B. F. Smith

Molecular Basis of Canine Muscle Type Phosphofructokinase Deficiency

Muscle type phosphofructokinase (M-PFK) deficiency is a rare inherited glycogen storage disease in humans that causes exertional myopathy and hemolysis. The molecular basis of canine M-PFK deficiency, the only naturally occurring animal homologue, was investigated. Lack of M-PFK enzyme activity was caused by a nonsense mutation in the penultimate exon of the M-PFK gene, leading to rapid degradation of a truncated (40 amino acids) and therefore unstable M-PFK protein. A polymerase chain reaction-based test was devised to identify M-PFK-deficient and carrier animals. This represents one of only a few inborn errors of metabolism where the molecular defect has been identified in a large animal model which can now be used to develop and assess novel therapeutic strategies.

Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

Retroviral vectors were used to transfer genes efficiently into rat and dog myoblasts in primary cultures under conditions which permitted the transduced myoblasts to differentiate into myotubes expressing the transferred genes. The transduced myotubes expressed normal markers of differentiation and were morphologically indistinguishable from uninfected myotubes. Retroviral vector-mediated gene transfer was also used to correct a genetic enzyme deficiency in mutant canine muscle cells.

A cDNA encoding canine muscle-type phosphofructokinase

The canine muscle-type-phosphofructokinase-encoding gene (M-PFK) was sequenced by using a combination of cDNA cloning and RT-PCR amplification. The canine M-PFK sequence shares 88 and 90% identity with rabbit and human M-PFK, respectively. The canine ORF was determined to be 6-bp longer than either human or rabbit M-PFK due to a 6-bp insertion at the end of exon 13.