Identification of Mutations Associated with Peroxisome-to-Mitochondrion Mistargeting of Alanine/Glyoxylate Aminotransferase in Primary Hyperoxaluria Type 1

• Published in: The Journal of cell biology Vol. 111; no. 6; pp. 2341 - 2351
• Main Authors: Purdue, P. Edward, Takada, Yoshikazu, Danpure, Christopher J.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01-12-1990; The Rockefeller University Press
• Subjects: Alanine Transaminase - genetics; Alleles; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cloning, Molecular; Complementary DNA; DNA; Enzymes; Errors of metabolism; Genes; Genetic mutation; Humans; Hyperoxaluria - enzymology; Hyperoxaluria - genetics; Introns; Liver; Liver - enzymology; Medical sciences; Metabolic diseases; Microbodies - enzymology; Miscellaneous hereditary metabolic disorders; Mitochondria; Mitochondria, Liver - enzymology; Molecular Sequence Data; Mutation; Nucleotides; Oligonucleotide Probes; Peroxisomes; Plasmids; Poly A - genetics; Poly A - isolation & purification; Polymerase Chain Reaction; Protein Conformation; Protein Processing, Post-Translational; Reference Values; Restriction Mapping; RNA - genetics; RNA - isolation & purification; RNA, Messenger; Transaminases; Human; Mitochondria; Complementary DNA; Nucleotide sequence; Enzyme; Point mutation; Peroxisome; Metabolic diseases; Hyperoxaluria; Alanine―glyoxylate aminotransferase; Nucleic acid
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.111.6.2341

We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria. We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly→Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro→Leu and Ile→Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.