Genetic linkage between Von Hippel--Lindau disease and three microsatellite polymorphisms refines the localisation of the VHL locus

Von Hippel—Lindau (VHL) disease is a dominantly inheritedfamilial cancer syndrome characterised by the development ofretinal and central nervous system haemangioblastomas, renalcell carcinoma and phaeochromocytoma. The gene for VHL diseasehas been mapped to chromosome 3p25–p26 and presymtomaticdiagnosis using linked DNA markers is available. We have previouslymapped the VHL disease gene to a 4 cM interval between D3S1250and D3S18. To increase access to presymptomatic diagnosis andto accelerate progress towards isolating the VHL disease genewe attempted to identify microsatellite DNA markers linked tothe disease gene by genetic linkage analysis in 29 families.We found significant linkage between the VHL disease gene anddinucleotide (CA) repeat polymorphisms at D3S1038 (Zmax = 22.24at     

Somatic mutations of the von Hippel -- Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma

Loss of heterozygosity (LOH) studies have suggested that somaticmutations of a tumour suppressor gene or genes on chromosome3p are a critical event In the pathogenesls of non-familialrenal cell carcinoma (RCC). Germllne mutations of the von Hippel— Lindau (VHL) disease gene predispose to early onsetand multifocal clear cell renal cell carcinoma, and the mechanismof tumorigenesls In VHL disease is consistent with a one-hitmutation model. To Investigate the role of somatic VHL genemutations in non-familial RCC, we analysed 99 primary RCC forVHL gene mutations by SSCP and heteroduplex analysis. SomaticVHL gene mutations were Identified In 30 of 65 (46%) sporadicRCC with chromosome 3p allele loss and one of 34 (3%) tumourswith no LOH for chromosome 3p. The VHL gene mutations were heterogeneous(17 frameshift deletions, eight missense mutations, four frameshiftinsertions, one nonsense and one splice site mutation), butno mutations were detected in the first 120 codons of clonedcoding sequence. Most RCCs with somatic VHL mutations (23 of27 (85%) informative cases) had chromosome 3p25 allele lossin the region of the VHL gene so that both alleles of the VHLgene had been inactivated as expected from a two–hit modelof tumorigenesis. Detailed histopathology was available for59 of the tumours investigated: 18 of 43 (42%) RCC with a clearcell appearance had a somatic VHL gene mutation but none of16 non–clear cell RCC (eight chromophilic, three chromophobeand five oncocytoma) (x²= 7.77, P<0.025). These results suggestthat somatic mutations of the VHL gene are a critical eventin the pathogenesis of non-familial clear cell renal carcinoma,but do not exclude a role for other chromosome 3p tumour suppressorgenes.     

Role of chromosome 3p12-p21 tumour suppressor genes in clear cell renal cell carcinoma: analysis of VHL dependent and VHL independent pathways of tumorigenesis.

AIMS: Chromosome 3p deletions and loss of heterozygosity (LOH) for 3p markers are features of clear cell renal cell carcinoma but are rare in non-clear cell renal cell carcinoma. The VHL tumour suppressor gene, which maps to 3p25, is a major gatekeeper gene for clear cell renal cell carcinoma and is inactivated in most sporadic cases of this disease. However, it has been suggested that inactivation of other 3p tumour suppressor genes might be crucial for clear cell renal cell carcinoma tumorigenesis, with inactivation (VHL negative) and without inactivation (VHL positive) of the VHL tumour suppressor gene. This study set out to investigate the role of non-VHL tumour suppressor genes in VHL negative and VHL positive clear cell renal cell carcinoma. METHODS: Eighty two clear cell renal cell carcinomas of known VHL inactivation status were analysed for LOH at polymorphic loci within the candidate crucial regions for chromosome 3p tumour suppressor genes (3p25, LCTSGR1 at 3p21.3, LCTSGR2 at 3p12 and at 3p14.2). RESULTS: Chromosome 3p12-p21 LOH was frequent both in VHL negative and VHL positive clear cell renal cell carcinoma. However, although the frequency of 3p25 LOH in VHL negative clear cell renal cell carcinoma was similar to that at 3p12-p21, VHL positive tumours demonstrated significantly less LOH at 3p25 than at 3p12-p21. Although there was evidence of LOH for clear cell renal cell carcinoma tumour suppressor genes at 3p21, 3p14.2, and 3p12, both in VHL negative and VHL positive tumours, the major clear cell renal cell carcinoma LOH region mapped to 3p21.3, close to the lung cancer tumour suppressor gene region 1 (LCTSGR1). There was no association between tumour VHL status and tumour grade and stage. CONCLUSIONS: These findings further indicate that VHL inactivation is not sufficient to initiate clear cell renal cell carcinoma and that loss of a gatekeeper 3p21 tumour suppressor gene is a crucial event for renal cell carcinoma development in both VHL negative and VHL positive clear cell renal cell carcinoma.     

