Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia.

Abstract

Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.
Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.
This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category.
As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.

Full text

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Distinct deregulation of the hypoxia inducible factor by PHD2
mutants identified in germline DNA of patients with polycythemia
by Charline Ladroue, David Hoogewijs, Sophie Gad, Romain Carcenac,
Federica Storti, Michel Barrois, Anne-Paule Gimenez-Roqueplo, Michel Leporrier,
Nicole Casadevall, Olivier Hermine, Jean-Jacques Kiladjian, Andre' Baruchel,
Fadi Fakhoury, Brigitte Bressac de Paillerets, Jean Feuteun, Nathalie Mazure,
Jacques Pouyssegur, Roland H. Wenger, Stephane Richard, and Betty Gardie
Haematologica 2011 [Epub ahead of print]
Citation: Ladroue C, Hoogewijs D, Gad S, Carcenac R, Storti F, Barrois M,
Gimenez-Roqueplo AP, Leporrier M, Casadevall N, Hermine O, Kiladjian JJ,
Baruchel A, Fakhoury F, Bressac-de Paillerets B, Feuteun J, Mazure N, Pouyssegur J,
Wenger RH, Richard S, and Gardie B. Distinct deregulation of the hypoxia inducible
factor by PHD2 mutants identified in germline DNA of patients with polycythemia.
Haematologica. 2011; 96:xxx
doi:10.3324/haematol.2011.044644
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Published Ahead of Print on September 20, 2011, as doi:10.3324/haematol.2011.044644.

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1
Distinct deregulation of the hypoxia inducible factor by PHD2 mutants
identified in germline DNA of patients with polycythemia

Charline Ladroue1*, David Hoogewijs2*, Sophie Gad1, Romain Carcenac1, Federica Storti2,
Michel Barrois3, Anne-Paule Gimenez-Roqueplo4, Michel Leporrier5, Nicole Casadevall6,
Olivier Hermine7, Jean-Jacques Kiladjian8, André Baruchel9, Fadi Fakhoury10,
Brigitte Bressac- de Paillerets3,11, Jean Feunteun12, Nathalie Mazure13, Jacques Pouysségur13,
Roland H. Wenger2, Stéphane Richard1,14 and Betty Gardie1

1Génétique Oncologique EPHE, INSERM U753, Institut de Cancérologie Gustave Roussy,
Villejuif et Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France; 2Institute of Physiology
and Zürich Center for Integrative Human Physiology ZIHP, University of Zürich, Zürich,
Switzerland; 3Service de Génétique, Institut de cancérologie Gustave Roussy, Villejuif, France;
4Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de
Génétique, Paris, France; INSERM, UMR970, Paris-Cardiovascular research Center at HEGP,
Paris, France; Université Paris Descartes, Faculté de Médecine, Paris, France; 5CHU de Caen,
service d’hématologie clinique, France; 6Hôpital Saint Antoine, Assistance Publique–Hôpitaux de
Paris and Pierre et Marie Curie University, Institut Gustave Roussy, Inserm, UMR790, Villejuif,
France; 7Service d’Hématologie, Hôpital Necker-Enfants Malades, AP-HP, Paris, France; 8Centre
d’Investigations Cliniques, Hôpital Saint-Louis, AP-HP, Paris, France; 9Service d’Hématologie,
Hôpital St. Louis, Paris, France; 10Service de Néphrologie, Hôpital Necker, Paris, France;
11Unité INSERM U946, Variabilité Génétique et Maladies Humaines, Fondation Jean
Dausset/CEPH Unité, Paris, France;
12Institut de cancérologie Gustave Roussy,
DOI: 10.3324/haematol.2011.044644

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CNRS-UMR8200, Villejuif, France; 13Institut de biologie du développement et cancer,
2
Université de Nice - Sophia Antipolis, CNRS UMR-6543, France, and 14Centre Expert
National Cancers Rares INCa “PREDIR” and Réseau National INCa, AP-HP, Service
d'Urologie, CHU, Le Kremlin-Bicêtre and Service de Néphrologie, Hôpital Necker, Paris,
France

*Equal contribution to the work

Correspondence
Betty Gardie, Laboratoire de Génétique Oncologique EPHE, INSERM U753, Institut de
Cancérologie Gustave Roussy, Villejuif, France. E-mail: betty.gardie@inserm.fr
Stéphane Richard, Laboratoire de Génétique Oncologique EPHE, INSERM U753, Institut de
Cancérologie Gustave Roussy, Villejuif et Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre,
France. E-mail:stephane.richard@u-psud.fr.

DOI: 10.3324/haematol.2011.044644

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Abstract
Background. Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible
factor resulting in overproduction of erythropoietin. The most common germline mutation identified
in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau
tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in
hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau
mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is marked
by the development of multiple tumors. Recently, a few mutations in the Prolyl Hydroxylase Domain
2 protein (PHD2) gene have been reported in cases of congenital erythrocytosis not associated with
tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.
Design and Methods. Five PHD2-variants, four of which were novel, were identified in patients with
erythrocytosis. These PHD2-variants were functionally analyzed and compared with the PHD2
mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of
PHD2 to regulate activity, stability and hydroxylation of hypoxia inducible factor α was assessed
using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.
Results. This functional comparative study shows that two categories of PHD2 mutants could be
distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a
second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second
category.
Conclusions. As observed with germline von Hippel-Lindau mutations, there are functional
differences between the PHD2 mutants regarding hypoxia inducible factor regulation. Therefore, the
PHD2 mutation carriers need careful medical follow-up, since some mutations must be considered as
potential candidates for tumor predisposition.

DOI: 10.3324/haematol.2011.044644

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Introduction
Secondary erythrocytosis is due to external factors such as increased production of erythropoietin
(EPO), the origin of which is variable and may result from germline mutations in genes encoding
factors involved in the oxygen-sensing pathway. The primary cellular component implicated in
oxygen homeostasis is the Hypoxia-Inducible transcription Factor (HIF). HIF operates as a
heterodimer composed of a constitutively expressed beta subunit (also known as aryl
hydrocarbon receptor nuclear translocator (ARNT) and an alpha subunit (1α, 2α or 3α) that is
tightly regulated by oxygen via post-translational modification. The prolyl-4-hydroxylase domain
(PHD) enzymes hydroxylate proline residues located in the oxygen-dependent degradation
(ODD) domain of HIF-α. This hydroxylation allows the binding of the VHL protein (pVHL), the
substrate recognition subunit of an E3 ubiquitin ligase complex that induces ubiquitination and
subsequent degradation of HIF-α by the proteasome.1, 2 In the absence of oxygen, HIF-α is
stabilized, heterodimerizes with HIF-1β and induces expression of hundreds of genes involved in
cell survival, angiogenesis, erythropoiesis and cell proliferation.2, 3 There is some restricted target
gene specificity depending on the HIF-α subunit of the HIF-α/β heterodimeric transcription
factor. For example, renal and hepatic erythropoietin is regulated by the HIF-2α subunit in vivo.4-
7 Germline mutations in genes involved in the HIF pathway have been reported in association
with syndromes that predispose patients to both neoplasms and/or congenital secondary
erythrocytosis.8 The most frequent mutations involve the VHL tumor suppressor gene.
Heterozygous germline mutations in this gene are responsible for von Hippel-Lindau (VHL)
disease, an autosomal dominant condition predisposing to multiple tumors including central
nervous system (CNS) and retinal hemangioblastomas (Hb), clear-cell renal cell carcinoma
(ccRCC), pheochromocytomas and pancreatic endocrine tumors.8 Established correlations
DOI: 10.3324/haematol.2011.044644