Molecular Basis of the Disease

The VHL tumor suppressor gene was isolated by positional cloning in 1993.3 The gene, which consists of three exons spanning about 10 kilobases (kb) of genomic DNA, is highly conserved among worms, flies, rodents, and humans (reviewed in Reference 4). Two transcripts, 6.0kb and 6.5kb in size, are almost ubiquitously expressed and encode proteins of 213 and 159 amino acid residues. The latter isoform is the major product in most tissues and results from initiation of translation from an internal methionine codon at position 54. Both protein isoforms appear to be functional.

The VHL protein has been implicated in a variety of functions including transcriptional regulation, posttran-scriptional gene expression, protein folding, extracellular matrix formation, and ubiquitylation (reviewed in Reference 4). In recent years, the role of VHL in the regulation of hypoxia-inducible genes through the targeted ubiquity-lation and degradation of hypoxia-inducible factor 1 alpha subunit (HIF1A) has been elucidated, leading to a model of how disruption of the VHL gene results in the production of highly vascularized tumors.

Normal VHL binds to the protein elongin C,which forms a complex with elongin B and cullin-2 (CUL2). This complex resembles the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase or E3 complex in yeast that catalyzes the polyubiquitylation of specific proteins and targets them for degradation by proteosomes. Under normoxic conditions, HIF1A is hydroxylated at a specific asparagine residue by a member of the egg-laying deficiency protein nine-like (EGLN) protein family of prolyl hydroxylase enzymes.VHL binds to hydroxylated HIF1A and targets it for degradation. Under hypoxic conditions, HIF1A is not hydroxylated, VHL does not bind, and HIF1A subunits accumulate. HIF1A forms heterodimers with HIF1B and activates transcription of a variety of hypoxia-inducible messenger RNAs (mRNAs) (i.e., VEGF, EPO, TGFA, PDGFB). Likewise, when VHL is absent or mutated, HIF1A subunits accumulate, resulting in cell proliferation and the neovasculariza-tion of tumors characteristic of VHLD.4

Predisposition to VHLD is inherited as an autosomal dominant trait. However, tumor formation requires loss of the second allele (loss of heterozygosity [LOH]), and so the disease is recessive at the level of the cell. Mutations known to result in predisposition to VHLD include partial or complete deletions of the gene and point mutations (missense, nonsense, frameshift, and splice site). Point mutations are predicted either to truncate the protein, to alter protein folding, or to interfere with the binding of VHL to elongin C, HIF1A, or other target proteins.4,5 Although there are a handful of "common" mutations and one mutation hotspot in exon 3, point mutations are distributed over all three exons of the gene from codon 54 (internal initiator methionine) to the stop codon.

Four VHLD phenotypes have been described based on the likelihood of pheochromocytoma or renal cell carci-noma.6 Type 1 is characterized by a low risk for pheochro-mocytoma. Truncating mutations or missense mutations that are predicted to grossly disrupt the folding of the VHL protein5 are associated with VHLD type 1. VHLD type 2 is characterized by a high risk for pheochromocytoma. Patients with VHLD type 2 almost invariably have a missense mutation. VHLD type 2 is further subdivided into those with a low risk (type 2A) and those with a high risk

(type 2B) of renal cell carcinoma, as well as individuals at risk for pheochromocytoma only (type 2C). Some missense mutations correlate with a specific type 2 VHLD phenotype.6

A novel genotype-phenotype correlation has been reported.7 Individuals with a complete deletion of the VHL gene are more likely to present with multiple heman-gioblastomas of the brain or spine or both. At present, it is not clear why a complete deletion of the VHL gene would result in a phenotype distinctly different from that caused by a partial deletion or truncating mutation (i.e., VHL type 1).

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