Missense mutation in PFKM associated with muscle-type phosphofructokinase deficiency in the Wachtelhund dog.

Hereditary muscle-type phosphofructokinase (PFK) deficiency causing intermittent hemolytic anemia and exertional myopathy due to a single nonsense mutation in PFKM has been previously described in English Springer and American Cocker Spaniels, Whippets, and mixed breed dogs. We report here on a new missense mutation associated with PFK deficiency in Wachtelhunds. Coding regions of the PFKM gene were amplified from genomic DNA and/or cDNA reverse-transcribed from RNA of EDTA blood of PFK-deficient and clinically healthy Wachtelhunds and control dogs. The amplicons were sequenced and compared to the published canine PFKM sequence. A point mutation (c.550C>T, in the coding sequence of PFKM expressed in blood) was found in all 4 affected Wachtelhunds. This missense mutation results in an amino acid substitution of arginine (Arg) to tryptophan (Trp) at position 184 of the protein expressed in blood (p.Arg184Trp). The mutation is located within an alpha-helix, and based on the SIFT analysis, this amino acid substitution is not tolerated. Amplifying the region around this mutation and digesting the PCR fragment with the restriction enzyme MspI, produces fragments that readily differentiate between PFK-deficient, carrier, and normal animals. Furthermore, we document 2 additional upstream PFKM exons expressed in canine testis but not in blood. Despite their similar phenotypic appearance and use for hunting, Wachtelhunds and English Springer Spaniels are not thought to have common ancestors. Thus, it is not surprising that different mutations are responsible for PFK deficiency in these breeds. Knowledge of the molecular basis of PFK deficiency in Wachtelhunds provides an opportunity to screen and control the spread of this deleterious trait.

Haemolytic anaemia and exercise intolerance due to phosphofructokinase deficiency in related springer spaniels.

Phosphofructokinase (PFK) deficiency is an autosomal recessive inherited disorder in dogs causing haemolytic crises and exertional myopathy. The clinical signs may be confused with those of recurrent immune-mediated haemolytic anaemia. The deficiency has been commonly observed in field trial (working) English springer spaniels (ESSPs), but also in the conformation line of ESSPs in the USA over the past two decades. This report documents the first family of ESSPs found with PFK deficiency in Europe. Two related adult ESSPs in Denmark had intermittent signs of pigmenturia after exercise (hunting) and had evidence of a regenerative haemolytic anaemia. Based upon DNA sequencing data, both dogs had the previously described nonsense point mutation in the muscle-type PFK gene (delta2228G-->A). Study of 17 related family members using a simple and accurate PFK-DNA test revealed one additional PFK-deficient dog (with minor exercise intolerance), nine carriers and seven normal (or 'clear') ESSPs. Recently, the authors have also identified PFK carriers and affected ESSPs in the UK. Screening for PFK deficiency is recommended for ESSPs with suspicious clinical signs and before using any for field trials or breeding in order to prevent the further spread of this hereditary disorder.

Polysaccharide storage myopathy in canine phosphofructokinase deficiency (type VII glycogen storage disease).

A severe, progressive myopathy developed in an 11-year-old, phosphofructokinase (PFK)-deficient, male, English Springer Spaniel dog. Results from a routine neurological examination were normal. Examination of histologic sections of skeletal muscle revealed large accumulations of material in some myofibers. These deposits were pale, basophilic, somewhat flocculent, and slightly granular with hematoxylin and eosin stain. Most fascicles examined in sections of limb and trunk muscles were affected to some degree, with up to 10% of muscle fibers being involved. Deposits stained strongly with periodic acid-Schiff and were resistant to digestion by alpha amylase but were removed by incubation with gamma amylase. Deposits were faintly positive with Gomori's methenamine silver technique and alcian blue (pH 2.5) and were brown-gray with Lugol's iodine solution but were negative with other stains. Based on staining characteristics, the deposits seemed to consist primarily of an amylopectin-like polysaccharide(s). Alcian blue staining (pH 2.5) was removed by treatment with neuraminidase but not with hyaluronidase, indicating that some sialic acid residues were also present. Electron microscopically, the deposits were composed of short granular filaments, small granules and amorphous material. They were not membrane bound. The morphologic appearance and staining characteristics of the deposits were remarkably similar to deposits previously described in human PFK-deficient myopathy. As expected, total PFK activities were markedly reduced when assayed in skeletal muscles of this dog. In contrast with other PFK-deficient dogs, muscle glycogen in this animal was not increased above that of normal dogs.