¹Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Disease (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, MD 20892, USA

²Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, MD 20892, USA

³Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

⁴Georgetown University, Washington DC, USA

Correspondence to: C A Koch, National Institutes of Health, NICHD, PREB, Building 10, Rm 9D42, Bethesda, MD 20892, USA; E-mail: kochc@exchange.nih.gov

^aThese two authors contributed equally to this study

Multiple endocrine neoplasia type 2 (MEN 2) is an inherited cancer syndrome that includes pheochromocytoma. Germline mutations in RET are responsible for MEN 2 but the precise pathogenetic mechanisms of tumorigenesis are unknown. We have recently identified possible mechanisms of tumor formation in patients with MEN 2A-related pheochromocytoma. Two of nine tumors investigated, however, did not reveal either of these mechanisms. In the present study, we therefore searched for other possible mechanisms underlying the pathogenesis of MEN 2A-related pheochromocytoma. Hereditary pheochromocytoma also occurs in patients with von Hippel-Lindau (VHL) disease, a syndrome consisting of tumors caused by inactivation of the VHL tumor suppressor gene. A subset of sporadic pheochromocytomas have somatic mutations in RET or VHL, suggesting that both genes contribute to pheochromocytoma pathogenesis in a subset of tumors. It is unknown, however, whether VHL gene alterations would be associated with tumorigenesis in hereditary, MEN 2-related pheochromocytoma. We therefore investigated four pheochromocytomas from patients with MEN 2A and RET germline mutations for the presence of allelic deletion and/or somatic mutation of the VHL gene. LOH analysis using the polymorphic markers D3S1038 and D3S1110 that map to the VHL gene locus 3p25/26, revealed evidence for somatic VHL gene deletion in all four MEN 2A-related pheochromocytomas. Mutation analysis of the VHL gene showed frameshift mutations in two tumors and a splice acceptor mutation in one tumor. The remaining tumor did show LOH but not mutation of the VHL gene. These results suggest that somatic genetic alterations of the VHL gene may play a role in the tumorigenesis of some MEN 2A-related pheochromocytomas.

Oncogene (2002) 21, 479-482 DOI: 10.1038/sj/onc/1205133

RET; MEN 2; VHL; pheochromocytoma; deletion

Pheochromocytomas are rare neuroendocrine tumors that arise from chromaffin tissue. Such tumors develop in patients with multiple endocrine neoplasia type 2A (MEN 2A), a rare familial cancer syndrome characterized by the presence of medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia adenoma (Ponder, 1999). RET is expressed in tissues derived from the neural crest cells such as the chromaffin cells of the adrenal medulla (Nakamura et al., 1994). Although germline mutations of RET, a proto-oncogene, are responsible for tumorigenesis in patients with MEN 2A, it is unclear why only selected cells undergo tumor formation in these patients. We have recently shown that duplication of the mutant RET allele in trisomy 10 or loss of the wild-type RET sequence, both leading to overrepresentation of mutant RET, may represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma (Huang et al., 2000). However, two of the nine pheochromocytomas investigated in our study, did not demonstrate either of these two mechanisms.

Hereditary pheochromocytoma also occurs in the hereditary syndromes von Hippel-Lindau (VHL) disease and neurofibromatosis type 1 (NF 1) (Walther et al., 1999; Choyke et al., 1995; Riccardi, 1991). Whereas the molecular pathogenesis of patients with VHL disease-related pheochromocytoma has been well characterized (Bender et al., 2000; Crossey et al., 1994; Zeiger et al., 1995; Prowse et al., 1997; Khosla et al., 1991), it is less clear in NF 1-related and sporadic pheochromocytomas. A subset (less than 10%) of sporadic pheochromocytomas have somatic mutations in RET or VHL (Brauch et al., 1997; Eng et al., 1995; Lindor et al., 1995; Beldjord et al., 1995; Crossey et al., 1995), suggesting that both genes contribute to pheochromocytoma pathogenesis in a subset of tumors. It is unknown, however, whether somatic VHL gene alterations would be associated with tumorigenesis in hereditary, MEN 2-related pheochromocytoma. We here sought to analyse pheochromocytomas from patients with MEN 2A and a single germline mutation of RET for the additional presence of allelic deletion and somatic mutation in the VHL gene, as possible tumor initiating events.

