A Comprehensive Study of the VHL-R200W Chuvash Polycythemia Mutation Reveals a Gradual Dysregulation of the Hypoxia Pathway in Oncogenesis
Authors: Gardie Betty , Couvé Sophie , Ladroue Charline , Laine Elodie , Mahtouk Karène , Guégan Justine , Gad Sophie , Le Jeune Hélène , Lecomte Bernard , Pagès Jean-Christophe , Benusiglio Patrick , Bressac-de Paillerets Brigitte , Feunteun Jean , Dessen Philippe , Hermouet Sylvie , Tchertanov Luba , Mole David R. , Kaelin William G. , Ratcliffe Peter J , Richard Stephane 
Published: October 2019

Abstract
Context : Germline mutations in genes involved in the hypoxia sensing pathway (VHL, PHD2/EGLN1, HIF2A/EPAS1) predispose patients to erythrocytosis associated with normal or high serum erythropoietin level. The more frequent mutation, VHL-R200W (R200W), has been identified in homozygous carriers with a congenital erythrocytosis named Chuvash polycythemia. Survival in the Chuvash patients was found to be reduced compared to control groups due to higher rates of arterial and venous thromboses, and to haemorrhagic events. Noteworthily, a characteristic of these patients and their parents, heterozygous for the mutation, is the total absence of tumor development as the opposite of heterozygous carriers of other VHL mutations. Indeed, VHL is a tumor suppressor gene and heterozygous carriers of VHLmutations have von Hippel-Lindau disease and are at high risk of multiple tumors (e.g. CNS hemangioblastomas, pheochromocytoma, renal cell carcinoma). The absence of tumor development in patients with congenital erythrocytosis remained unexplained. The R200W mutation is transmitted within a 340-kilobases haplotype inherited 14,000 to 62,000 years ago from a single founder event. The absence of tumor development in patients carrying the R200W mutation raised the possibility of the presence of a protective element within this core haplotype.
Objective: The purpose of this study was to determine the mechanisms underlying the absence of tumor development in patients carrying the R200W mutation, healthy (heterozygous carriers) or with congenital erythrocytosis (homozygous).
Methods: We report here one particular family carring a heterozygous R200W mutation associated, for the first time, with a typical von Hippel-Lindau disease (with pheochromocytoma, hemangioblastoma and renal cell carcinoma) (Olswang et al., 1998). We performed a comprehensive study with genotyping, phenotypic analysis, structural modeling, functional and transcriptomic studies of the R200W mutant in comparison to classical mutants involved in von Hippel-Lindau disease. For the first time, the use of inducible expression vectors encoding untagged VHL in the functional studies allowed the observation of very subtle effects.
Results: We show that the R200W mutation alone is definitely not associated with the von Hippel Lindau disease because this particular family actually carries another pathogenic mutation, VHL-R161Q, on the same VHL allele, and the double mutated allele segregates with disease. Our genotyping showed that the doubled mutations lies on the Chuvash core haplotype transmitted with the R200W mutation. We can thus conclude that this haplotype does not contain any protective elements against cancer development and that the VHLmutations are, by themselves, predominantly responsible for manifestations of the von Hippel-Lindau disease. Functional studies demonstrate that the function of the R200W mutation is close to the wild type protein and the severity of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of pVHL dysfunction in hypoxia signaling pathways.
Conclusion: We show that the R200W mutation associated with congenital erythrocytosis is at the bottom of an oncogenic gradient. These results validate for the first time in humans the recently proposed “continuum” model of tumor suppression, which differs from the classical Knudson’s two-hit model and implies that subtle gene dosage-sensitivity may underlie tumor predisposition. This model may be of major importance in our understanding of tumor risk in patients with erythrocytosis carrying germline mutations in other genes belonging the HIF pathway. Indeed, in rare cases, PHD2 and HIF2A pathogenic mutations also predispose patients to pheochromocytomas or paragangliomas development.

Disclosures
No relevant conflicts of interest to declare.