Abstract
Objective
To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus.
Sample Population
Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPS VII.
Procedure
The canine ß-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPS VII locus by 2 independent methods. For the first method, PCR products were used for heteroduplex analysis, using conformation-sensitive gel electrophoresis. In the second method, an allele-specific restriction site was created by use of a mismatch primer in PCR. The amplified DNA fragment was digested with a restriction enzyme (Eag I) to enable identification of the wild-type and mutant alleles.
Results
Conformation-sensitive gel electrophoresis resulted in a single DNA band representing homoduplex when the sample contained a wild-type or MPS VII allele, but 2 bands representing hetero- and homoduplexes when both alleles were in the sample. Restriction digestion of the DNA fragment obtained by use of a mismatch primer was cleaved only when the template was a wild-type allele. Thus, samples from phenotypically normal carrier dogs that contained wild-type and MPS VII alleles were partially digested by the enzyme.
Conclusions
The diagnostic test used 2 strategies for independently ascertaining the wild-type or mutant MPS VII alleles in dogs. Thus, test results can distinguish phenotypically normal MPS Vll-carrier dogs from homozygous normal dogs. (Am J Vet Res 1998;59:1092-1095)
