ISOLATION AND NUCLEOTIDE-SEQUENCE OF CANINE GLUCOSE-6-PHOSPHATASE MESSENGER-RNA - IDENTIFICATION OF MUTATION IN PUPPIES WITH GLYCOGEN-STORAGE-DISEASE TYPE IA

Two Maltese puppies with massive hepatomegaly and failure to thrive ha d isolated deficient glucose-6-phosphatase (G-6-Pase) activity in live r and kidney and pathological findings compatible with GSD-Ia. To iden tify the mutation, we cloned G-6-Pase canine cDNA by RT-PCR with prime rs from the murine G-6-Pase gene sequence. The canine G-6-Pase cDNA is 2346 bp, with a 5' untranslated region of 87 bp, a coding region of 1 071 bp, and a 3' untranslated region of 1185 bp. The difference betwee n the canine and human sequences is in the 3' untranslated region. A g reater than 90% amino acid sequence homology was seen with canine, hum an, murine, and rat G-B-Pase. G-6-Pase cDNA from affected and control puppies revealed complete homology except at nt position 450, which sh owed a guanine to cytosine (G to C) transversion resulting in substitu tion of a methionine by isoleucine at codon 121 (M121I) in all five cl ones studied. The loss of an NcoI restriction site on genomic DNA ampl ified with primers flanking the mutation allowed us to prove that affe cted puppies were homozygous for the mutation and parents were heteroz ygous carriers. The mutant G-6-Pase cDNA had 15 times less enzyme acti vity than wild-type cDNA following transient transfection. Northern bl ot analysis of puppies with GSD-Ia revealed increased G-6-Pase mRNA, c ompared to normal controls. Increased G-6-Pase mRNA was also seen in n ormal fasted puppies compared to littermates in the fed state, suggest ing that the increased G-6-Pase mRNA is a physiologic response to fast ing. This is the first report of a molecularly confirmed naturally occ urring animal model of GSD-Ia. The establishment of a breeding colony of this dog strain will facilitate studies on the role of G-6-Pase gen e in glucose homeostasis, in pathophysiology of disease, and developme nt of novel therapeutic approaches such as gene therapy. (C) 1997 Acad emic Press.