Molecular diagnostic tests for ascertainment of genotype at the mucopolysaccharidosis type VII locus in dogs

Am J Vet Res. 1998 Sep;59(9):1092-5.

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 MPSVII.

Procedure: The canine beta-glucuronidase gene (exon 3) was amplified by polymerase chain reaction (PCR), using 2 pairs of primers to determine the genotype of the MPSVII 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 VII-carrier dogs from homozygous normal dogs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Animals
  • Base Pair Mismatch
  • DNA / chemistry
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Dog Diseases / diagnosis*
  • Dog Diseases / enzymology
  • Dog Diseases / genetics
  • Dogs
  • Exons
  • Genotype
  • Glucuronidase / genetics
  • Mucopolysaccharidosis VII / diagnosis
  • Mucopolysaccharidosis VII / enzymology
  • Mucopolysaccharidosis VII / genetics
  • Mucopolysaccharidosis VII / veterinary*
  • Nucleic Acid Conformation
  • Pedigree
  • Polymerase Chain Reaction / veterinary

Substances

  • DNA
  • endodeoxyribonuclease XmaIII
  • Deoxyribonucleases, Type II Site-Specific
  • Glucuronidase