VHL gene mutations and their effects on hypoxia inducible factor HIF{alpha}: Identification of potential driver and passenger mutations

Abstract

Mutations of the von Hippel-Lindau gene (VHL) 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 had missense mutations. Location and destabilizing effects of VHL missense mutations were determined in silico. The majority of thermodynamically destabilizing missense mutations were located in exon 1 in the core of pVHL, while 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 re-introducing VHL missense mutations into a VHL null cell line and by monitoring the GFP signals after the transfection of a HIFα-GFP expression vector. pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1α and HIF2α degradation. In sum, 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 HIFα, elongin B, or elongin C, and a third with pVHL functions comparable to 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.pVHL's functionality ranged from no effect to complete HIF stabilization. Interestingly, Asn78Ser, Asp121Tyr, and Val130Phe selectively influenced HIF1α and HIF2α degradation. In sum, 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 HIFα, elongin B, and elongin C, and a third with pVHL functions comparable to 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.