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.     

Cryptic von Hippel-Lindau disease: germline mutations in patients with haemangioblastoma only

OBJECTIVES— Central nervous system haemangioblastoma (HAB) is a major feature of von Hippel-Lindau (VHL) disease, and it is estimated that about 30% of HAB patients have VHL disease. Consequently, it is widely recommended that sporadic HAB patients are screened for clinical and radiological features of VHL disease because of the risk of multiple tumours. We investigated the frequency of VHL germline mutations in patients with HAB only with no clinical or radiological evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients.
METHODS—Eighty four patients with a single HAB (23 Dutch, 61 UK) and four with multiple HAB (two Dutch, two UK) were studied by direct sequencing of the coding region and quantitative Southern blotting.
RESULTS—A VHL germline mutation was found in three of 69 (4.3%) single HAB patients aged 50 years or less (three of 84 (3.6%) total single HAB patients). A germline VHL mutation was detected in a 44 year old woman with a solitary cerebellar HAB, as well as in four clinically unaffected close relatives, and in two single HAB cases presenting at the ages of 29 and 36 years. Germline VHL mutations were detected in two of four cases with multiple HAB.
CONCLUSIONS—Early detection of VHL disease is important to reduce morbidity and mortality and therefore we recommend that, in addition to conventional clinical and radiological investigations, VHL gene mutation analysis should be offered to all HAB patients younger than 50 years. HAB patients aged >50 years will have a lower a priori risk of VHL disease and further data are required to evaluate the role of routine molecular genetic investigations in late onset HAB cases. The failure to detect germline VHL mutations in some patients with multiple HAB may indicate the presence of somatic mosaicism or additional HAB susceptibility genes.


Keywords: haemangioblastoma; von Hippel-Lindau disease; VHL; germline mutation     

Germline mutations of the VHL gene in seven Chinese families with von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions within the VHL tumor-suppressor gene, but VHL germline mutations in the Chinese have rarely been studied. To investigate the genetic profile of VHL mutations in the Chinese population, we evaluated the clinical characteristics of seven Chinese families suffering from VHL disease and determined the particular germline mutations in their VHL genes. Direct sequencing and real-time quantitative PCR was carried out. Disease-associated genetic abnormalities were identified in all of the seven families examined. Two novel intragenic germline mutations (645 G insertion and 417 G deletion) were identified and are reported for the first time. Partial VHL gene deletions in exon 1 were found in two of the seven families. Three clinically asymptomatic mutation carriers were also identified. The spectrum of VHL gene abnormalities in our group is distinct from that observed in North America, Europe and Japan. These mutations are also different from those previously identified in other Chinese VHL patients. Future meta-analysis will provide greater perspective on the Chinese VHL genetic profile. VHL gene screening can play a key role in identifying asymptomatic patients who are carriers of VHL-predisposing genetic abnormalities.     

Phenotypic expression in von Hippel-Lindau disease: correlations with germline VHL gene mutations.

Von Hippel-Lindau disease is an autosomal dominantly inherited familial cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. VHL disease shows variable expression and interfamilial differences in predisposition to phaeochromocytoma. In a previous study of 65 VHL kindreds with defined VHL mutations we detected significant differences between VHL families with and without phaeochromocytoma such that missense mutations were more common and large deletions or protein truncating mutations less frequent in phaeochromocytoma positive families. To investigate the significance and cause of this association further, we studied 138 VHL kindreds for germline mutations and calculated the age related tumour risks for different classes of VHL gene mutations. Using SSCP, heteroduplex and Southern analysis we identified a germline VHL gene mutation in 101 families (73%). Direct sequencing of the VHL coding region further increased the mutation detection rate to 81%. In addition to precise presymptomatic diagnosis, identification of a VHL gene mutation can provide an indication of the likely phenotype. We found that large deletions and mutations predicted to cause a truncated protein were associated with a lower risk of phaeochromocytoma (6% and 9% at 30 and 50 years, respectively) than missense mutations (40% and 59%, respectively) and that missense mutations at codon 167 were associated with a high risk of phaeochromocytoma (53% and 82% at ages 30 and 50 years). Cumulative probabilities of renal cell carcinoma did not differ between the two groups (deletion/ truncation mutations: 8% and 60%, and missense mutations: 10% and 64% at ages 30 and 50 years, respectively). Age related risks for haemangioblastoma were similar in the two mutation groups, with the age related risks of cerebellar haemangioblastoma slightly less (35% and 64% v 38% and 75% at ages 30 and 50 years) and retinal haemangioblastoma slightly higher (45% and 72% v 37% and 64% at ages 30 and 50 years) in the missense mutation group than in the deletion/protein truncation group. These results provide valuable data for counselling VHL families and indicate that specific VHL mutations may be associated with different tumour susceptibility risks. There was no evidence of a generalised increase in age related tumour risks for missense mutations, suggesting that missense mutations predisposing to phaeochromocytoma have tissue specific effects, possibly because the VHL protein has several functions, the importance of which varies from tissue to tissue, or because the proteins which interact with VHL differ between different tissues.     

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.     

