Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the deficient activity of galactocerebrosidase (GALC). This enzyme is responsible for the lysosomal catabolism of certain galactolipids, including galactocerebroside and a toxic metabolite, psychosine. There are several animal models for Krabbe disease, including the twitcher mouse and the West Highland White and Cairn terriers. We describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in this animal model. The 2007 nt open reading frame is 88% identical to the human and the deduced amino acid sequence is 90% identical. Two changes were found in the affected dog, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells showed that the A to C change at position 473 is the disease-causing mutation. A rapid test to identify the genotype at this position has been developed. As a preliminary study in gene therapy, we show that an amphotropic retroviral vector containing the human GALC cDNA can infect GALC-deficient canine fibroblasts and other cell types and raise GALC activity to levels higher than found in normal cells.
The murine GALC cDNA was also cloned. Its coding area is 2004 nt long, 3 nt shorter than the human or the dog. At the nucleotide level, the murine coding region is 83% identical to both the human and the dog, and at the amino acid level, the murine enzyme is 84% and 82% identical to the human and the dog respectively. Transient expression in the COS-1 cell system of the murine GALC cDNA resulted in GALC activity averaging 25 fold above that of mock transfected cells. The effects of two human polymorphisms, R168C and I546T, on murine GALC activity were also tested in the COS-1 cell expression system. These studies showed that cells containing the vector with Cys$\sp{168}$ have 12% of the activity of the control cells transfected with the normal GALC expression vector, cells with Thy$\sp{545}$ have 24% of the activity of the control normal cells, and those transfected with the vector containing the two changes have 36% of the activity of the control cells. In the future, it is our purpose to make transgenic mice homozygous for both changes to study the response of a demyelinating stress in an animal model with low GALC activity.
