Molecular diagnostic tests for ascertainment of genotype at the mucopolysaccharidosis type VII locus in dogs.
OBJECTIVE
To develop a molecular diagnostic test to ascertain genotype of the mucopolysaccharidosis type VII (MPS VII) locus.


METHODS
Blood samples from 45 mixed-breed (German Shepherd Dog X Beagle) dogs that were phenotypically normal or affected with MPSVII.


METHODS
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.
Ray
J
J
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Haskins
M E
ME
Ray
K
K
D000483:Alleles; D000818:Animals; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004283:Dog Diseases / Q000175:diagnosis* / Q000201:enzymology / Q000235:genetics; D004285:Dogs; D005091:Exons; D005838:Genotype; D005966:Glucuronidase / Q000235:genetics; D016538:Mucopolysaccharidosis VII / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics / Q000662:veterinary*; D009690:Nucleic Acid Conformation; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000662:veterinary
D004247:DNA; C046751:endodeoxyribonuclease XmaIII; D015252:Deoxyribonucleases, Type II Site-Specific; D005966:Glucuronidase