Familial Erythrocytosis Associated with a Mutation in the HIF Prolyl Hydroxylase, PHD2.

Normal erythropoiesis is precisely regulated by a negative feedback mechanism based on the oxygen status of the body. Whenever a deficit in oxygen supply is detected erythropoietin (Epo) is rapidly synthesised to increase circulating red cells. This process is under the control of the HIF transcription complex, which is composed of one alpha and one beta subunit. Although the alpha subunit is constitutively synthesized, it is maintained at a low level by continual targeting to the proteasome. Prolines 402 and 564 in the oxygen dependent degradation (ODD) domain of HIF-1α are hydroxylated in the presence of oxygen by members of the PHD family of prolyl hydroxylases. Once hydroxylated HIF-1α is captured by the von Hippel Lindau tumour suppressor gene product (VHL), ubiquitylation follows and HIF-1α is then targeted for proteasomal degradation. Defects in the hypoxia response pathway appear to be the most common cause of erythrocytosis associated with inappropriately normal or elevated serum Epo.

Several different mutations in the VHL gene have been detected in erythrocytosis individuals. Although these mutations explain a significant number of cases there remains a large cohort where the molecular defect is undefined. Thus we screened a group of such individuals for base changes in PHD 1–3. Two affected siblings and their deceased father were found to possess a heterozygous C to G change at base 950 in the coding sequence of PHD2, causing loss of proline at codon 317 and replacement with arginine. This base change was not present in their normal mother nor in 200 normal controls. Residue 317 is located in the enzyme’s active site and at a position two residues C-terminal to Asp315, which chelates the active site iron moiety. Functional analysis indicated that the Pro317Arg PHD2 variant bound to HIF-1α and HIF-2α more weakly and displayed substantially less HIF hydroxylase activity than the wild type protein. These results indicate that the Pro317Arg PHD2 mutant protein’s function will be impaired, less hydroxylation of HIF will occur, allowing more HIF to escape proteasomal degradation and to increase transcription of HIF target genes. Consequently, Epo gene expression will be elevated thereby allowing increased erythrocyte production and the development of erythrocytosis.