Germline mutations in the von Hippel–Lindau disease tumor suppressor gene: Correlations with phenotype

Von Hippel-Lindau disease (VHL) is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatie cysts, and pheochromocytomas. The VHL gene was recently isolated by positional cloning. The cDNA encodes 852 nucleotides in 3 exons. The VHL gene is unrelated to any known gene families. We identified germline mutations in 85/114 (75%) of VHL families. Clinical heterogeneity is a well-known feature of VHL. VHL families were classified into 2 types based on the presence or absence of pheochromocytoma. The types of mutations responsible for VHL without pheochromocytoma (VHL type 1) differed from those responsible for VHL with pheochromocytoma (VHL type 2). Fifty-six % of the mutations responsible for VHL type l were microdeletions/insertions, nonsense mutations, or deletions; 96% of the mutations responsible for VHL type 2 were missense mutations. Specific mutations in codon 238 accounted for 43% of the mutations responsible for VHL type 2. The mutations identified in these families will be useful in presymptomatic diagnosis. The identification of mutations associated with phenotypes contributes to the understanding of fundamental genetic mechanisms of VHL disease. © 1995 Wiley-Liss, Inc.     

Allelic Deletion of VHL Gene Detected in Papillary Tumors of the Broad Ligament, Epididymis, and Retroperitoneum in von Hippel-Lindau Disease Patients

Although clinically associated with von Hippel-Lindau (VHL) disease, the pathogenesis of papillary tumors of the broad ligament, epididymis, and peritoneum arising in patients with VHL disease is not clear. The "classic" VHL-associated neoplasms, including hemangioblastoma and renal ceil carcinoma, have been found to be associated with the inactivation of both VHL gene copies. It is not known whether a similar inactivation of the VHL gene is also responsible for the development of these uncommon VHL-associated lesions. The authors performed PCR (polymerase chain reaction) and PCR-based SSCP (single-strand conformation polymorphism) analysis on five predominantly papillary tumors in five VHL patients (one papillary cystadenoma of the broad ligament, one endometrioid cystadenoma of the broad ligament, two papillary cystadenomas of the epididymis, one papillary tumor of the retroperitoneum) with four polymorphic markers of VHL gene (D3S1038, D3S1110, D3S2452, 104/105). All five tumors showed allelic loss of VHL gene. The results provide the first genetic evidence for the role of VHL gene in the tumorigenesis of these rare benign neoplasms and confirm these tumors as phenotypic manifestations of VHL disease. Int J Surg Pathol 8(3):207-212, 2000     

中国人von Hippel-Lindau病家系致病基因大片段缺失研究

目的 研究中国人von Hippel-Lindau病(von Hippel-Lindau syndrome,VHL)家系致病基因大片段缺失情况及其表型特点.方法 采用通用引物荧光定量PCR(universal primer quantitative fluorescent multiplex-polymerase chain reaction,UPQFM-PCR)技术结合GeneSean分析系统对新发现的20个VHL病家系的先证者及部分自愿者进行VHL基因胚系突变大片段缺失检测.结果 20例先证者中检测到VHL基因大片段缺失6例,其中第1外显子缺失3例、第3外显子缺失1例、VHL基因完全缺失2例.进一步检测其中2个家系其他患者和部分成员也存在与先证者相同的VHL基因缺失.2个VHL基因完全缺失的家系,其患者表现为中枢神经系统血管母细胞瘤和视网膜血管瘤但无肾癌表型.结论 中国汉族人VHL患者基因种系突变存在VHL基因大片段缺失;VHL病诊断应常规开展VHL基因大片段缺失检测;VHL基因完全缺失和中枢系统血管母细胞瘤和视网膜血管瘤可能存在一定关系并需进一步研究.     

