Associations between endocrine disrupting chemicals and equine metabolic syndrome phenotypes.

Equine Metabolic Syndrome (EMS) is characterized by abnormalities in insulin regulation, increased adiposity and laminitis, and has several similarities to human metabolic syndrome. A large amount of environmental variability in the EMS phenotype is not explained by commonly measured factors (diet, exercise, and season), suggesting that other environmental factors play a role in EMS development. Endocrine disrupting chemicals (EDCs) are associated with metabolic syndrome and other endocrine abnormalities in humans. This led us to hypothesize that EDCs are detectable in horse plasma and play a role in the pathophysiology of EMS. EDCs acting through the aryl hydrocarbon and estrogen receptors, were measured in plasma of 301 horses from 32 farms. The median (range) TEQ (2,3,7,8-TCDD equivalent) and EEQ (17ß-estradiol equivalent) were 19.29 pg/g (0.59-536.36) and 10.50 pg/ml (4.35-15000.00), respectively. TEQ was negatively associated with plasma fat extracted and batch analyzed. EEQ was positively associated with pregnancy and batch analyzed, and negatively associated with being male and superfund score ≤100 miles of the farm. Of particular interest, serum glucose and insulin, glucose and insulin post oral sugar challenge, and leptin concentrations were associated with EEQ, and serum triglyceride concentration was associated with TEQ. Overall, we demonstrated that EDCs are present in the plasma of horses and may explain some of the environmental variability in measured EMS phenotypes. This is the first example of EDCs being associated with clinical disease phenotype components in domestic animals.

Coat color genotypes and risk and severity of melanoma in gray quarter horses.

BACKGROUND: Both graying and melanoma formation in horses have recently been linked to a duplication in the STX17 gene. This duplication, as well as a mutation in the ASIP gene that increases MC1R pathway signaling, affects melanoma risk and severity in gray horses. OBJECTIVE: To determine if melanoma susceptibility in gray Quarter Horses (QH) is lower than gray horses from other breeds because of decreased MC1R signaling resulting from a high incidence of the MC1R chestnut coat color allele in the QH population. ANIMALS: A total of 335 gray QH with and without dermal melanomas. METHODS: Blood or hair root samples were collected from all horses for DNA extraction and genotyping for STX17, ASIP, and MC1R genotypes. Age, sex, and external melanoma presence and grade were recorded. The effect of age and genotype on melanoma presence and severity was evaluated by candidate gene association. RESULTS: Melanoma prevalence (16%) and grade (0.35) in this QH cohort was lower than that reported in other breeds. Age was significantly associated with melanoma prevalence (P = 5.28 × 10(-11)) and severity (P = 2.2 × 10(-13)). No significant effect of MC1R genotype on melanoma prevalence or severity was identified. An effect of ASIP genotype on melanoma risk was not detected. Low STX17 homozygosity precluded evaluation of the gray allele effect. CONCLUSION AND CLINICAL IMPORTANCE: Melanoma prevalence and severity is lower in this population of gray QH than what is reported in other breeds. This could be because of the infrequent STX17 homozygosity, a mitigating effect of the MC1R mutation on ASIP potentiation of melanoma, other genes in the MC1R signaling pathway, or differences in breed genetic background.

A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds.

REASONS FOR PERFORMING STUDY: A glycogen synthase (GYS1) mutation has been described in horses with histopathological evidence of polysaccharide storage myopathy (PSSM) in the USA. It is unknown whether the same mutation is present in horses from the UK. OBJECTIVES: To determine whether the GYS1 mutation occurs in UK horses with histopathological evidence of PSSM and exertional rhabdomyolysis. HYPOTHESIS: The R309H GYS1 mutation is present in a variety of UK horse breeds and that the mutation is commonly associated with exertional rhabdomyolysis. METHODS: DNA was extracted from 47 muscle or blood samples from UK horses with histories of exertional rhabdomyolysis in which muscle biopsy diagnosis had been pursued. The proportions of GYS1 mutation positive cases were compared among histopathologically defined groups. In addition, breeds that carried the GYS1 mutation were identified from a total of 37 grade 2 (amylase-resistant) PSSM cases. RESULTS: Of 47 horses with exertional rhabdomyolysis in which a muscle biopsy diagnosis was pursued, 10 (21%) carried the GYS1 mutation. The mutation was only found in horses with grade 2 PSSM (i.e. not in horses with normal, idiopathic myopathy or grade 1 PSSM biopsy samples). In total, the GYS1 mutation was found in 24/37 (65%) of grade 2 PSSM cases. A variety of breeds, including Quarter Horse, Appaloosa, Warmblood, Connemara-cross, Cob, Polo Pony and Thoroughbred cross carried the mutation. CONCLUSIONS: The GYS1 mutation is an important cause of exertional rhabdomyolysis of UK horse breeds but does not account for all forms of PSSM. POTENTIAL RELEVANCE: Genotyping is recommended in cases of exertional rhabdomyolysis, prior to or in combination with, muscle biopsy. However a significant proportion of horses with histopathological evidence of PSSM and/or exertional rhabdomyolysis have different diseases.

