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14 February 2002, Volume 21, Number 8, Pages 1167-1170
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Original Paper
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 Oldfield1 and Zhengping Zhuang1

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

Correspondence to: Z Zhuang;, Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, Building 10, Room 5D37, Bethesda, Maryland, MD 20892, USA. E-mail: zhuangp@ninds.nih.gov

Abstract

The majority of patients with Von Hippel-Lindau (VHL) disease are affected by a VHL germline mutation involving one copy of the VHL gene. Loss of heterozygosity of the second VHL allele can be consistently demonstrated in tumor tissue from these patients, suggesting that allelic deletion is a very early or even initiating event for tumorigenesis. Approximately 20% of VHL disease patients, however, exhibit germline deletion of one entire copy or at least a substantial part of the VHL gene. To investigate the nature of the 'second genetic hit' in this patient population, we analysed two renal cell carcinomas and one CNS hemangioblastoma from three unrelated patients for genetic changes of the second copy of the VHL gene. All three tumors showed retention of one VHL allele by FISH. Single-strand conformation polymorphism and mutation analysis of microdissected tumor DNA revealed somatic point mutations of the wild-type VHL copies in each of the three tumors. The results indicate that the 'two hit model' is equally applicable to patients with VHL germline mutation and VHL germline deletion. In contrast to tumors from patients with VHL germline mutation, however, point mutations of the wild-type allele can be detected in tumors from patients with VHL germline deletion.

Oncogene (2002) 21, 1167-1170 DOI: 10.1038/sj/onc/1205121

Keywords

VHL disease; VHL gene; mutation; deletion

Introduction

Von Hippel-Lindau (VHL) disease, an autosomal dominant genetic disorder, is characterized by the development of a variety of tumors including renal cell carcinomas, CNS hemangioblastomas, pheochromocytomas, and pancreatic microcystic adenomas (Choyke et al., 1995; Maher and Kaelin, 1997). The VHL tumor suppressor gene has been linked to chromosome 3p25 (Seizinger et al., 1988) and subsequently identified (Latif et al., 1993).

The majority of patients with VHL disease carry a VHL germline mutation on one VHL gene copy. It has been suggested that a 'second hit' at the remaining wild-type VHL allele would initiate tumor growth in susceptible cells in these patients (Knudson, 1985). In agreement with this hypothesis, previous analyses of VHL disease-associated neoplasms have consistently demonstrated both VHL gene mutations and deletions in tumors that are associated with the syndrome (Gnarra et al., 1994).

In 20% of patients with VHL disease, however, germline mutations of the coding region of the VHL gene are absent. Instead, most of these patients exhibit deletion of one entire copy or at least a substantial part of the VHL gene (Pack et al., 1999). Since the nature of the second genetic hit is unknown in this patient population we analysed three tumors from three patients with VHL germline deletion for genetic alterations in comparison to the patients' germline DNA.

Results

Fluorescent in situ hybridization (FISH) using genomic probes P1, c3, c11 and g7 cDNA for the VHL gene (Pack 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 the open-reading frame using the cDNA probe g7. The two other cases not only showed loss of the open-reading frame, but more extended deletions by using c11 and P1 demonstrating complete loss of the VHL gene sequence (>35 kb, but <90 kb). For comparison, five blood samples from patients with known VHL gene mutation were studied and consistently revealed two copies of the VHL allele (Figure 1, d-f).

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 indicative of retention of one VHL allele in both normal and tumor tissue of these patients and did not suggest a deletion of the VHL allele as a pathogenetic mechanism underlying development of these tumors. In contrast, tumors from patients with VHL germline mutation had consistently lost one VHL gene copy (Figure 1e).

The FISH results were confirmed by PCR amplification of blood and microdissected tumor DNA with highly polymorphic markers flanking the VHL gene revealing presence of a single amplification band. Single-strand conformation polymorphism (SSCP) and mutation analysis of microdissected tumor DNA, however, revealed evidence of point mutation in the wild-type copy of the VHL gene as compared to the germline DNA: first, SSCP analysis demonstrated aberrant bands in tumor DNA as compared to normal control DNA from the same patients (Figure 1c). Second, sequencing analysis revealed a single missense mutation in each of the three tumors: exon 2, codon 151 (ATC to AGC; ILE to SER), exon 3, codon 200 (CGG to TGG; ARG to TRY), and exon 1, codon 69 (CGC to TGC; ARG to CYS) (Figure 2).

Discussion

Deletion of the wild-type VHL allele is almost universally found in tumors from patients with germline VHL gene mutation, suggesting a common pathogenetic pathway. Furthermore, VHL gene deletion can be demonstrated in numerous, independently occurring morphologic precursor lesions of these tumors (Lubensky et al., 1996). The large number of precursor lesions and the almost consistent presence of allelic wild-type deletion in these lesions suggest that the 'second hit', deletion of the wild-type VHL allele, represents a very early or even initiating event. It is not clear at what time the second hit occurs and what the nature of the susceptible cell population is, although the slow growth of VHL-associated tumors and the presence of these tumors in infants indicate that tumor initiation may occur in early childhood or even during embryogenesis.

Wild-type allelic deletion is known to represent a hallmark event associated with tumorigenesis in patients with germline tumor suppressor gene mutations; however, the nature of the 'second hit' in tumors occurring in association with germline deletion has never been investigated. Our findings demonstrate that VHL wild-type deletion does not play a pathogenetic role in tumors of patients with VHL germline deletion. Instead, all three investigated tumors revealed wild-type VHL point mutation. It appears therefore that the common 'mutation-deletion sequence' in patients with VHL germline mutation is replaced by a 'deletion-mutation sequence' in patients with VHL germline deletion. A possible explanation for this finding is positive selection of cells with somatic point mutation of the VHL wild-type allele in patients with VHL germline deletion. In contrast, somatic wild-type deletion would result in homozygous VHL gene deletion, which may be incompatible with cell survival as inactivation of both copies of the VHL gene in mice have been shown to cause death in utero (Gnarra et al., 1997).

