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.