Comparative skeletal muscle histopathologic and ultrastructural features in two forms of polysaccharide storage myopathy in horses.

Polysaccharide storage myopathy (PSSM) has been found in more than 35 different horse breeds through identification of abnormal storage of polysaccharide in muscle biopsies. A dominant mutation in the glycogen synthase 1 gene (GYS1) accounts for a substantial proportion of PSSM cases in at least 17 breeds, including Quarter Horses, but some horses diagnosed with PSSM by muscle histopathologic analysis are negative for the mutation. We hypothesized that a second distinct form of glycogen storage disease exists in GYS1-negative horses with PSSM. The objectives of this study were to compare the histopathologic features, ultrastructure of polysaccharide, signalment, history, and presenting complaints of GYS1-negative Quarter Horses and related breeds with PSSM to those of GYS1-positive horses with PSSM. The total histopathologic score in frozen sections of skeletal muscle stained with hematoxylin and eosin, periodic acid Schiff (PAS) and amylase-PAS stains from 53 GYS1-negative horses did not differ from that of 52 GYS1-positive horses. Abnormal polysaccharide was fine granular or homogenous in appearance (49/53; 92%), often amylase-sensitive (28/53; 53%), more commonly located under the sarcolemma, and consisting of beta glycogen particles in GYS1-negative horses. However, in GYS1-positive horses, abnormal polysaccharide was usually coarse granular (50/52; 96%), amylase-resistant (51/52; 98%), more commonly cytoplasmic, and consisting of beta glycogen particles or, in some myofibers, filamentous material surrounded by beta glycogen particles. Retrospective analysis found that GYS1-negative horses (n = 43) were younger at presentation (4.9 +/- 0.6 years vs. 6.7 +/- 0.3 years for GYS1-positive horses) and were more likely to be intact males than GYS1-positive horses (n = 160). We concluded that 2 forms of PSSM exist and often have distinctive abnormal polysaccharide. However, because evaluation of the histologic appearance of polysaccharide can be subjective and affected by age, the gold standard for diagnosis of PSSM at present would appear to be testing for the GYS1 mutation followed by evaluating muscle biopsy for characteristic abnormal polysaccharide in those horses that are negative for the mutation.

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses.

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene (GBE1). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene (GYS1) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.

Polysaccharide storage myopathy phenotype in quarter horse-related breeds is modified by the presence of an RYR1 mutation.

In this study we examined a family of Quarter Horses with Polysaccharide Storage Myopathy (PSSM) with a dominant mutation in the skeletal muscle glycogen synthase (GYS1) gene. A subset of horses within this family had a more severe and occasionally fatal PSSM phenotype. The purpose of this study was to identify a modifying gene(s) for the severe clinical phenotype. A genetic association analysis was used to identify RYR1 as a candidate modifying gene. A rare, known equine RYR1 mutation, associated with malignant hyperthermia (MH), was found to segregate in this GYS1 PSSM family. Retrospective analysis of patient records (n=179) demonstrated that horses with both the GYS1 and RYR1 mutations had a more severe clinical phenotype than horses with the GYS1 mutation alone. A treadmill trial (n=8) showed that serum creatine kinase activity was higher and exercise intolerance greater in horses with both mutations compared to the GYS1 mutation alone.

Glycogen synthase 1 (GYS1) mutation in diverse breeds with polysaccharide storage myopathy.

BACKGROUND: A missense mutation in the GYS1 gene was recently described in horses with polysaccharide storage myopathy (PSSM). OBJECTIVES: The first objective was to determine the prevalence of the GYS1 mutation in horses with PSSM from diverse breeds. The second objective was to determine if the prevalence of the GYS1 mutation differed between horses diagnosed with PSSM based on grade 1 (typically amylase-sensitive) or grade 2 (typically amylase-resistant) polysaccharide. ANIMALS: Eight hundred and thirty-one PSSM horses from 36 breeds. PROCEDURES: Horses with PSSM diagnosed by histopathology of skeletal muscle biopsy samples were identified from the Neuromuscular Disease Laboratory database. Eight hundred and thirty-one cases had blood or tissue that was available for DNA isolation; these 831 cases were genotyped for the GYS1 mutation by restriction fragment length polymorphism. RESULTS: The PSSM mutation was identified in horses from 17 different breeds. The prevalence of the GYS1 mutation in PSSM horses was high in Draft- (87%) and Quarter Horse-related breeds (72%) and lower in Warmbloods (18%) and other light horse breeds (24%), when diagnosis was based on grade 2 diagnostic criteria. Overall, the PSSM mutation was present in 16% of grade 1 and 70% of grade 2 PSSM horses. CONCLUSIONS AND CLINICAL IMPORTANCE: GYS1 mutation causes PSSM in diverse breeds and is the predominant form of PSSM in Draft- and Quarter Horse-related breeds. False-positive diagnosis, as well as the possibility of a second glycogenosis in horses with neuromuscular disease (type 2 PSSM), might explain the absence of the GYS1 mutation in horses diagnosed with excessive glycogen accumulation in muscle.

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system.

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.

Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from Thoroughbreds with recurrent exertional rhabdomyolysis.

