A gain-of-function mutation in the HIF2A gene in familial erythrocytosis

Abstract

Hypoxia-inducible factor (HIF) alpha, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-alpha is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2alpha protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2alpha protein and suggest that wild-type HIF-2alpha regulates erythropoietin production in adults.

Figure 1. HIF2A Genotypes in a Family with Hereditary Erythrocytosis and Identification of the G1609→T Mutation

The pedigree of the family with hereditary erythrocytosis and mutant HIF2A is shown in Panel A. Squares represent male family members, circles represent female family members, and solid symbols represent family members with erythrocytosis; genotypes are shown under each symbol. The index patient is indicated with an arrow. Not shown are an unaffected younger sister of the index patient whose genotype was unavailable and two maternal aunts and two maternal uncles of the index patient, from whom neither medical histories nor genotypes could be obtained. Panel B shows nucleotides 1599 to 1621 in exon 12 of HIF2A. On sequencing, a heterozygous G→T change at base 1609 was detected in the index patient (top plot, asterisk and arrow), as compared with the wild-type sequence (bottom). The maternal grandmother of the index patient also had the mutation (middle plot, arrow). Panel C shows the results of amplification-refractory mutation-system (ARMS)–polymerase chain reaction (PCR) performed on peripheral-blood mononuclear cells for detection of the G1609→T mutation. Samples with a G, or wild-type, allele yielded a PCR product of 184 bp, whereas the T, or mutant, allele is 204 bp. The affected index patient and his mother and grandmother had both the wild-type and mutant alleles (lanes 2, 3, and 5, respectively). The T allele is absent in the unaffected father and brother of the index patient (lanes 4 and 6, respectively). Lane 1 contains a 100-bp DNA size marker, and all other lanes contain the PCR control band of 333 bp. Lane 7 contains a control sample that is negative for the G1609→T mutation. Panel D lists the amino acid sequences (shown with single-letter symbols) of the hypoxia-inducible factor (HIF) α residues 526 to 549 (human HIF-2α nomenclature) in human, mouse, chicken, Xenopus laevis, and zebrafish, as well as the single HIFs from Drosophila melanogaster and Caenorhabditis elegans. Shading indicates conserved residues, the asterisk indicates the hydroxyl acceptor proline (Pro531 in HIF-2α), and the inverted triangle indicates Gly537 of human HIF-2α.

Figure 2. Functional Characterization of the Gly537→Trp Hypoxia-Inducible Factor (HIF) 2α Mutant

Panel A shows the results of binding assays. Input represents 1% of the total amount of wild-type Gly537→Trp GAL4–HIF-2α (516–549) incubated with (His)₆FlagPHD2. Panel B shows the results of hydroxylase assays, involving matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry. The positions of the unmodified peptides were 1877 amu for wild type and 2007 amu for Gly537→Trp and for hydroxylated peptides were 1893 atomic mass units for wild type and 2023 amu for Gly537→Trp (as potassium adducts). The small peaks at 1893 and 2023 in the untreated samples (asterisks) probably represent the oxidation of methionine in the peptide. Panel C shows the results of experiments examining the capacity of wild-type or Gly537→Trp HIF-2α (527–542) peptide to compete with hydroxylated GST–HIF-1α (531–575) for VHL binding. Input represents 25% of the total amount of VHL added in these experiments. Panels D and F show the results of Western blotting. Panel D shows the steady-state protein levels of wild-type and mutant HIF-2α, whereas Panel F shows the protein levels after protein synthesis has been arrested with the use of cycloheximide. Panel E shows the results of real-time PCR assays. The levels of messenger-RNA transcripts from the adrenomedullin gene (ADM), N-myc downstream regulated gene 1 (NDRG1), and the vascular endothelial growth factor gene (VEGF) were measured with the use of real-time PCR. Means from three separate experiments are shown. T bars indicate standard deviations. The P values are for the comparison with doxycycline-induced levels of wild-type HIF-2α. In Panel F, for each construct, the relative recovery was normalized to the value for 0 minutes, and the first lane shows results for the parental cell line. Hyp denotes hydroxylproline, and PHD2 prolyl hydroxylase domain protein 2.