells with wild-type VHL in orthotopic xenografts in nude mice, an indication that FLCN plays a role in the tumor-suppressing activity of VHL. Interestingly, FLCN, similarly to VHL, is necessary for the activity of LC3C-mediated autophagic program that we have previously characterized as contributing to the tumor suppressing activity of VHL. The results show the existence of functional crosstalk between two major tumor suppressors in renal cancer, VHL and FLCN, converging on regulation of autophagy.     

Downregulation of integrins by von Hippel-Lindau (VHL) tumor suppressor protein is independent of VHL-directed hypoxia-inducible factor alpha degradation

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene occurs in the majority of clear-cell renal cell carcinomas (RCCs). It was previously shown that VHL decreased the abundance of integrins alpha2, alpha5, and beta1, which is consistent with VHL-associated changes in cell-cell and cell - extracellular matrix adhesions. We investigated the mechanism by which VHL downregulates integrins. Although VHL can target hypoxia-inducible factor alpha (HIFalpha) subunits for degradation, VHL-dependent reduction of integrins was independent of O2 concentration and HIFalpha levels. VHL reduced the half-lives of integrins, and this activity was blocked by proteasomal inhibition. Although ectopically expressed FLAG-VHL retained HIFalpha degradation activity, it neither downregulated integrins nor promoted adherens and tight intercellular junctions, in contrast to expressed wild-type VHL. Moreover, integrins co-immunoprecipitated with wild-type VHL, but not FLAG-VHL. These data indicate that the downregulation of integrins by VHL is distinct from the regulation of HIFalpha subunits by VHL, and suggests that the loss of this activity contributes to VHL-associated RCC development through disruption of adherens and tight junctions.