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the Von Hippel Lindau disease gene

Von Hippel LIndau disease (VHL) is a rare autosomal dominantdisease associated with tumors and cysts in multiple organ systems.The VHL disease gene is tightly linked to the polymorphic DNAmarker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomericand RAF1 on its centromeric side. Two additional markers, D3S1038and D3S601, have also been identified, and these markers, likeD3S720, are very tightly linked to VHL. Previously 93 cosmidclones were mapped to the larger region, 3p24.2 - pter, surroundingthe VHL disease gene (1). Using a Southern-based screening strategyon pools of YAC clones we have Isolated a contig of overlappingYAC clones that extends about 0.7 megabase centromeric, andabout 1.3 megabases telomeric of D3S720 and contains all threetightly linked VHL markers. Individual YACs In this contig werehybridized to grids containing cosmids localized between 3p24.2-pterand to several cosmids localized by fluorescent in situ hybridization(FISH) to 3p25. A total of 28 cosmids were positioned on thiscontig of overlapping YAC clones. We have also identified homologousYAC clones to many additional cosmid clones localized between3p24.2–p25, although these have not yet been preciselylocalized relative to the contig of YAC clones. This contigof YAC clones probably contains the VHL disease gene and shouldfacilitate the isolation and characterization of this gene.     

Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Papillary cystadenoma of the epididymis is an uncommon benign lesion that may occur sporadically or as a manifestation of von Hippel—Lindau (VHL) disease. Neither immunohistochemical studies nor molecular genetic analyses of the VHL gene have been reported previously for this lesion. The authors describe two cases of clear cell papillary cystadenoma of the epididymis, both of which were initially confused with metastatic renal cell carcinoma. Both lesions showed positive immunohistochemical staining for low and intermediate molecular weight keratins (Cam 5.2 and AE1/AE3), EMA, vimentin, α₁-antitrypsin, and α₁-antichymotrypsin. Each was negative for CEA. Because clear cell papillary cystadenoma is similar to renal cell carcinoma histologically, and because both occur as components of the von Hippel—Lindau disease complex, the authors analyzed both cases for the presence of mutations in the VHL gene. A somatic VHL gene mutation was detected in one of the two tumors by polymerase chain reaction followed by single-strand conformation polymorphism analysis. Direct sequencing revealed a cytosine to thymine transition at nucleotide 694, resulting in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. As the VHL gene is believed to function as a tumor suppressor gene, VHL gene mutations may play a role in the initiation of tumorigenesis in sporadic cystadenomas of the epididymis.     

Detailed genetic mapping of the von Hippel-Lindau disease tumour suppressor gene.

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited familial cancer syndrome characterised by a predisposition to the development of retinal, cerebellar, and spinal haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. The gene for VHL disease has been mapped to chromosome 3p25-p26 and flanking markers identified. We report the detailed genetic mapping of the VHL disease locus in 38 families. Significant linkage was detected between VHL disease and D3S601 (Zmax = 18.86 at theta = 0.0, CI 0.0-0.025), D3S18 (Zmax = 11.42 at theta = 0.03, CI 0.005-0.08), RAF1 (Zmax = 11.02 at theta = 0.04, CI 0.007-0.01), and D3S1250 (Zmax = 4.73 at theta = 0.05, CI 0.005-0.15). Multipoint linkage analysis mapped the VHL disease locus between D3S1250 and D3S18 close to D3S601. There was no evidence of locus heterogeneity. This study has (1) confirmed the tight linkage between VHL disease and D3S601, (2) identified D3S1250 as the first marker telomeric to RAF1 which maps centromeric to the VHL disease gene, and (3) narrowed the target region for isolation of the VHL disease gene by positional cloning techniques to a 4 cM interval between D3S1250 and D3S18. These findings will improve the clinical management of families with VHL disease by improving the accuracy of presymptomatic diagnosis using linked DNA markers, and will enhance progress towards isolating the VHL disease gene.     

Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in pancreatic microcystic adenomas.

