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     

Analysis of Germline Variants in CDH1, IGFBP3, MMP1, MMP3, STK15 and VEGF in Familial and Sporadic Renal Cell Carcinoma

Background

The investigation of rare familial forms of kidney cancer has provided important insights into the biology of sporadic renal cell carcinoma (RCC). In particular, the identification of the von Hippel Lindau (VHL) familial cancer syndrome gene (VHL) provided the basis for the discovery that VHL is somatically inactivated in most sporadic clear cell RCC. Many cases of familial RCC do not have mutations in known RCC susceptibility genes and there is evidence that genetic modifiers may influence the risk of RCC in VHL disease patients. Hence we hypothesised that low-penetrance functional genetic variants in pathways related to the VHL protein (pVHL) function might (a) modify the phenotypic expression of VHL disease and/or (b) predispose to sporadic RCC.

Methodology/Principal Findings

We tested this hypothesis for functional polymorphisms in CDH1 (rs16260), IGFBP3 (rs2854744), MMP1 (rs1799750), MMP3 (rs679620), STK15 (rs2273535) and VEGF (rs1570360). We observed that variants of MMP1 and MMP3 were significant modifiers of RCC risk (and risks of retinal angioma and cerebellar haemangioblastoma) in VHL disease patients. In addition, higher frequencies of the MMP1 rs1799750 2G allele (p = 0.017, OR 1.49, 95%CI 1.06–2.08) and the MMP1/MMP3 rs1799750/rs679620 2G/G haplotype (OR 1.45, 95%CI 1.01–2.10) were detected in sporadic RCC patients than in controls (n = 295).

Conclusions/Significance

These findings (a) represent the first example of genetic modifiers of RCC risk in VHL disease, (b) replicate a previous report of an association between MMP1/MMP3 variants and sporadic RCC and (c) further implicate MMP1/MMP3-related pathways in the pathogenesis of familial and sporadic RCC.     

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.     

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.     

Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors.

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing to retinal and CNS hemangioblastomas, renal cell carcinoma (RCC), pheochromocytoma, and pancreatic tumors. Interfamilial differences in predisposition to pheochromocytoma reflect allelic heterogeneity such that there is a strong association between missense mutations and risk of pheochromocytoma. We investigated the mechanism of tumorigenesis in VHL disease tumors to determine whether there were differences between tumor types or classes of germ-line mutations. Fifty-three tumors (30 RCCs, 15 hemangioblastomas, 5 pheochromocytomas, and 3 pancreatic tumors) from 33 patients (27 kindreds) with VHL disease were analyzed. Overall, 51% of 45 informative tumors showed loss of heterozygosity (LOH) at the VHL locus. In 11 cases it was possible to distinguish between loss of the wild-type and mutant alleles, and in each case the wild-type allele was lost. LOH was detected in all tumor types and occurred in the presence of both germ-line missense mutations and other types of germline mutation associated with a low risk of pheochromocytoma. Intragenic somatic mutations were detected in three tumors (all hemangioblastomas) and in two of these could be shown to occur in the wild-type allele. This provides the first example of homozygous inactivation of the VHL by small intragenic mutations in this type of tumor. Hypermethylation of the VHL gene was detected in 33% (6/18) of tumors without LOH, including 2 RCCs and 4 hemangioblastomas. Although hypermethylation of the VHL gene has been reported previously in nonfamilial RCC and although methylation of tumor-suppressor genes has been implicated in the pathogenesis of other sporadic cancers, this is the first report of somatic methylation in a familial cancer syndrome.     

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.     

Familial non-syndromic clear cell renal cell carcinoma

The diagnosis of familial non-syndromic clear cell renal cell carcinoma is one of exclusion. In families presenting with clear cell RCC a germline VHL mutation and a constitutional translocation of chromosome 3 must be excluded before familial non-syndromic clear cell RCC can be diagnosed. Large familial non-syndromic clear cell RCC kindreds are uncommon and a predisposing gene has not been identified. However inheritance is autosomal dominant in most cases and age at onset is earlier than in sporadic cases. Recognition and appropriate screening of familial non-syndromic clear cell RCC cases will reduce morbidity and mortality. Large scale collaborative linkage studies may provide a basis for the identification of familial non-syndromic clear cell RCC susceptibility gene(s).     

