The molecular basis of canine pyruvate kinase deficiency.

Inherited hemolytic anemia due to pyruvate kinase (PK) deficiency is an autosomal recessive disease of the Basenji dog that closely resembles human PK deficiency. Characterization of transcriptional and translational expression of PK isozymes and sequencing of DNA from normal and mutant dogs were performed to identify the genetic defect in Basenji dogs. Measurement of erythrocytic PK activity by ion exchange chromatography, substrate kinetics, immunologic reactivity, and electrophoretic mobility suggests that M2-type PK is the major form of PK activity in erythrocytes of PK-deficient dogs, in contrast to normal dogs having only R-type PK activity. Both R-type and M2-type PK mRNA are detectable in reticulocytes of PK-deficient dogs, suggesting that the aberrant isozyme expression is not due to a failure in the erythroid maturational switch from M2- to R-type isozymes. Nucleotide sequence data from wild-type and mutant R-type PK cDNA identified a single nucleotide deletion, delta C433, in the mutant cDNA. The deduced amino acid sequence predicts a truncated mutant protein devoid of all residues contributing to the catalytic site of the wild-type protein. In the absence of R-type PK activity, there is anomalous compensatory expression of M2-type PK in erythroid cells of PK-deficient Basenjis. The PK-deficient Basenji dog may be valuable in somatic cell gene therapy trials involving manipulation of hematopoietic stem cells.