An association between pancreatic microcystic (serous) adenomas (MCAs) and von Hippel-Lindau (VHL) disease has been suggested. However, genetic alterations of the VHL gene in MCAs of the pancreas have never been reported. In this study, we performed genetic analysis of 12 pancreatic MCAs. In 2 cases, VHL disease was documented clinically, and 10 cases were sporadic. For LOH analysis, tumor and normal pancreatic cells were procured from formalin-fixed, paraffin-embedded material using tissue microdissection. After DNA extraction, the samples were amplified by polymerase chain reaction using the polymorphic markers D3S2452, D3S1110, D3S192, and D3S656. In addition, the sporadic tumors were analyzed for VHL gene mutations using probes 3b/10b and K55/K56. Both MCAs associated with VHL disease showed LOH with at least one of the microsatellite markers tested. Among the 10 sporadic cases, 7 tumors showed LOH at the VHL gene locus. A somatic VHL gene mutation on exon 2 was documented in one sporadic case. The study provides the first direct genetic evidence for the role of the VHL gene in MCA tumorigenesis. Furthermore, VHL gene alterations may be detected in both VHL-associated and sporadic pancreatic MCAs.     

Expression of HIF-1 and ubiquitin in conventional renal cell carcinoma: relationship to mutations of the von Hippel-Lindau tumor suppressor gene

Conventional clear cell renal cell carcinomas (cRCC) have mutations of the von Hippel-Lindau (VHL) tumor suppressor gene at 3p25 in approximately 50% of cases. The VHL gene normally regulates ubiquitin-mediated proteolysis of hypoxia-inducible factor 1alpha (HIF-1alpha); in cell lines, VHL inactivation blocks HIF-1alpha proteolysis, resulting in increased HIF-1 expression. This study was undertaken to investigate the relationship between VHL mutations and the expression of ubiquitin and HIF-1alpha in cRCC. Eleven cRCC were studied with microsatellite analysis for 3p deletions and with sequencing for VHL mutations. Immunohistochemistry was performed for HIF-1alpha and ubiquitin. Deletions at 3p25 were found in 10 tumors, and VHL mutations were identified in 6 of these cases. There was staining for ubiquitin and HIF-1alpha in all tumors with VHL mutations. Among the five cases without VHL mutations, staining for ubiquitin or HIF-1alpha was not present in three cases but was present in two tumors, both of which had 3p deletions. The findings support a role for VHL mutations promoting cRCC development by an impairment of HIF-1alpha proteolysis. The findings also suggest that a 3p tumor suppressor gene other than VHL may also influence HIF-1alpha degradation and that there is an additional tumorigenic pathway for cRCC that does not involve VHL or HIF-1.     

散发性肾癌中VHL基因的两个单核苷酸多态位点分析和杂合性缺失检测

目的检测79例散发性肾癌中抑癌基因VHL内部的两个单核苷酶多态(single nucleotide polymorphism，SNP)位点并分析杂合性缺失(loss of heterozygosity，LOH)发生情况，探讨VHL基因LOH与肾癌临床病理特征的关系。方法从肿瘤和正常肾组织中提取DNA，应用聚合酶链反应-限制性片段长度多态性方法检测VHL基因5′端SNP位点rs779805和3′端SNP位点rs1642742的基因型。在两个位点的杂合子中进行LOH检测，并分析VHL基因LOH与临床病理特征的关系。结果我们计算了两个位点的基因型、基因频率、杂合度、多态信息含量等遗传学参数。综合两个位点发现杂合子29例．其中12例(41．4％)存在LOH。VHL基因LOH与肾癌发生年龄、性别、临床分期、病理分级无显著相关性。结论在散发性肾癌中，VHL基因LOH是肿瘤发生的重要机理，其发生率达41．4％，VHL基因LOH与肾癌分期、分级无关。     

Expression of fibronectin and HIF-1alpha in renal cell carcinomas: relationship to von Hippel-Lindau gene inactivation