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 calcium. ANIMALS: 4 Thoroughbred mares with RER and 4 clinically normal (control) Thoroughbreds. PROCEDURES: Single chemically-skinned type-I (slow-twitch) 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 activity. CONCLUSIONS 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.

Autosomal dominant canine malignant hyperthermia is caused by a mutation in the gene encoding the skeletal muscle calcium release channel (RYR1).

BACKGROUND: Malignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. METHODS: To determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane-succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. RESULTS: Pedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a theta = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at theta = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. CONCLUSIONS: These results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

Calcium regulation by skeletal muscle membranes of horses with recurrent exertional rhabdomyolysis.

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) horses. 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.

Abnormal regulation of muscle contraction in horses with recurrent exertional rhabdomyolysis.

OBJECTIVE: To determine whether abnormal regulation of muscle contraction similar to that associated with malignant hyperthermia (MH) was evident in intact external intercostal muscle cells from Thoroughbreds with recurrent exertional rhabdomyolysis (RER). ANIMALS: 5 adult Thoroughbred horses with RER and 7 clinically normal adult Thoroughbred or mixed-breed horses. PROCEDURES: Twitch time course variables and contracture responses to various concentrations of potassium, caffeine, and halothane were measured in small bundles of intact external intercostal muscle cells from clinically normal horses and horses with RER. RESULTS: Threshold for significant contracture induced by potassium depolarization was lower for RER-affected muscles, compared with normal muscles, although the relationship between potassium concentration and membrane potential were not different. Thresholds for contracture induced by caffeine and halothane were also lower for RER-affected muscles, compared with normal muscles. Lower thresholds for caffeine- and halothane-induced contractures, as well as depolarization-elicited contractures, in RER-affected muscles suggest a defect in myoplasmic calcium regulation. CONCLUSIONS AND CLINICAL RELEVANCE: Regulation of muscle contraction is abnormal in Thoroughbreds with RER. The specific defect may be attributable to abnormal intracellular calcium regulation. Knowledge of the specific defect involved in RER may lead to improved prevention and treatment of RER-affected horses.

A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers.

To generate a domestic horse genome map we integrated synteny information for markers screened on a somatic cell hybrid (SCH) panel with published information for markers physically assigned to chromosomes. The mouse-horse SCH panel was established by fusing pSV2neo transformed primary horse fibroblasts to either RAG or LMTk mouse cells, followed by G418 antibiotic selection. For each of the 108 cell lines of the panel, we defined the presence or absence of 240 genetic markers by PCR, including 58 random amplified polymorphic DNA (RAPD) markers and 182 microsatellites. Thirty-three syntenic groups were defined, comprised of two to 26 markers with correlation coefficient (r) values ranging from 0.70 to 1.0. Based on significant correlation values with physically mapped microsatellite (type II) or gene (type I) markers, 22 syntenic groups were assigned to horse chromosomes (1, 2, 3, 4, 6, 9, 10, 11, 12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 26, 30, X and Y). The other 11 syntenic groups were provisionally assigned to the remaining chromosomes based on information provided by heterologous species painting probes and work in progress with type I markers.

Skeletal muscle metabolic response to exercise in horses with 'tying-up' due to polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a distinct cause of exertional rhabdomyolysis in Quarter Horses that results in glycogen and abnormal polysaccharide accumulation. The purpose of this study was to determine if excessive glycogen storage in PSSM is due to a glycolytic defect that impairs utilisation of this substrate during exercise. Muscle biopsies, blood lactates and serum CK were obtained 1) at rest from 5 PSSM Quarter Horses, 4 normal Quarter Horses (QH controls) and 6 Thoroughbreds with recurrent exertional rhabdomyolysis (TB RER) and 2) after a maximal treadmill exercise test in PSSM and QH controls. In addition, 3 PSSM horses performed a submaximal exercise test. At rest, muscle glycogen concentrations were 2.4x and 1.9x higher in PSSM vs. QH controls or TB RER, respectively. Muscle lactates at rest were similar between PSSM and QH controls but significantly higher in PSSM vs. TB RER. Muscle glucose-6-phosphate concentrations were also higher in PSSM horses than controls combined. During maximal exercise, mean muscle glycogen concentrations declined 2.7x more and mean lactate increased 2x more in PSSM vs. QH controls; however, differences were not statistically significant. Blood lactate concentrations after maximal exercise did not reflect generally higher muscle lactate in PSSM vs. QH controls. No change in blood lactate concentrations occurred in PSSM horses with submaximal exercise. Serum CK activity increased significantly 4 h after maximal and submaximal exercise and was significantly higher in PSSM vs. QH controls. These results show that during maximal exercise, PSSM horses utilised muscle glycogen and produce lactic acid via a functional glycolytic pathway and that during submaximal exercise oxidative metabolism was unimpaired. The excessive glycogen storage and formation of abnormal polysaccharide in PSSM horses therefore appear to reflect increased glycogen synthesis rather than decreased utilisation. The specific subset of horses with exertional rhabdomyolysis due to PSSM would likely benefit clinically from a diet low in soluble carbohydrates like grain with fat added as well as gradually increasing daily exercise to reduce excessive glycogen accumulation and enhance utilisation.