Molecular analysis of de novo germline mutations in the von Hippel-Lindau disease gene

VHL disease is a dominantly inherited familial cancer syndromewith variable expression and age-dependent penetrance. The diagnosisof isolated cases is often delayed compared with familial cases,and estimates of the new mutation rate have varied more than20-fold. To investigate the frequency and origin of de novoVHL gene mutations we have analysed: (i) families with identicalmutations to determine if there is a common haplotype, and (ii)apparent new mutation cases to determine whether the clinicaldiagnosis of such cases is reliable and to define the parentalorigin of de novo VHL gene mutations. Haplotyping of 12 VHLmutations occurring in two or more families (total 42 kindreds)revealed that for most mutations there was no evidence of afounder effect. A marked bias for a paternal origin of new mutationshas been reported in other familial cancer syndromes such asneurofibromatosis type 1 (NF1), multiple endocrine neoplasia(MEN) 2B and bilateral retinoblastoma, but it is unclear whetherthis bias results from a greater susceptibility for mutagenesisduring male gametogenesis because of the larger number of celldivisions compared with that in oogenesis, or from genomic imprintingeffects. Analysis of 13 de novo VHL mutations in which the parentof origin could be established, showed no evidence for a biasfor a paternal origin (seven paternal, six maternal), and differedsignificantly from that reported in NF1, MEN2B and bilateralretinoblastoma. This result demonstrates that an increased susceptibilityto paternal allele mutation is not a universal finding in autosomalgenetic diseases and that the origin of new mutations may beinfluenced by both genomic imprinting effects and the increasednumber of cell divisions in spermatogenesis compared with oogenesis.     

单细胞水平检测von Hippel-Lindau病基因突变的实验研究

目的建立单细胞水平检测vonHippel-Lindau病基因(VHL)突变的实验方法。方法单个淋巴细胞基于多重置换扩增(multiple displacement amplification,MDA)的全基因组扩增后进行常规PCR后测序和实时荧光定量PCR,结合各荧光的终点变化判断对应的等位基因存在与否。结果MDA后单细胞扩增效率为90.91%,污染率为0;通过测序,患者VHL等位基因的脱扣率为26.67%,诊断正确率为73.33%;通过结合相应荧光的终点变化判断患者VHL基因的等位基因的脱扣率为16.67%,正确率为83.33%。结论单细胞MDA后常规PCR后测序及实时荧光定量PCR能够特异、准确地检测单个淋巴细胞的VHL基因型,两者的联合应用有助于提高检测的准确性。     

Detection of germline mutations in the von Hippel-Lindau disease gene by the primer specified restriction map modification method.

Von Hippel-Lindau disease (VHL) is an inherited disorder characterised by a predisposition to develop tumours in the eyes, central nervous system, kidneys, and adrenal glands. Recently the VHL gene was cloned and shown to be mutated in 75% of US and Canadian VHL families. To develop simple, rapid methods for the detection of mutations found in large numbers of affected people, we designed based on the primer specified restriction site modification method. These tests have proved useful in identifying asymptomatic mutated VHL gene carriers who have the nt 505 T to C mutation or the nt 686 T to C mutation. Together with an MspI digestion test which can detect a mutation hot spot in codon 238, polymerase chain reaction/restriction endonuclease based tests can now detect VHL mutations in more than 50% of VHL type 2 families.     

Papillary neoplasm of the endolymphatic sac in a patient with von Hippel-Lindau disease.

Glandular tumours involving the middle ear and the mastoid are rare, and distinguishing between adenoma and adenocarcinoma is difficult. Two distinct histopathological patterns are accepted. While their clinical presentation differs, both require primary surgical treatment and both have a high rate of local recurrence. The papillary form takes a more aggressive course and wider regional spread. This pattern occurs predominantly in women. Its microscopic appearance and clinical course have been extensively described by Heffner who considered it as "low-grade adenocarcinoma of probable endolymphatic sac origin". A few cases have been associated with von Hippel-Lindau disease. The case of a 32 year old black woman is described. It is suggested that papillary adenocarcinoma of the endolymphatic sac should be considered in the spectrum of neoplasms seen in von Hippel-Lindau disease.     

Effects of iron-chelators on ion-channels and HIF-1alpha in the carotid body

Acute hypoxia instantaneously increases the chemosensory discharge from the carotid body, increasing ventilation mostly by inhibiting the oxygen sensitive ion channels and exciting the mitochondrial functions in the glomus cells. On the other hand, Fe2+-chelation mimics hypoxia by inhibiting the prolyl hydroxylases and the degradation of HIF-1alpha in non-excitable cells. Whether Fe2+-chelation can inhibit the ion channels giving rise to the sensory responses in excitable cells was the question. We characterized the responses to Fe2+-chelators on excitable glomus cells of the rat, and found that they instantaneously blocked the ion-channels, exciting the chemosensory discharge, and later causing a gradual accumulation of HIF-1alpha. Although initiated by the same stimuli, the two effects (on ion channels and cytosolic HIF-1alpha) possibly occurred by two different mechanisms.     