The von Hippel-Lindau (VHL) tumor suppressor gene (TSG) at 3p25 is mutated in approximately 50% of conventional (clear cell) renal cell carcinomas (cRCC). VHL normally regulates the ubiquitin-mediated proteolysis of hypoxia-inducible factor 1alpha (HIF-1alpha), and VHL inactivation results in increased cellular HIF-1alpha expression. VHL protein (pVHL) also interacts with fibronectin (Fn) and VHL inactivation results in defective Fn extracellular matrix assembly. The present study investigated the immunohistochemical (IHC) staining for Fn and HIF-1alpha in 11 cRCC and the relationship of the staining to VHL inactivation by gene deletion, mutation, or hypermethylation. Evidence for VHL inactivation by 3p deletions and VHL mutations were found in six tumors. Fn-positive IHC staining of tumor cells and negative to weak staining of extracellular stroma was found in five cases having exon 1 or exon 2 mutations. In contrast, Fn staining was absent in tumor cells and positive in the stroma of five tumors without VHL inactivation and in one tumor with a C-terminal exon 3 mutation. HIF-1alpha tumor cell staining was present in the cRCC with VHL inactivation but was also present in two tumors having 3p deletions but neither mutation nor hypermethylation of VHL. These two cRCC showed a tumor cell-negative and stroma-positive pattern of Fn staining. The findings indicate that VHL inactivation plays a role in the development of some cRCC by altering Fn cell--stroma relationships. They also suggest that some C-terminal mutations may not interfere with Fn assembly and that a 3p TSG in addition to VHL influences HIF-1alpha degradation.     

Mutational analysis of the von hippel lindau gene in clear cell renal carcinomas from tuberous sclerosis complex patients.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by seizures, mental retardation, autism, and tumors of multiple organs. Renal disease in TSC includes angiomyolipomas, cysts, and renal cell carcinomas. It is known that somatic mutations in the von Hippel Lindau (VHL) tumor suppressor gene occur in most clear cell renal carcinomas. To determine whether TSC-associated clear cell carcinomas also contain VHL mutations, we analyzed six tumors for loss of heterozygosity in the VHL gene region of chromosome 3p and for mutations in the VHL gene. Four of the patients were women between the ages of 34 and 68 years, and two were males under the age of 21 years. The loss of heterozygosity analysis was performed using polymorphic microsatellite markers, and the mutational analysis was performed using direct sequencing. Chromosome 3p loss of heterozygosity was not detected, and no VHL mutations were identified. These findings suggest that mutations in the TSC1 and TSC2 genes lead to clear cell renal carcinogenesis via an alternate pathway not involving VHL mutations.     

The VHL-dependent regulation of microRNAs in renal cancer

Background  

The commonest histological type of renal cancer, clear cell renal cell carcinoma (cc RCC), is associated with genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumour suppressor. VHL inactivation leads to induction of hypoxia-inducible factors (HIFs) and a hypoxic pattern of gene expression. Differential levels of specific microRNAs (miRNAs) are observed in several tumours when compared to normal tissue. Given the central role of VHL in renal cancer formation, we examined the VHL-dependent regulation of miRNAs in renal cancer.     

Presymptomatic diagnosis of von Hippel-Lindau disease with flanking DNA markers.

Von Hippel-Lindau (VHL) disease is a dominantly inherited cancer syndrome characterised by the development of retinal, cerebellar, and spinal haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. The gene for VHL disease has been mapped to chromosome 3p25-p26 and flanking markers identified. We have investigated the usefulness of currently available DNA markers for the presymptomatic diagnosis of VHL disease. In the first part of this investigation, genetic linkage data from two previously published studies were updated and reanalysed to provide accurate estimates of sex specific recombination fractions and to confirm that there is no evidence of locus heterogeneity. In the second part of this study, 14 families containing 23 asymptomatic subjects at 50% prior risk of VHL disease were investigated with closely linked DNA markers (RAF1, D3S18, D3S732). Seventeen subjects were informative with one or more markers, six of whom were informative at markers flanking the VHL disease gene. By combining age related and DNA based risk information the carrier risk for 11 subjects was reduced to < 2%.     

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters

To delineate more precisely the somatic von Hippel-Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA-SSCP followed by sequencing, methylation-specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear-cell or variant-type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear-cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear-cell RCCs. The VHL gene therefore seems to be inactivated in a two-hit manner by intragenic mutation or hypermethylation plus allelic loss in clear-cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7-19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear-cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (< or = 55 years) than that in older (> or = 56 years) patients. These data suggested that the inactivation of the VHL tumor-suppressor gene is a specific genetic change in clear-cell RCC, and that it may occur at an early or first step in the clear-cell tumorigenic pathway rather than as a late event.