The VHL tumor suppressor in development and disease: functional studies in mice by conditional gene targeting

The von Hippel-Lindau tumor suppressor pVHL plays a critical role in the pathogenesis of familial and sporadic clear cell carcinomas of the kidney and hemangioblastomas of the retina and central nervous system. pVHL targets the oxygen sensitive alpha subunit of hypoxia-inducible factor (HIF) for proteasomal degradation, thus providing a direct link between tumorigenesis and molecular pathways critical for cellular adaptation to hypoxia. Cell type specific gene targeting of VHL in mice has demonstrated that proper pVHL mediated HIF proteolysis is fundamentally important for survival, proliferation and differentiation of many cell types and furthermore, that inactivation of pVHL may, unexpectedly, inhibit tumor growth under certain conditions. Mouse knock out studies have provided novel mechanistic insights into VHL associated tumorigenesis and established a central role for HIF in the development of the VHL phenotype.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

Expression of p53 in renal carcinoma cells is independent of pVHL

Renal clear-cell carcinoma (RCC) is the predominant form of kidney cancer and is highly refractory to conventional anti-cancer therapies. The status of p53 tumor suppressor gene has been correlated with the efficacy of radio- and chemotherapies, where presence of mutant p53 is associated with reduced responsiveness to treatment. However, p53 itself is rarely mutated in RCC, rather suggesting that the p53 pathway might be compromized in RCC cells. In support of this notion, the transactivation property of normal p53 was shown to be repressed in various transformed kidney epithelial cells via an unknown dominant-negative mechanism. Mutation of the von Hippel-Lindau (VHL) gene causes familial VHL disease, which includes predisposition to RCC. Moreover, biallelic inactivation of VHL has been observed in the vast majority of sporadic RCC. Recently, the expression of pVHL in RCC cells was demonstrated to elevate the expression of p53 by inducing the binding of RNA-stabilizing protein HuR to the 3'untranslated region of p53 mRNA. Contrary to this finding, we report here that the reconstitution of a variety of VHL(-/-) RCC lines including 786-O, RCC4, and A498 or non-RCC cells with wild-type pVHL does not influence the expression of p53 and fails to induce p53-responsive gene p21CIP1/WAF1 or p53-responsive reporters. These results suggest that the expression of p53 in RCC cells is independent of pVHL.     

Molecular pathology and targeted therapy of clear cell renal cancer

The golden standard of care for advanced renal cell cancer (RCC) was until now the cytokine therapy with relatively low response rates. Advances of molecular genetics in RCC revealed several molecular targets such as VHL and angiogenic genotype or EGFR in the clear cell variant. Among the novel targeted agents, multiple tyrosine kinase inhibitors were proved to be clinically effective against advanced clear cell renal cancer, changing the standard of care. It is a further question how molecular diagnostics can improve these results by the detection of these targets or gene defects in individual tumors.     

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.     

Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer

Insulin-like growth factor-I (IGF-I)-mediated signaling is thought to be involved in the regulation of multiple cellular functions in different tumors including renal cell carcinoma (RCC). Blocking IGF-I signaling by any of the several strategies abolishes or delays the progression of a variety of tumors in animal models. Herein, we demonstrate that in RCC cell lines, IGF-I-mediated signaling is found to be inhibited in the presence of wild type von Hippel-Lindau (VHL) tumor suppresser gene. Moreover, molecular modeling and biochemical approaches have revealed that beta-domain of the VHL gene product by interacting directly with protein kinase Cdelta inhibits its association with IGF-IR for downstream signaling. We also demonstrated that RCC has IGF-I-mediated invasive activity where protein kinase Cdelta is an important downstream molecule, and this invasiveness can be blocked by wild type VHL. These experiments thus elucidate a novel tumor suppresser function of VHL with its unique kinase inhibitory domain.     

