Objective—To determine incidence of the Ile118Lys
endothelin receptor B (EDNRB) mutation responsible
for overo lethal white syndrome (OLWS) and its association
with specific types of white patterning.
Animals—945 horses of white-patterned bloodlines
and 55 solid-colored horses of other breeds.
Procedure—Horses were genotyped by use of allelespecific
polymerase chain reaction to determine incidence
of the Ile118Lys EDNRB mutation.
Results—Genotypes detected were homozygous
Ile118, homozygous Lys118, and heterozygous. All
foals with OLWS were homozygous for the Ile118Lys
EDNRB mutation, and adults that were homozygous
were not found. White patterning was strongly associated
with EDNRB genotype. Color patterns with
highest incidence (> 94%) of heterozygotes were
frame overo, highly white calico overo, and frame
blend overo. White-patterned bloodlines with lowest
incidence of heterozygotes (< 21%) were tobiano,
sabino, minimally white calico overo, splashed white
overo, nonframe blend overo, and breeding-stock
solid. The mutation was not detected in solid-colored
horses from breeds without white patterning.
Conclusions and Clinical Relevance—In homozygotes,
the Ile118Lys EDNRB mutation causes OLWS.
In heterozygotes, the mutation is usually responsible
for a frame overo phenotype. The frame pattern can
be combined with other white patterns, making accurate
estimation of EDNRB genotype by visual inspection
difficult. Wide range of incidence of heterozygotes
in various subtypes of white-patterned horses
indicates different genetic control of these color patterns.
Determination of EDNRB genotype by use of a
DNA-based test is the only way to determine with
certainty whether white-patterned horses can produce
a foal affected with OLWS. ( Am J Vet Res
Objective—To determine whether an alteration in calcium
regulation by skeletal muscle sarcoplasmic reticulum,
similar to known defects that cause malignant
hyperthermia (MH), could be identified in membrane
vesicles isolated from the muscles of Thoroughbreds
with recurrent exertional rhabdomyolysis (RER).
Sample Population—Muscle biopsy specimens
from 6 Thoroughbreds with RER and 6 healthy (control)
Procedures—RER was diagnosed on the basis of a history
of > 3 episodes of exertional rhabdomyolysis confirmed
by increases in serum creatine kinase (CK) activity.
Skeletal muscle membrane vesicles, prepared by
differential centrifugation of muscle tissue
homogenates obtained from the horses, were characterized
for sarcoplasmic reticulum (SR) activities, including
the Ca2+ release rate for the ryanodine receptor-Ca2+
release channel, [3H]ryanodine binding activities, and
rate of SR Ca2+-ATPase activity and its activation by Ca2+.
Results—Time course of SR Ca2+-induced Ca2+
release and [3H]ryanodine binding to the ryanodine
receptor after incubation with varying concentrations
of ryanodine, caffeine, and ionized calcium did not differ
between muscle membranes obtained from control
and RER horses. Furthermore, the maximal rate
of SR Ca2+-ATPase activity and its affinity for Ca2+ did
not differ between muscle membranes from control
horses and horses with RER.
Conclusions and Clinical Relevance—Despite clinical
and physiologic similarities between RER and MH,
we concluded that RER in Thoroughbreds does not
resemble the SR ryanodine receptor defect responsible
for MH and may represent a novel defect in muscle
excitation-contraction coupling, calcium regulation,
or contractility. (Am J Vet Res 2000;61:242–247)
Objective—To determine whether alterations in
myoplasmic calcium regulation can be identified in
muscle cell cultures (myotubes) and intact muscle
fiber bundles derived from Thoroughbreds affected
with recurrent exertional rhabdomyolysis (RER).
Animals—6 related Thoroughbreds with RER and 8
clinically normal (control) Thoroughbred or crossbred
Procedures—Myotube cell cultures were grown from
satellite cells obtained from muscle biopsy specimens
of RER-affected and control horses. Fura-2 fluorescence
was used to measure resting myoplasmic
calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in
myoplasmic calcium. In addition, intact intercostal
muscle fiber bundles were prepared from both types
of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined.
Results—Myotubes of RER-affected and control
horses had identical resting myoplasmic calcium concentrations.
Myotubes from RER-affected horses had
significantly higher myoplasmic calcium concentrations
than myotubes from control horses following
the addition of ≥ 2mM caffeine; however, there was
no difference in their response to 4-CMC (≥ 1mM).
Caffeine contracture thresholds for RER and control
intact muscle cell bundles (2 vs 10mM, respectively)
were significantly different, but 4-CMC contracture
thresholds of muscle bundles from RER-affected and
control horses (500µM) did not differ.
Conclusions and Clinical Relevance—An increase
in caffeine sensitivity of muscle cells derived from a
family of related RER-affected horses was detected in
vitro by use of cell culture with calcium imaging and
by use of fiber bundle contractility techniques. An
alteration in muscle cell calcium regulation is a primary
factor in the cause of this heritable myopathy.
(Am J Vet Res 2002;63:1724–1731)
Objective—To identify characteristics of exercise-induced collapse in Labrador Retrievers and compare characteristics for dogs with various dynamin 1 gene (DNM1) mutation statuses.
Design—Retrospective cross-sectional study.
Animals—109 Labrador Retrievers with a history of recurrent exercise-induced collapse, clinically normal behavior and gait between episodes, and no reason for collapse identified via medical evaluation.
Procedures—Data were collected via surveys from owners of dogs that were tested for an autosomal recessive DNM1 mutation causing DNM1-associated exercise-induced collapse (d-EIC). Dogs were identified as having d-EIC (homozygous for the mutation) or not having d-EIC (heterozygous for or without the mutation). Survey data were reviewed by an investigator unaware of the genotypes of dogs, and collapse characteristics were compared between groups.
