A Novel HIFalpha Mutation Associated with Familial Erythrocytosis Supports This Isoform Being the Major Regulator of Erythropoietin in Humans.

Red cell homeostasis is crucial to maintain a sustained tissue oxygen supply. Oxygen is sensed by the kidneys and hypoxia induces renal erythropoietin (Epo) to stimulate red cell production. At the molecular level this process is controlled by the hypoxia inducible factor (HIF) complex, comprised of a labile a-subunit and a constitutively expressed b subunit. Key prolines present in the oxygen-dependent degradation (ODD) domain of the a subunit are hydroxylated by prolyl hydroxylase domain proteins (PHD1-3). The von Hippel Lindau (VHL) protein then binds and targets HIFa to the proteasome. There are 3 isoforms of the a subunit, each of which shows differential tissue distribution. The HIF-1, HIF-2 and HIF-3a subunit, in association with the b subunit, exhibit diverse affinities for the hypoxia response elements (HREs) located in the promoters and enhancers of HIF target genes. It is unclear whether Epo synthesis is under the control of HIF-1 or HIF-2 in humans although recent studies in mice support HIF-2 as the critical regulator. Dysregulation of Epo production can give rise to the red cell disorder of erythrocytosis. Defects in the oxygen sensing pathway are commonly associated with erythrocytosis and mutations in both the VHL and PHD2 genes have been described. Previously, screening the ODD domain of HIF-1a detected a polymorphism, Pro582Ser, that did not impair the function of HIF-1a. Subsequently, when sequencing HIF-2a, a novel heterozygous c.G1609T mutation was uncovered in 3 generations of one family with erythrocytosis. All affected individuals exhibited greatly elevated serum Epo levels. The c.G1609T mutation was absent in other unaffected family members as well as a cohort of 200 normal control samples. The mutation predicts the replacement of Gly by Trp at amino acid 537, which is close to the main site of proline hydroxylation at 531. Functional assays confirmed the Gly537Trp variant was less hydroxylated and exhibited reduced affinity for PHD2. Furthermore, the increased stability in normoxia of the Gly537Trp variant would result in elevated transcription of HIF-2 target genes. Consequently, these findings support a critical role for HIF-2a in control of Epo production in humans.