A detailed analysis of von Hippel Lindau (VHL) mutations in sporadic clear cell renal carcinoma (ccRCC), VHL syndrome, and Chuvash polycythaemia.

Abstract

e15024 Background: Germline mutations in the VHL gene cause VHL syndrome. Functional loss of the VHL gene product, pVHL is also seen in the majority of cases of sporadic ccRCC and homozygosity for the germline R200W VHL mutation causes Chuvash polycythaemia, a benign congenital erythrocytosis. pVHL has many functions including the ability to target hypoxia-inducible factor (HIF) for polyubiquitylation and proteasomal degradation by forming the substrate recognition component of a ubiquitin-ligase complex. The development of ccRCC has been attributed to impaired HIF-α downregulation by pVHL mutants. However many point mutations within VHL which are associated with ccRCC are external to the HIF-α binding site. The structural and functional effects of the R200W mutation are unknown. Methods: We present a detailed biochemical analysis of pVHL mutant proteins identified in patients with sporadic ccRCC, type 2A (low risk of ccRCC) and 2B (high risk of ccRCC) VHL syndrome and Chuvash polycythaemia. Results: Type 2A mutations Y98H and Y112H are not globally destabilising, but affect the binding site for the HIF-α hydroxyproline, as revealed by localised chemical shift changes in NMR. Mutations at S65, N78, G93 and T157 have been described in both sporadic ccRCC and type 2B VHL syndrome and are not predicted to directly impact upon the HIF-α binding site. The G93V and S65L mutations cause extensive structural perturbations of pVHL which are likely to affect both HIF-α related and HIF-α unrelated functions of pVHL. In contrast N78D, T157I and R200W mutations cause minimal structural perturbations of pVHL and do not impair HIF-α binding, implying potential novel mechanisms of pathogenicity. Truncating mutations identified in patients with sporadic ccRCC result in fundamentally unstable proteins which retain the ability to bind HIF-α, supporting previous assertions suggesting a possible dominant negative role for truncated pVHL mutants. Conclusions: Increased knowledge with respect to the functional effects of mutational events within VHL will allow us to develop informative ways of categorising these events in a way which may ultimately provide novel therapeutic opportunities. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cancer Research UK, Medical Research Council