Results—74 dogs had d-EIC; 35 dogs did not have d-EIC. Dogs with d-EIC were young (median age, 12 months) at the time of the first collapse episode; collapse in such dogs typically originated in the hind limbs and was characterized by low muscle tone, clinically normal mentation, and rapid recovery. Dogs without d-EIC were older (median age, 23 months) than dogs with d-EIC; such dogs had various characteristics of collapse that were not consistent with a single disease.
Conclusions and Clinical Relevance—Characteristics of exercised-induced collapse in Labrador Retrievers with various DNM1 genotypes were identified in this study; findings may help distinguish dogs with d-EIC from those with other types of collapse conditions. Characteristics of collapse in Labrador Retrievers that were not homozygous for the DNM1 mutation differed substantially among dogs and may have been attributable to multiple causes.
Objective—To determine whether the basis for recurrent
exertional rhabdomyolysis (RER) in Thoroughbreds
lies in an alteration in the activation and regulation
of the myofibrillar contractile apparatus by ionized
Animals—4 Thoroughbred mares with RER and 4
clinically normal (control) Thoroughbreds.
Procedure—Single chemically-skinned type-I (slowtwitch)
and type-II (fast-twitch) muscle fibers were
obtained from punch biopsy specimens, mounted to
a force transducer, and the tensions that developed in
response to a series of calcium concentrations were
measured. In addition, myofibril preparations were
isolated from muscle biopsy specimens and the maximal
myofibrillar ATPase activity, as well as its sensitivity
to ionized calcium, were measured.
Results—Equine type-I muscle fibers were more
readily activated by calcium than were type-II muscle
fibers. However, there was no difference between
the type-II fibers of RER-affected and control horses
in terms of calcium sensitivity of force production.
There was also no difference between muscle
myofibril preparations from RER-affected and control
horses in calcium sensitivity of myofibrillar ATPase
Conclusion and Clinical Relevance—An alteration
in myofibrillar calcium sensitivity is not a basis for
pathologic contracture development in muscles from
RER-affected horses. Recurrent exertional rhabdomyolysis
in Thoroughbreds may represent a novel heritable
defect in the regulation of muscle excitation-contraction
coupling or myoplasmic calcium concentration.
(Am J Vet Res 2001;62:1647–1652)
Objective—To determine clinical characteristics and
mode of inheritance of idiopathic epilepsy (IE) in
English Springer Spaniels.
Animals—45 dogs with IE and 74 siblings and their
Procedure—IE was diagnosed on the basis of age at
the time of seizure onset and results of laboratory
testing and neurologic examinations. Simple segregation
analysis was performed with the Davie method.
Results—Median age at the onset of seizures was 3
years; however, 9 (20%) dogs were between 5 and 6
years old at the time of the onset of seizures. Twentyone
dogs (47%) had generalized seizures, and 24
(53%) had focal onset seizures. Results of segregation
analysis were consistent with partially penetrant
autosomal recessive or polygenic inheritance.
Simulated linkage indicated that there was a 58%
chance of obtaining suggestive linkage with the available
Conclusions and Clinical Relevance—Results of the
present study suggest that in English Springer Spaniels,
IE segregates in a manner that is consistent with partially
penetrant autosomal recessive inheritance (ie, a
single major locus with modifying genes) or polygenic
inheritance. Given enough families with accurate phenotypic
information and available DNA, it should be possible
to use genetic linkage analysis to identify chromosomal
segments containing the causative gene or
genes. (J Am Vet Med Assoc 2005;226:54–58)
Objective—To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation.
Animals—34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed.
Procedures—Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype.
Results—Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait.
Conclusions and Clinical Relevance—It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.
Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.
Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.
Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.
Objective—To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses.
Animals—30 PSSM-affected and 30 unaffected (control) Quarter Horses.
Procedures—By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK γ subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA.
Results—No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK γ subunit genes were also not associated with PSSM.
Conclusions and Clinical Relevance—These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.
Objective—To estimate allele frequencies of the hyperkalaemic periodic paralysis (HYPP), lethal white foal syndrome (LWFS), glycogen branching enzyme deficiency (GBED), hereditary equine regional dermal asthenia (HERDA), and type 1 polysaccharide storage myopathy (PSSM) genes in elite performance subgroups of American Quarter Horses (AQHs).
Design—Prospective genetic survey.
Animals—651 elite performance AQHs, 200 control AQHs, and 180 control American Paint Horses (APHs).
Procedures—Elite performance AQHs successful in 7 competitive disciplines (barrel racing, cutting, halter, racing, reining, western pleasure, and working cow horse) were geno- typed for 5 disease-causing alleles. Age-matched control AQHs and APHs were used to establish comparative whole-breed estimates of allele frequencies.
Results—Highest allele frequencies among control AQHs were for type 1 PSSM (0.055) and GBED (0.054), whereas HERDA (0.021) and HYPP (0.008) were less prevalent. Control APHs uniquely harbored LWFS (0.107) and had high prevalence of HYPP (0.025), relative to AQHs. Halter horse subgroups had significantly greater allele frequencies for HYPP (0.299) and PSSM (0.155). Glycogen branching enzyme deficiency, HERDA, and PSSM were found broadly throughout subgroups; cutting subgroups were distinct for HERDA (0.142), and western pleasure subgroups were distinct for GBED (0.132). Racing and barrel racing subgroups had the lowest frequencies of the 5 disease genes.
Conclusions and Clinical Relevance—Accurate estimates of disease-causing alleles in AQHs and APHs may guide use of diagnostic genetic testing, aid management of genetic diseases, and help minimize production of affected foals.