Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.
OBJECTIVE
To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available.


METHODS
The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD.


METHODS
Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD.


METHODS
DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly.


CONCLUSIONS
Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia.


CONCLUSIONS
Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.
Bartlett
R J
RJ
Department of Neurology, School of Medicine, University of Miami, FL 33136, USA.
Winand
N J
NJ
Secore
S L
SL
Singer
J T
JT
Fletcher
S
S
Wilton
S
S
Bogan
D J
DJ
Metcalf-Bogan
J R
JR
Bartlett
W T
WT
Howell
J M
JM
Cooper
B J
BJ
Kornegay
J N
JN
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D003402:Creatine Kinase / Q000097:blood; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004283:Dog Diseases / Q000175:diagnosis / Q000235:genetics*; D004285:Dogs; D005091:Exons; D005260:Female; D006580:Genetic Carrier Screening; D008040:Genetic Linkage; D005838:Genotype; D008297:Male; D008969:Molecular Sequence Data; D009137:Muscular Dystrophy, Animal / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012150:Polymorphism, Restriction Fragment Length; D014960:X Chromosome
D017931:DNA Primers; D004247:DNA; D003402:Creatine Kinase