Identification of the 1112 G > A mutation in the PHD2 gene. (A) Detection of the G1112A mutation by PCR-direct sequencing. PCR was performed on total peripheral blood DNA using a set of primers to specifically amplify exon 3. Sequencing detected a heterozygous G-to-A change at base 1112 as indicated by an arrow (top panel) as compared with wild-type sequence (bottom panel). Shown are nucleotides 1093 to 1131. Bases are as follows: G, black; A, green; T, red; and C, blue. (B) Screening family members for G1112A mutation. The presence of A at base 1112 creates a restriction site for the enzyme Tsp45I to give 2 products of 246 bp and 273 bp from an exon 3 PCR product from the patient (lane 4). The mother of the patient (lane 3) was screened, and the absence of the 246 bp and 273 bp bands indicated she did not possess the G1112A mutation. Lane 1 contains a 100-bp DNA size marker; lane 2, nondigested exon 3 PCR product of 519 bp; and lane 5, Tsp45I-digested exon 3 PCR product from a control sample negative for the mutation as detected by sequencing. (C) Comparison of amino acid sequence from residues 366-379 (numbering according to hPHD2 nomenclature) in human HIF prolyl hydroxylases with those from D. melanogaster (DmHPH) and C. elegans (CeEGL9). Sequence shading indicates completely conserved residues. *Iron-chelating residue His-374. ▼ indicates Arg371 of human PHD2, which is predicted to be changed to His by the G1112A mutation. Also shown is hPHD2 sequence from residues 307-323. *Iron-chelating residues His313 and Asp315. ● indicates Pro317. The positions of these sequences in full-length hPHD2 is shown, with shading indicating prolyl hydroxylase domain. Numbers at bottom indicate residues.

Functional characterization of the Arg371His PHD2 mutant. (A) Association of HIF-2α with Arg371His PHD2. ³⁵S-labeled, in vitro–translated wild-type or Arg371His FlagPHD2 was incubated with 1 μg of either GST or GST–HIF-2α (516-549) immobilized on GSH-agarose. The resins were washed and eluted, and the eluates were subjected to SDS-PAGE and autoradiography. Input represents 10% of the total. The relative recovery of wild-type PHD2 from 3 replicates is 100 ± 24 AU (arbitrary units ± SD), whereas that of Arg371His PHD2 is 2.4 ± 4 AU (P < .005). (B) Association of HIF-1α with Arg371His PHD2. Binding assays with GST–HIF-1α (531-575) were performed as described in panel A. Input represents 5% of total. The relative recovery of wild-type PHD2 from 3 replicates is 100 ± 0.1 AU, whereas that of Arg371His PHD2 is 0.8 ± 0.0001 AU (P < .005). (C) Hydroxylase activity of Arg371His PHD2. Equal quantities (as determined by phosphorimager analysis) of in vitro–translated wild-type or Arg371His FlagPHD2, or mock in vitro translation reaction, was incubated with 0.75 μg of GST–HIF-2α (516-549) for 1 hour in the presence of 2-oxoglutarate, ascorbic acid, and FeCl₂. The GST–HIF-2α (516-549) was isolated using GSH-agarose, washed, and then the degree of HIF hydroxylation was assessed by subsequent incubation with ³⁵S-labeled, in vitro–translated VHL. Input represents 5% of the total. Under the conditions of the assay, the recovery of ³⁵S-labeled, in vitro–translated VHL using wild-type PHD2 from 3 independent experiments is 100 ± 38 AU, while that using Arg371His PHD2 is 12 ± 7.6 AU (P < .05). (D) HIF-inhibitory activity of Arg371His PHD2. HEK293 cells were cotransfected with 150 ng of (eHRE)₃-Luc, 150 ng of pRL-TK, 300 ng of either pcDNA3 or pSV-Sport-HA-hHIF-2α, and either 0, 0.3, or 0.6 ng of pcDNA3-FlagPHD2 (wild-type or P317R). The total DNA dose was normalized with pcDNA3. At 48 hours after transfection, the cells were harvested and assayed for luciferase activity. Activities were normalized to that of the Renilla luciferase internal transfection control. Shown are results performed in triplicate ± SD. *P < .05; **P < .01 when comparing results of wild-type and mutant PHD2 at the same dose. In separate experiments, HEK293 cells were transfected with 2 μg of wild-type or Arg371His pcDNA3-FlagPHD2; 48 hours later, extracts (15 μg) were analyzed by Western blotting with anti-Flag (M2; Sigma, St Louis, MO) or anti–β tubulin (H-235; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. The position of PHD2, β-tubulin, and a molecular weight marker are as indicated. (E) 3D structure of PHD2. The structure was generated using Cn3D from Protein Data Bank coordinates (2G1M) deposited by McDonough et al.²³ Arg371 and Pro317 are highlighted in yellow. Compound A (a 2-oxoglutarate competitive inhibitor) is shown in brown.