Isolated erythrocytosis: study of 67 patients and identification of three novel germ-line mutations in the prolyl hydroxylase domain protein 2 (PHD2) gene

Abstract

The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway.

Figure 1.

Mutational analysis results and localization of the PHD2 mutations. (A) Sequencing results of wild-type and mutated alleles in 3 patients. Nucleotide positions (GenBank accession, NM 022051), nucleotide changes and corresponding amino acid changes are indicated below. (B) Confirmation of the new mutations by allele-specific PCR (AS-PCR) and agarose gel electrophoresis. AS-PCR fragment lengths agree with the respective primer positions (Online Supplementary Table S1). Lane M: molecular weight marker, lane pat #: patient’s DNA amplified with mutation specific primer, lane wt: control DNA amplified with mutation specific primer, lane RC: reaction control (no template). (C) Pedigree of the family with erythrocytosis. Squares represent males, circles females, affected individuals are indicated in black and slashes indicate deceased members. Genetically tested individual is indicated by an asterisk.

Figure 2.

(A) Schematic diagram representing the human PHD2 protein. Znf_MYND, Zinc finger MYND-like domain, 2OG_FeII_oxy, 2-oxoglutarate and Fe(II)-dependent oxygenase-type domain. Diamonds indicate the location of the mutations, the novel genetic variations described in this study are underscored. Numbers indicate amino acid residue positions. (B) Three dimensional ribbon representation of the catalytic domain of the human PHD2, highlighting the locations of the mutated residues. The structure was generated using Swiss-PdbViewer v3.7 software from Protein Data Bank coordinates of the X-ray structure, 2G1M. Note that the C-terminal K423 is not visible as it falls outside the available PHD2 X-ray maps (residues from 188 to 403). (C) Active site contacts. P317 and R371 are close to the iron-chelating residues H313 and H374, respectively, both in the active site. Hydrogen bonds are shown as dashed lines.