Frequency of Von Hippel-Lindau germline mutations in classic and non-classic Von Hippel-Lindau disease identified by DNA sequencing, Southern blot analysis and multiplex ligation-dependent probe amplification

The current clinical diagnosis of Von Hippel-Lindau (VHL) disease demands at least one specific [corrected] VHL manifestation in a patient with familial VHL disease, or, in a [corrected] sporadic patient, at least two or more hemangioblastomas or a single hemangioblastoma in combination with a typical visceral lesion. To evaluate this definition, we studied the frequency of germline VHL mutation in three patients groups: (i) multi-organ involvement (classic VHL), (ii) limited VHL manifestations meeting criteria (non-classic VHL) and (iii) patients with VHL-associated tumors not meeting current diagnostic VHL criteria. In addition, we validated multiplex ligation-dependent probe amplification (MLPA) as a rapid and reliable quantitative method for the identification of germline VHL deletions. The frequency of germline VHL mutations was very high in classic VHL cases with multi-organ involvement (95%), lower in non-classic cases that meet current diagnostic criteria but have limited VHL manifestations or single-organ involvement (24%) and low (3.3%), but tangible in cases not meeting current diagnostic VHL criteria. The detection of germline VHL mutations in patients or families with limited VHL manifestations, or single-organ involvement is relevant for follow-up of probands and early identification of at-risk relatives.     

Genotype-phenotype correlations of pheochromocytoma in two large von Hippel-Lindau (VHL) type 2A kindreds with different missense mutations

Von Hippel–Lindau (VHL) disease type 2A is an inherited tumor syndrome characterized by predisposition to pheochromocytoma (pheo), retinal hemangioma (RA), and central nervous system hemangioblastoma (HB). Specific VHL subtypes display genotype–phenotype correlations but, unlike other familial syndromes such as MEN‐2, the phenotype in VHL has not yet been stratified at the codon level. Over decades, we have managed two very large VHL type 2A regional kindreds with nearly adjacent but distinct VHL missense mutations. We determined the phenotype of Family 2 and compared the clinical and pathologic parameters of pheo between 30 members of Family 1 (Y112H mutation) and 33 members of Family 2 (Y98H mutation) with mean follow‐up of 15.5 and 12.1 years, respectively (P = 0.24). In Family 2, pheo was the most frequent VHL manifestation (79%) and all pheo diagnoses occurred by age 50. Age at first diagnosis was younger in Family 2 than in Family 1 (mean 19.7 vs. 28.8 years; P = 0.02). Pheo expressivity differed by genotype: Family 1 pheo was more likely to be multifocal (P = 0.04), as well as malignant (P < 0.01) and lethal (P = 0.02). Family 1 pheo was also more likely to secrete vanillylmandelic acid (VMA) alone (P = 0.05). This analysis of 130 pheochromocytomas in 63 VHL type 2A patients demonstrates that mutation‐specific malignancy and expression patterns exist within the VHL type 2A subtype, and provides information that may help tailor the screening and management algorithms of affected members and those at risk. © 2010 Wiley‐Liss, Inc.     

Long-term effect of external beam radiotherapy of optic disc hemangioma in a patient with von Hippel-Lindau disease

An 18-year-old woman with a 2-year history of hypertension and headache was diagnosed with noradrenalin-secreting bilateral adrenal pheochromocytomas with paragangliomas in the background of von Hippel-Lindau disease with family histories and a missense mutation, 712C to T (Arg167Trp) in the VHL gene. She had optic disc hemangioma in the left eye which gradually enlarged and caused serous retinal detachment on the macula in one year. Low-dose external beam radiation (20 Gy) was administered to the left eye using a lens-sparing single lateral technique. She underwent craniotomy for cerebellar hemangioblastoma at the age of 22 years and total pancreatectomy for multiple neuroendocrine tumors at the age of 24 years. In the 6-year follow-up period after the radiotherapy, the optic disc hemangioma gradually reduced in size and its activity remained low, allowing good central vision to be maintained. External beam radiation is recommended as a treatment option for the initial therapy for optic disc hemangioma.     

Metastasis of renal cell carcinoma to central nervous system hemangioblastoma in two patients with von Hippel-Lindau disease

Here we report tumor-to-tumor metastases identified in two patients with von Hippel-Lindau (VHL) disease. The first patient had bilateral renal carcinomas and multiple cerebellar hemangioblastomas, and the second patient had a renal carcinoma and multiple hemangioblastomas in the retina, cerebellum and spinal cord. A cerebellar lesion from the first patient and a spinal lesion from the second patient contained two distinct components. The inner part of these tumors consisted of a nested mass of polygonal clear cells that expressed cytokeratin and epithelial membrane antigen, while the outer part of the tumors showed proliferation of capillaries and intervening foamy stromal cells that were negative for cytokeratin and epithelial membrane antigen. The tumors were thus considered to be hemangioblastomas complicated by metastatic lesions of renal cell carcinoma of clear cell type. These cases indicate that tumor-to-tumor metastasis should be considered when hemangioblastoma contains a clear cell carcinoma component in the setting of VHL disease, and that immunohistochemical staining for cytokeratin and epithelial membrane antigen is useful for the diagnosis.