The role of PHD2 mutations in the pathogenesis of erythrocytosis

Abstract

The transcription of the erythropoietin (EPO) gene is tightly regulated by the hypoxia response pathway to maintain oxygen homeostasis. Elevations in serum EPO level may be reflected in an augmentation in the red cell mass, thereby causing erythrocytosis. Studies on erythrocytosis have provided insights into the function of the oxygen-sensing pathway and the critical proteins involved in the regulation of EPO transcription. The α subunits of the hypoxia-inducible transcription factor are hydroxylated by three prolyl hydroxylase domain (PHD) enzymes, which belong to the iron and 2-oxoglutarate-dependent oxygenase superfamily. Sequence analysis of the genes encoding the PHDs in patients with erythrocytosis has revealed heterozygous germline mutations only occurring in Egl nine homolog 1 (EGLN1, also known as PHD2), the gene that encodes PHD2. To date, 24 different EGLN1 mutations comprising missense, frameshift, and nonsense mutations have been described. The phenotypes associated with the patients carrying these mutations are fairly homogeneous and typically limited to erythrocytosis with normal to elevated EPO. However, exceptions exist; for example, there is one case with development of concurrent paraganglioma (PHD2-H374R). Analysis of the erythrocytosis-associated PHD2 missense mutations has shown heterogeneous results. Structural studies reveal that mutations can affect different domains of PHD2. Some are close to the hypoxia-inducible transcription factor α/2-oxoglutarate or the iron binding sites for PHD2. In silico studies demonstrate that the mutations do not always affect fully conserved residues. In vitro and in cellulo studies showed varying effects of the mutations, ranging from mild effects to severe loss of function. The exact mechanism of a potential tumor-suppressor role for PHD2 still needs to be elucidated. A knockin mouse model expressing the first reported PHD2-P317R mutation recapitulates the phenotype observed in humans (erythrocytosis with inappropriately normal serum EPO levels) and demonstrates that haploinsufficiency and partial deregulation of PHD2 is sufficient to cause erythrocytosis.

Keywords: EGLN1; HIF; PHD2; erythrocytosis; erythropoietin; hypoxia.

Figure 1

Hypoxia and red cell production signalization pathway with associated mutations responsible for erythrocytoses. Notes: In the presence of oxygen (normoxia), the α subunit of the hypoxia inducible factor (HIF) is hydroxylated and binds the von Hippel–Lindau (VHL) protein that induces its ubiquitination and degradation in the proteasome. In the absence of oxygen (hypoxia), HIF-2α is stabilized, translocates into the nucleus, associates with HIF-1β, and induces transcription of a number of target genes, including erythropoietin (EPO). EPO is released in the circulation and binds its receptor, EPOR, on the surface of erythroid progenitors in the bone marrow. This binding induces a cascade of phosphorylation and signal transduction, leading to proliferation and differentiation of the progenitors toward mature red cells. Genetic alteration of genes encoding some of these factors induces overproduction of red cells and erythrocytosis. Abbreviations: JAK2, Janus kinase 2; SHP1, Src homology 2 domain-containing phosphatase-1 region; STAT5, signal transducer and activator of transcription factor 5; ECYT, erythrocytosis type; HBB, hemoglobin-β; HBA1, hemoglobin-α1; HBA2, hemoglobin-α2; BPGM, bisphosphoglycerate mutase; PHD2, prolyl hydroxylase domain 2.

Figure 2

Erythrocytosis-associated mutation sites in human prolyl hydroxylase domain 2 (PHD2) and HIF-2α. Notes: Domain architectures of PHD2 and HIF-α are shown in the color-coded bars. Ribbon representation of view from a crystal structure of PHD2 (residues 181–426, blue) in complex with HIF-1α. CODD (556–574, salmon), Mn (an Fe surrogate, violet), and N-oxalylglycine/NOG (a 2-OG mimic, not shown for clarity) (Protein Data Bank ID: 3HQR). Three important mutation sites in PHD2 (Pro317, Arg371, and His374) are highlighted in yellow sticks. Adapted from Lee FS, Percy MJ. The HIF pathway and erythrocytosis. Annu Rev Pathol. 2011:165–192. Abbreviations: MYND, myeloid, Nervy and DEAF1; PAS, Per Arnt Sim; ODD, oxygen-dependent degradation domain; CAD, carboxy-terminal activation domain; HIF, hypoxia-inducible factor; ID, identification.

Figure 3

Multiple sequence alignment of PHD2 homologs. Notes: Conservation of prolyl hydroxylase domain enzyme 2 sequences throughout various taxonomic classes. Gray-scale shading indicates conservation of isofunctional amino acid residues. Erythrocytosis-associated prolyl hydroxylase domain enzyme 2 mutations are labeled alternating in red and green. Abbreviation: PHD2, prolyl hydroxylase domain 2.