In contrast, a wide spectrum of wild-type alterations can induce tumorigenesis in patients with VHL germline mutation, whereas tumorigenesis in patients with VHL germline deletion may in fact be restricted to somatic VHL wild-type point mutation. It is possible that the proposed mechanism may apply to the pathogenesis of other tumor suppressor syndromes. For example, in tumor tissue from a patient with MEN1 germline deletion retention of MEN1 has been demonstrated by amplification of exon sequences using DNA from microdissected tumor cells (A Calender, personal communication). Finally, 'deletion-mutation sequences' may play pathogenetic roles in the development of sporadic tumors in which random genetic deletions appear to be more frequent than random mutations (Lengauer et al., 1998).

Materials and methods

Frozen and paraffin-embedded, formalin-fixed tumor tissue was retrieved from three patients with VHL germline deletion. Two patients had presented with renal cell carcinoma, one patient with hemangioblastoma as manifestations of VHL disease. The patients had been cared for by the Urologic Oncology Branch, NCI, and the Surgical Neurology Branch, NINDS, respectively. Tissues were obtained as part of an institutional review board-approved protocol for which informed consent was obtained.

Fluorescent in situ hybridization (FISH) was performed using touch preparations from frozen tumor tissue. Genomic probes included P1-191 (90 kb in size) containing the entire VHL locus; cosmid c3 (~30 kb) which includes the 3' portion of the VHL gene (a part of the reading frame and 3'-UTR); cosmid c11 (~35 kb), which overlaps the exon 1 and 5'-UTR; and the cDNA 'group 7' (1.65 kb) which contains the entire open reading frame and some 5'- and 3'-UTR sequences (Pack et al., 1999). As controls, normal blood samples and blood samples from patients with known VHL mutation were studied.

For further genetic analysis, tumor cells were microdissected from tissue sections as described previously (Zhuang and Vortmeyer, 1998). After DNA extraction, PCR amplification of blood and microdissected tumor DNA was performed with highly polymorphic markers flanking VHL; the amplification products were separated on a 8% polyacrylamide sequencing gel. In addition, single strand conformation polymorphism (SSCP) and mutation analysis of microdissected tumor DNA was performed to screen for somatic mutations.

References

Choyke PL, Glenn GM, Walther MM, Patronas NJ, Linehan WM, Zbar B. (1995). Radiology, 194: 629-642. MEDLINE

Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, Latif F, Liu S, Chen F, Duh FM, Lubensky I, Duan DR, Forence C, Pozzati R, Walther MM, Bander NH, Grossman HB, Brauch H, Pomer S, Brooks JD, Isaacs WB, Lerman MI, Zbar B, Linehan WM. (1994). Nat. Genet., 7: 85-90. MEDLINE

Gnarra JR, Ward JM, Porter FD, Wagner JR, Devor DE, Grinberg A, Emmert-Buck MR, Westphal H, Klausner RD, Linehan WM. (1997). Proc. Natl. Acad. Sci. USA, 94: 9102-9107. Article MEDLINE

Knudson JrAG. (1985). Cancer Res., 45: 1437-1443. MEDLINE

Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt ML, Stackhouse T, Kuzmin I, Modi W, Geil L, Schmidt L, Zhou FW, Li H, Wei MH, Chen F, Glenn G, Choyke P, Walther MM, Weng YK, Duan DSR, Dean M, Glavac D, Richards FM, Crossey PA, Ferguson-Smith MA, Lepaslier D, Chumakov I, Cohen D, Chinault AC, Maher ER, Linehan WM, Zbar B, Lerman MI. (1993). Science, 260: 1317-1320. MEDLINE

Lengauer C, Kinzler KW, Vogelstein B. (1998). Nature, 396: 643-649. Article MEDLINE

Lubensky IA, Gnarra JR, Bertheau P, Walther MM, Linehan WM, Zhuang Z. (1996). Am J Pathol, 149: 2089-2094. MEDLINE

Maher ER, Kaelin JrWG. (1997). Medicine (Baltimore), 76: 381-391. Article MEDLINE

Pack SD, Zbar B, Pak E, Ault DO, Humphrey JS, Pham T, Hurley K, Weil RJ, Park WS, Kuzmin I, Stolle C, Glenn G, Liotta LA, Lerman MI, Klausner RD, Linehan WM, Zhuang Z. (1999). Cancer Res., 59: 5560-5564. MEDLINE

Seizinger BR, Rouleau GA, Ozelius LJ, Lane AH, Farmer GE, Lamiell JM, Haines J, Yuen JW, Collins D, Majoor-Krakauer D, Bonner T, Mathew C, Rubenstein A, Halperin J, McKonkierosell A, Green JS, Trofatter JA, Ponder BA, Eierman L, Bowmer MI, Schimke R, Oostra B, Aronin N, Smith DI, Drabkin H, Waziri MH, Hobbs WJ, Martuza RL, Conneally PM, Hsia YE, Gusella JF. (1988). Nature, 332: 268-269. MEDLINE

Zhuang Z, Vortmeyer AO. (1998). Cell. Vis., 5: 43-48. MEDLINE

Figures

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 amplification of microdissected tumor DNA with primers flanking 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

Figure 2 Sequencing analysis. Three tumors from three different 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)

Received 13 July 2001; revised 2 October 2001; accepted 29 October 2001
14 February 2002, Volume 21, Number 8, Pages 1167-1170
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