The Positive Regulation of p53 by the Tumor Suppressor VHL

The ubiquitin-mediated degradation of hypoxia-inducible factor-α (HIF-α) by a von Hippel-Lindau tumor suppressor protein (pVHL) is mechanistically responsible for controlling gene expression due to oxygen availability. Germline mutations in the VHL gene cause dysregulation of HIF and induce an autosomal dominant cancer syndrome referred to as VHL disease. However, it is unclear whether HIF accumulation caused by VHL mutations is sufficient for tumorigenesis. Recently, we found that pVHL directly associates and positively regulates the tumor suppressor p53 by inhibiting Mdm2-mediated ubiquitination, and by subsequently recruiting p53-modifying enzymes. Moreover, VHL-deleted RCC cells showed attenuated apoptosis or abnormal cell-cycle arrest upon DNA damage, but became normal when pVHL was restored. Thus, pVHL appears to play a pivotal role in tumor suppression by participating actively as a component of p53 transactivation complex during DNA damage response.     

Systemic VHL gene functions and the VHL disease

The von Hippel-Lindau tumor suppressor gene (VHL) is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor (HIF). VHL mutations are the genetic defects underlying several human diseases including polycythemia, familial VHL tumor syndrome and sporadic renal cell carcinoma. VHL mutations can lead to cell-autonomous phenotypes in the tumor cells. However, non-tumor cell-autonomous functions of VHL have also been noted. VHL tumor-derived cytokines can promote inflammation and induce mobilization of endothelial progenitor cells. Up-regulation of HIF caused by VHL loss-of-function mutants, including heterozygotes, has been shown to increase the activities of hematopoietic stem cells, endothelial cells and myeloid cells. As such, systemic functions of VHL likely play important roles in the development of VHL disease.     

Protective Function of von Hippel-Lindau Protein against Impaired Protein Processing in Renal Carcinoma Cells

The absence of functional von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of neoplasias characteristic of VHL disease, including renal cell carcinoma (RCC). Here, we compared the sensitivity of RCC cells lacking VHL gene function with that of RCC cells expressing the wild-type VHL gene (wtVHL) after exposure to various stresses. While the response to most treatments was not affected by the VHL gene status, glucose deprivation was found to be much more cytotoxic for RCC cells lacking VHL gene function than for wtVHL-expressing cells. The heightened sensitivity of VHL-deficient cells was not attributed to dissimilar energy requirements or to differences in glucose uptake, but more likely reflects a lesser ability of VHL-deficient cells to handle abnormally processed proteins arising from impaired glycosylation. In support of this hypothesis, other treatments which act through different mechanisms to interfere with protein processing (i.e., tunicamycin, brefeldin A, and azetidine) were also found to be much more toxic for VHL-deficient cells. Furthermore, ubiquitination of cellular proteins was elevated in VHL-deficient cells, particularly after glucose deprivation, supporting a role for the VHL gene in ubiquitin-mediated proteolysis. Accordingly, the rate of elimination of abnormal proteins was lower in cells lacking a functional VHL gene than in wtVHL-expressing cells. Thus, pVHL appears to participate in the elimination of misprocessed proteins, such as those arising in the cell due to the unavailability of glucose or to other stresses.     

Localization of point mutations in the coding part of the VHL gene in clear cell renal cancer

VHL gene is often inactivated in sporadic clear cell renal cancer (CCRC) due to somatic mutations, and it's germline mutations cause hereditary CCRC--von Hippel-Lindau syndrome. Localization of mutations in VHL, identification of new mutations and their influence on CCRC progression and sensitivity to targeted therapy are actual problems in modern oncogenetics. We have provided search and characterization of mutations in 248 primary CCRC using SSCP-analysis and sequencing. Somatic mutations were detected in 37.5% of samples, 72% of mutations were identified for the first time. New missense-mutations were analyzed by alignment programs and three-dimensional structure modeling. Mutation frequency was compared in different groups of patients in respect to stage, grade, and metastases. It was demonstrated that 39.1% samples with stage I harbor somatic mutations, however, no association with progression or metastases was found. We also have investigated localization of mutations in the VHL coding part and positions of missense-mutations and inframe deletions/insertions focusing on VHL critical sequences. VHL mutation analysis performed in this study improve the possibilities of laboratory diagnostics of familial and sporadic CCRC.