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Somatic Point Mutation of the Wild-Type Allele Detected in Tumors of Patients with VHL Germline Deletion

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Somatic Point Mutation of the Wild-Type Allele Detected in Tumors of Patients with VHL Germline Deletion Oncogene (2002) 21, 1167 ± 1170 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Somatic point mutation of the wild-type allele detected in tumors of patients with VHL germline deletion Alexander O Vortmeyer1, Steve C Huang1, Svetlana D Pack1, Christian A Koch2, Irina A Lubensky1, Edward H Old®eld1 and Zhengping Zhuang*,1 1Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, Building 10, Room 5D37, Bethesda, Maryland, MD 20892, USA; 2Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, MD 20892, USA The majority of patients with Von Hippel-Lindau (VHL) (Seizinger et al., 1988) and subsequently identi®ed disease are aected by a VHL germline mutation (Latif et al., 1993). involving one copy of the VHL gene. Loss of The majority of patients with VHL disease carry a heterozygosity of the second VHL allele can be VHL germline mutation on one VHL gene copy. It has consistently demonstrated in tumor tissue from these been suggested that a `second hit' at the remaining patients, suggesting that allelic deletion is a very early or wild-type VHL allele would initiate tumor growth in even initiating event for tumorigenesis. Approximately susceptible cells in these patients (Knudson, 1985). In 20% of VHL disease patients, however, exhibit germline agreement with this hypothesis, previous analyses of deletion of one entire copy or at least a substantial part VHL disease-associated neoplasms have consistently of the VHL gene. To investigate the nature of the demonstrated both VHL gene mutations and deletions `second genetic hit' in this patient population, we in tumors that are associated with the syndrome analysed two renal cell carcinomas and one CNS (Gnarra et al., 1994). hemangioblastoma from three unrelated patients for In 20% of patients with VHL disease, however, genetic changes of the second copy of the VHL gene. germline mutations of the coding region of the VHL All three tumors showed retention of one VHL allele by gene are absent. Instead, most of these patients exhibit FISH. Single-strand conformation polymorphism and deletion of one entire copy or at least a substantial part mutation analysis of microdissected tumor DNA revealed of the VHL gene (Pack et al., 1999). Since the nature somatic point mutations of the wild-type VHL copies in of the second genetic hit is unknown in this patient each of the three tumors. The results indicate that the population we analysed three tumors from three `two hit model' is equally applicable to patients with patients with VHL germline deletion for genetic VHL germline mutation and VHL germline deletion. In alterations in comparison to the patients' germline contrast to tumors from patients with VHL germline DNA. mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion. Results Oncogene (2002) 21, 1167 ± 1170. DOI: 10.1038/sj/ onc/1205121 Fluorescent in situ hybridization (FISH) using genomic probes P1, c3, c11 and g7 cDNA for the VHL gene (Pack Keywords: VHL disease; VHL gene; mutation; deletion et al., 1999) demonstrated a single VHL allele copy in blood lymphocytes of all three patients (Figure 1, a ± c). In one case, VHL deletion was demonstrated by loss of Introduction the open-reading frame using the cDNA probe g7. The two other cases not only showed loss of the open- Von Hippel-Lindau (VHL) disease, an autosomal reading frame, but more extended deletions by using c11 dominant genetic disorder, is characterized by the and P1 demonstrating complete loss of the VHL gene development of a variety of tumors including renal cell sequence (435 kb, but 590 kb). For comparison, ®ve carcinomas, CNS hemangioblastomas, pheochromocy- blood samples from patients with known VHL gene tomas, and pancreatic microcystic adenomas (Choyke mutation were studied and consistently revealed two et al., 1995; Maher and Kaelin, 1997). The VHL tumor copies of the VHL allele (Figure 1, d ± f). suppressor gene has been linked to chromosome 3p25 FISH of tumor tissue (two renal cell carcinomas and one cerebellar hemangioblastoma) from the patients with VHL germline deletion was identical with normal tissue (Figure 1 a,b). Therefore, the results were *Correspondence; Z Zhuang; E-mail: [email protected] Received 13 July 2001; revised 2 October 2001; accepted 29 indicative of retention of one VHL allele in both October 2001 normal and tumor tissue of these patients and did not Genetic changes in tumors in VHL germline deletion AO Vortmeyer et al 1168 Figure 1 FISH and SSCP analysis of blood and tumor tissue from patients with VHL germline deletion (a±c) and patients with VHL germline mutation (d±f). FISH analysis of (a) white blood cells