We studied four pheochromocytomas from four unrelated patients with MEN 2A. In all patients, we identified RET germline mutations. VHL gene deletion analysis revealed loss of heterozygosity (LOH) of the VHL gene locus in all four MEN 2A-related pheochromocytomas (Figure 1).

To further investigate whether inactivation of the VHL tumor suppressor gene may play a role in tumorigenesis of MEN 2A-related pheochromocytoma, we performed mutation analysis of the VHL gene and found mutations in three of the four pheochromocytomas (Figure 2, Table 1).

In case 1, sequencing analysis of the VHL gene revealed a single base (del G) deletion at nucleotide 376 in exon 1, leading to a shift in the translational frame of the mRNA from codon 55 to a premature stop codon at position 66. Sequencing analysis of case 2 showed a two base deletion (del TT) at nucleotide 656-657 in exon 2 of the VHL gene, resulting in a frameshift from codon 148 to a premature stop codon at position 172.

In case 3, sequencing analysis revealed an A to C base change at the -2 position of the exon 3 splice acceptor sequence of VHL, disrupting the highly conserved consensus sequence and resulting in abnormal splicing of the encoded transcript.

Case 4 showed no evidence of VHL mutation but of VHL gene deletion by microsatellite marker analysis.

In this study, we show that somatic VHL mutation and deletion (knockout) may be involved in tumorigenesis of MEN 2A-related pheochromocytoma. In both sporadic and familial forms of pheochromocytoma, allelic losses at 1p, 3p (up to 45%), 17p, and 22q have been reported (Bender et al., 2000; Vargas et al., 1997; Moley et al., 1992; Mulligan et al., 1993; Dannenberg et al., 2000; Benn et al., 2000). Specific tumor suppressor or oncogenes at these chromosomal loci, however, have rarely been analysed. In sporadic pheochromocytomas, somatic mutations of VHL or RET are rarely found (<10%) and thus have no major role in tumorigenesis (Brauch et al., 1997; Eng et al., 1995; Lindor et al., 1995; Beldjord et al., 1995; Crossey et al., 1995). VHL disease consists of a variety of neoplasms including hemangioblastomas of the central nervous system, renal cell carcinomas, pheochromocytomas, and cysts involving the kidney, pancreas, and epididymis (Choyke et al., 1995; Knudson, 1986). The gene responsible for VHL disease is the VHL tumor suppressor gene, located on chromosome 3p25/26. Neoplasms in VHL disease including pheochromocytoma typically develop according to Knudson's two-hit model, an inherited germline mutation of VHL and loss of function of the wild-type allele of the VHL gene (Bender et al., 2000; Maher and Kaelin, 1997).

We detected inactivating somatic mutations of VHL in three of four MEN 2A-related pheochromocytomas. Furthermore, genetic analysis revealed evidence for VHL gene deletion in all four selected cases. It is possible that in case 4 additional genetic changes involving the VHL gene may have contributed to tumor initiation and/or progression. For instance, VHL gene inactivation secondary to CpG island hypermethylation of the VHL gene promoter may represent another mechanism of VHL gene inactivation (Herman et al., 1994). We were not able to perform studies of epigenetic silencing of the remaining VHL gene in case 4 because we only had microdissected DNA available which is not sufficient for such an analysis. In all these cases of MEN 2A, regular wild-type VHL alleles were detected in normal control tissue confirming the presence of two wild-type alleles and excluding the presence of VHL disease.

Mutations in the VHL gene result in constitutive expression of many hypoxia-inducible genes, in part related to increasing levels of hypoxia-inducible transcription factor (HIF) 1-alpha which in normal cells is rapidly ubiquitinated and degraded (Kamura et al., 2000). Furthermore, VHL protein, a ubiquitinase adapter, has been implicated in a variety of processes that are central to carcinogenesis including cell-cycle control, differentiation, extracellular matrix formation and turnover, and angiogenesis (Kondo and Kaelin, 2001). Therefore, mutations in VHL may lead to an absent or reduced VHL protein function with reduced degradation of proteins including RET protein. In addition to other events, RET protein accumulation secondary to absent or reduced VHL protein, may then cause transformation of selected (hyperplastic) chromaffin cells to pheochromocytoma.

