VHL gene mutations and their effects on hypoxia inducible factor HIF? : identification of potential driver and passenger mutations

Mutations of the von Hippel-Lindau (VHL) gene are frequent in clear cell renal cell carcinomas (ccRCC). Nonsense and frameshift mutations abrogate the function of the VHL protein (pVHL), whereas missense mutations can have different effects. To identify those missense mutations with functional consequences, we sequenced VHL in 256 sporadic ccRCC and identified 187 different VHL mutations of which 65 were missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico. The majority of the thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, whereas protein surface mutations in exon 3 affected the interaction domains of elongin B and C. Their impact on pVHL's functionality was further investigated in vitro by stably reintroducing VHL missense mutations into a VHL null cell line and by monitoring the green fluorescent protein (GFP) signals after the transfection of a hypoxia inducible factor (HIF)?-GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1? and HIF2? degradation. In summary, we obtained three different groups of missense mutations: one with severe destabilization of pVHL; a second without destabilizing effects on pVHL but relevance for the interaction with HIF?, elongin B, and elongin C; and a third with pVHL functions comparable with wild type. We therefore conclude that the specific impact of missense mutations may help to distinguish between driver and passenger mutations and may explain responses of ccRCC patients to HIF-targeted therapies.