doi: 10.1111/j.1469-1809.2011.00647.x Identification and In Silico Analysis of Novel von Hippel-Lindau (VHL) Gene Variants from a Large Population Emanuela Leonardi 1,2 , Maddalena Martella 1 , Silvio C.E. Tosatto 2 and Alessandra Murgia 1∗ 1 Department of Pediatrics, University of Padua, Italy 2 Department of Biology, University of Padua, Italy Summary Mutational inactivation of the VHL gene is the cause of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary cancer syndrome predisposing to haemangioblastomas, pheochromocytomas and clear-cell renal carcinomas. The gene product (pVHL) functions as an adapter in cellular processes including cell growth and apoptosis. VHL mutation analysis was carried out in 426 unrelated subjects with phenotypes ranging from VHL syndrome, to isolated VHL-related tumours that could represent the first manifestation of the disease. A total of 111 individuals were found to carry alterations, with large deletions representing 40% of the variants. Eighteen of the 95 detected variants were novel, seemingly disease-causing mutations; their pathogenic role has been evaluated in silico for effects on protein folding and interactions. Putative regions of interaction between pVHL and proteins involved in common pathways have been identified, assessing possible implications for the presence of mutations in these regions. All new variants predicted to truncate or cause complete pVHL loss of structure were associated with phenotypes consistent with VHL type 1. Seven of the new amino acid substitutions are disease-causing mutations, one is a neutral variant, whereas the results for two remain ambiguous. Our combined molecular and in silico approach for the evaluation of putative disease-causing mutations contributes to the interpretation of the potential pathogenicity of these novel variants. Keywords: VHL, structural bioinformatics, molecular genetics, angiogenesis, oncology Introduction von Hippel-Lindau disease (VHL; MIM No. 193300) is a fa- milial cancer syndrome due to mutations of the VHL gene (Kaelin & Maher, 1998). It is characterised by predisposi- tion to the development of highly vascularised tumours such as retinal and central nervous system haemangiobalstomas, pheochromocytomas (PH), and clear-cell renal carcinoma (RCC) (Lonser et al., 2003). VHL is clinically classified as type 1 and type 2 based on the absence or presence of PH, one of the early onset features of the disease. Type 2 VHL is subclassified based on lower (type 2A) or higher (type 2B) sus- ceptibility to RCC. Despite extreme phenotypic variability between and within families, important genotype–phenotype correlations have emerged for different classes of pathogenic mutations (Crossey et al., 1994; Hes et al., 2007; Ong et al., ∗ Corresponding author: Alessandra Murgia, University of Padua, Department of Pediatrics Via Giustiniani, 3, 35128 Padua, Italy. Tel: +39 049 821 1430; Fax: +39 049 821 3502; E-mail: murgia@pediatria.unipd.it 2007; Franke et al., 2009; Nordstrom-O’Brien et al., 2010). Mutant copies of the VHL gene that completely abolish its normal function are found virtually only in VHL type 1 dis- ease, with low risk of PH. VHL type 2 is almost invariably associated with missense mutations, and a limited number of these mutations have been specifically associated with a PH- only subtype, VHL type 2C (Crossey et al., 1995; Ritter et al., 1996). pVHL is a substrate recognition component of an E3 ubiq- uitin ligase complex (VCB), including Elongin C, Elongin B, Cullin2, and Rbx1/Roc1, targeting proteins for ubiquitin- mediated degradation (Lisztwan et al., 1999). It contains two functional domains: the C-terminal α-domain allows the pro- tein to adopt its native 3D conformation after binding to Elongin C. The β -domain forms a substrate docking inter- face for target proteins. The best known target of this complex is hypoxia-inducible factor-1α (HIF1α) (Min et al., 2002), but several other substrates have also been identified (Kaelin, 2007). Multiple HIF-dependent and HIF-independent func- tions are known, all contributing to the VHL-defective oxygen-sensing response and tumorigenesis (Kaelin, 2008). Annals of Human Genetics (2011) 75,483–496 483 C  2011 The Authors Annals of Human Genetics C  2011 Blackwell Publishing Ltd/University College London