Tumor formation in these selected MEN 2A-related pheochromocytomas may have occurred by first, a RET germline mutation, leading to hyperplasia of chromaffin cells and second, subsequent somatic VHL gene deletion and mutation in selected cells, thereby transforming those into pheochromocytoma. One of the four MEN 2A-related pheochromocytomas we investigated did not have somatic VHL gene mutation but only deletion. It is possible that the second VHL allele in this tumor may have been intact and tumor formation may have occurred by other mechanisms such as gross genomic changes involving other tumor suppressor or proto-oncogenes, or DNA repair genes (Lengauer et al., 1998).

Acknowledgements

We thank Dr Nieman, Clinical Director, NIH, NICHD, for support of this study.

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Figure 1 LOH analysis of the VHL gene locus of four cases of MEN 2A-related pheochromocytoma with marker D3S1038. All cases show loss of one allele in microdissected tumor tissue (T), whereas heterozygosity is retained in normal non-neoplastic tissue (N). Arrow indicates lost allele. Tissue was obtained from four patients (three men, one woman) under an Internal Review Board (IRB)-approved protocol (00-CH-0093) at the National Institutes of Health. All of them had MEN 2A with a germline mutation of RET, and have been operated on (uni- or bilateral adrenalectomy) for intraadrenal pheochromocytoma. Two of these four tumors (cases 1 and 2) stem from our former study (Huang et al., 2000). Blood was drawn for DNA extraction and tumors from all patients were removed at the time of surgery and frozen at -80°C. DNA was extracted from lymphoblasts and tumor tissue by standard methods. Six-micron sections were obtained from each frozen tumor and briefly stained with H&E. One tumor was paraffin-embedded and prepared for microdissection. Under direct light microscopic visualization using a 30-gauge needle, a modified microdissection procedure was performed, as previously described (Zhuang and Vortmeyer, 1998). In all cases, we also obtained samples of nontumor control tissue from the same slides. We performed LOH analysis, using polymorphic markers/primers D3S1038 and D3S1110 mapped to the VHL gene locus 3p25/26 in the presence of [-³²P]dCTP (0.1 Ci/l) (Dupont). PCR conditions using AmpliTaq Gold DNA polymerase (Perkin Elmer Roche) in a Hybaid Omnigene thermal cycler were as follows: initial denaturation at 95°C for 10 min, then 35 cycles, each with 1 min of denaturation at 95°C, 1 min of annealing at 60°C, and 1 min of extension at 72°C; PCR was completed with a final extension at 72°C for 10 min. The amplicons were resolved on a 6% polyacrylamide gel. Gels were dried and exposed to Kodak XAR film. All PCR reactions were performed in triplicate

Figure 2 DNA sequence analysis of MEN 2 pheochromocytoma samples with mutations in the VHL gene. (a) Portion of the sequence of exon 1 from case 1 demonstrating a deletion of a single nucleotide (del G) in one allele at the position indicated by the arrow. (b) Sequence of a portion of exon 2 from case 2 demonstrating the deletion of two nucleotides (del TT) at the positions indicated by the arrows. The mutant sequence is faint, but discernible as minor peaks under the normal sequence at several positions after the deletion. The tissue from which the DNA was extracted contained normal cells. (c) Portion of the sequence of exon 3 demonstrating an A to C mutation (arrow) at the -2 position at the splice acceptor sequence. The location of the intron-exon boundary is marked. Exons 1-3 of the VHL gene were amplified from genomic DNA using polymerase chain reaction conditions described elsewhere (Stolle et al., 1998). Mutation scanning by conformation sensitive gel electrophoresis (CSGE) was performed on the PCR products as described by Ganguly et al. (1993). DNA sequence analysis was performed using a cycle sequencing kit with dye-labeled terminators (PE Advanced Biosystems, Inc., Foster City, CA, USA). Sequences were analysed on an ABI 377 automated DNA sequencer. The primers for sequencing analysis of RET were as follows: exon 10 (IIF, 5'-GGG GGA TTA AAG CTG GCT AT and IR, 5'-CTC AGA TGT GCT GTT GAC AC), and exon 11 (IF, 5'-TCA CAC CAC CCC CAC CCA CAG and IIR, 5'-TGG TAG CAG TGG ATG CAG AA). The AmpliCycle sequencing kit (Perkin Elmer Roche) was used according to the manufacturer's protocol

Table 1 Somatic VHL gene deletion and mutation in MEN 2A-associated pheochromocytoma