and (b) tumor cells from renal cell carcinoma of patient with VHL germline deletion using c11 probe for VHL gene (red signal). Both blood cells and tumor cells reveal only one copy of the VHL gene; two signals are seen with an alpha satellite probe for chromosome 3 (green signal) in both normal and tumor tissue; (c) SSCP analysis of tumor of patient with VHL germline deletion after ampli®cation of microdissected tumor DNA with primers ¯anking exon 2 of the VHL gene. Sense and antisense strands of the VHL gene wild-type VHL allele (WT) are in regular position; mutation of exon 2 in tumor tissue (T) results in aberrant migration pattern of both sense and antisense strands. FISH analysis of (d) white blood cells and (e) tumor cells from renal cell carcinoma of patient with VHL germline mutation using P1 probe for VHL gene (red signal). White blood cells show two signals, tumor cells show loss of one copy of the VHL gene; two signals are seen with an alpha satellite probe for chromosome 3; (f) SSCP analysis of normal (N) and tumor cells (T) of patient with VHL germline mutation using polymorphic marker for VHL promoter area (104/105). Tumor tissue shows loss of wild-type band consistent with deletion suggest a deletion of the VHL allele as a pathogenetic occurring morphologic precursor lesions of these mechanism underlying development of these tumors. In tumors (Lubensky et al., 1996). The large number of contrast, tumors from patients with VHL germline precursor lesions and the almost consistent presence of mutation had consistently lost one VHL gene copy allelic wild-type deletion in these lesions suggest that (Figure 1e). the `second hit', deletion of the wild-type VHL allele, The FISH results were con®rmed by PCR ampli®ca- represents a very early or even initiating event. It is not tion of blood and microdissected tumor DNA with clear at what time the second hit occurs and what the highly polymorphic markers ¯anking the VHL gene nature of the susceptible cell population is, although revealing presence of a single ampli®cation band. the slow growth of VHL-associated tumors and the Single-strand conformation polymorphism (SSCP) presence of these tumors in infants indicate that tumor and mutation analysis of microdissected tumor DNA, initiation may occur in early childhood or even during however, revealed evidence of point mutation in the embryogenesis. wild-type copy of the VHL gene as compared to the Wild-type allelic deletion is known to represent a germline DNA: ®rst, SSCP analysis demonstrated hallmark event associated with tumorigenesis in aberrant bands in tumor DNA as compared to normal patients with germline tumor suppressor gene muta- control DNA from the same patients (Figure 1c). tions; however, the nature of the `second hit' in tumors Second, sequencing analysis revealed a single missense occurring in association with germline deletion has mutation in each of the three tumors: exon 2, codon never been investigated. Our ®ndings demonstrate that 151 (ATC to AGC; ILE to SER), exon 3, codon 200 VHL wild-type deletion does not play a pathogenetic (CGG to TGG; ARG to TRY), and exon 1, codon 69 role in tumors of patients with VHL germline deletion. (CGC to TGC; ARG to CYS) (Figure 2). Instead, all three investigated tumors revealed wild- type VHL point mutation. It appears therefore that the common `mutation-deletion sequence' in patients with Discussion VHL germline mutation is replaced by a `deletion- mutation sequence' in patients with VHL germline Deletion of the wild-type VHL allele is almost deletion. A possible explanation for this ®nding is universally found in tumors from patients with germ- positive selection of cells with somatic point mutation line VHL gene mutation, suggesting a common of the VHL wild-type allele in patients with VHL pathogenetic pathway. Furthermore, VHL gene dele- germline deletion. In contrast, somatic wild-type tion can be demonstrated in numerous, independently deletion would result in homozygous VHL gene Oncogene Genetic changes in tumors in VHL germline deletion AO Vortmeyer et al 1169 Figure 2 Sequencing analysis. Three tumors from three dierent patients with germline VHL deletion. Sequencing analysis of tumor #1 (renal cell carcinoma, case 1) shows missense mutation of exon 2, codon 151 (ATC to AGC; ile to ser); tumor #2 (hemangioblastoma, case 2) reveals missense mutation of exon 3, codon 200 (CGG to TGG; arg to try); tumor #3 (renal cell carcinoma, case 3) shows missense mutation of exon 1, codon 69 (CGC to TGC; arg to cys) deletion, which may be incompatible with cell survival deletion. Two patients had presented with renal cell as inactivation of both copies of the VHL gene in mice carcinoma, one patient with hemangioblastoma as manifesta- have been shown to cause death in utero (Gnarra et al., tions of VHL disease. The patients had been cared for by the 1997).
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