Polysaccharide Storage Myopathy Research Articles

Allele frequency of muscular genetic disorders in bull-catching (vaquejada) quarter horses

Quarter horses (QH), a prominent athletic breed in Brazil, are affected by muscular genetic disorders such as myosin-heavy chain myopathy (MYHM), polysaccharide storage myopathy (PSSM1), hyperkalemic periodic paralysis (HyPP), and malignant hyperthermia (MH). Bull-catching (vaquejada), primarily involving QH, is a significant equestrian sport in Brazil. Since the allele frequencies (AF) of MYHM, PSSM1, HyPP, and MH in vaquejada QH remain unknown, this study evaluated the AF in 129 QH vaquejada athletes, specifically from the Brazilian Northeast. These variants were exclusively observed in heterozygosity. The MYHM exhibited the highest AF (0.04 ±0.01), followed by PSSM1 (0.01 ±0.01) and the HyPP variant (0.004 ±0.01), while the MH variant was not identified in this study. This study represents the first identification of these variants in vaquejada QH, emphasizing the need to implement measures to prevent the transmission of pathogenic alleles and reduce the occurrence of clinical cases of these genetic diseases.

A nutritional review of polysaccharide storage myopathy

Polysaccharide storage myopathy is characterised as the dissolution of skeletal muscle in association with exercise. Genetic testing has uncovered two forms of polysaccharide storage myopathy: PSSM1 and PSSM2, and a subtype known as myofibrillar myopathy. Horses with these conditions can display a variety of symptoms. Nutritional management of PSSM1 and PSSM2 cases hinges on ensuring that rations are low in both starch and sugar, and daily exercise is also important for these horses.

Estudo da mutação causadora da miopatia por acúmulo de polissacarídeo tipo 1 em uma população de cavalos Mangalarga Marchador utilizados em programas de melhoramento

The Mangalarga Marchador (MM) breed, which originated in Brazil, constitutes the largest number of horses in the country. The animals are versatile and used in several sports because of major investments made for the genetic improve-ment of the breed. In recent decades, advances in molecular techniques enabled the identification of genetic diseases in hor-ses. Conducting molecular tests and determining the occurrence of mutations are fundamental for the early identification and prevention of abnormalities. Among the known genetic diseases that occur in horses, the c.926G>A mutation in the GYS1gene that causes type 1 polysaccharide storage myopathy (PSSM1) stands out, because it has been identified in several breeds of horses. Although myopathy is common in MM horses, the occurrence of the c.926G>A mutation in the GYS1 gene has not yet been evaluated. The lack of knowledge about the possible presence of PSSM1 averts the adoption of control measures to prevent the spread of the disease in MM horses. Therefore, the aim of this study was to verify the occurrence of the muta-tion that causes PSSM1 in MM horses used in breeding programs. Blood DNA was extracted and the region of the GYS1gene containing the mutation was amplified and sequenced. No mutation in the GYS1 gene was found in the evaluated sam-ples. However, since clinical signs of myopathy are frequently observed in MM horses, further studies, including histological analysis, are necessary to establish the underlying causes. In addition, if there is a genetic pattern of occurrence, molecular studies should be considered.

Polysaccharide storage myopathy in two Percheron horses

Polysaccharide storage myopathy (PSSM) is a genetic disorder or by undetermined cause characterized by the abnormal accumulation of glycogen and polysaccharides in skeletal muscle fibers. The present report describes two Percheron horses, from different properties in the municipality of Pouso Redondo - SC, with locomotor clinical signs that started after work. The clinical signs observed were rigid gait, mainly of the pelvic limbs, muscle weakness, and recumbency. In the first case, the animal was diagnosed with severe multifocal rhabdomyonecrosis, moderate multifocal myoglobin nephrosis and severe diffuse transmural fibrinonecrotic gastritis. PSSM was confirmed by the presence of polysaccharide inclusions in myofiber sarcolemma visualized by periodic acid-Schiff (PAS) staining. In the second case, the horse presented two episodes of muscle disorder after work. The therapeutic protocol was based on penicillin, flunixin meglumine and fluid therapy with ringer lactate. An increase in creatine kinase (669.0 UI/L) and aspartate aminotransferase (669.0 UI/L) was observed in the serum biochemical evaluation. Research for mutation in the GYS1 gene was performed, with a positive heterozygote result. After rest and gradual recovery, the horse was only submitted to light work, with no return of the clinical condition since then. Thus, this report describes two cases of PSSM with distinct clinical evolution and diagnostic methods.

Prevalence of Genetic Mutations in Horses With Muscle Disease From a Neuromuscular Disease Laboratory

Deleterious genetic variants are an important cause of skeletal muscle disease. Immunohistochemical evaluation of muscle biopsies is standard for the diagnosis of muscle disorders. The prevalence of alleles causing hyperkalemic periodic paralysis (HYPP), malignant hyperthermia (MH), polysaccharide storage myopathy 1 (PSSM1), glycogen branching enzyme deficiency (GBED), myotonia congenita (MC), and myosin heavy chain myopathy (MYHM) in horses with muscle disease is unknown. Archived slides processed for immunohistochemical analysis from 296 horses with muscle disease were reviewed blinded and clinical information obtained. DNA isolated from stored muscle samples from these horses were genotyped for disease variants. Histological findings were classified as myopathic in 192, neurogenic in 41, and normal in 63 horses. A third of the population had alleles that explained disease which constituted 45% of the horses with confirmed histological myopathic process. Four of six muscle disease alleles were identified only in Quarter horse breeds. The allele causing PSSM1 was detected in other breeds, and MC was not detected in these samples. The My allele, associated with susceptibility for MYHM, was the most common (62%) with homozygotes (16/27) presenting a more severe phenotype compared to heterozygotes (6/33). All cases with the MH allele were fatal upon triggering by anesthesia, stress or concurrent myopathy. Both, muscle histological and genetic analyses are essential in the investigation of muscle disease, since 10% of the horses with muscle disease and normal histology had a muscle disease causing genetic variant, and 63% of histologically confirmed muscle with alterations had no known genetic variants.

Type 2 polysaccharide storage myopathy in Quarter Horses is a novel glycogen storage disease causing exertional rhabdomyolysis.

Both type 1 (PSSM1) and type 2 polysaccharide storage myopathy (PSSM2) are characterised by aggregates of abnormal polysaccharide in skeletal muscle. Whereas the genetic basis for PSSM1 is known (R309H GYS1), the cause of PSSM2 in Quarter Horses (PSSM2-QH) is unknown and glycogen concentrations not defined. To characterise the histopathological and biochemical features of PSSM2-QH and determine if an associated monogenic variant exists in genes known to cause glycogenosis. Retrospective case control. Sixty-four PSSM2-QH, 30 PSSM1-QH and 185 control-QH were identified from a biopsy repository and clinical data, histopathology scores (0-3), glycogen concentrations and selected glycolytic enzyme activities compared. Coding sequences of 12 genes associated with muscle glycogenoses were identified from whole genome sequences and compared between seven PSSM2-QH and five control-QH. Exertional rhabdomyolysis in PSSM2-QH occurred predominantly in barrel racing and working cow/roping performance types and improved with regular exercise and a low starch/fat-supplemented diet. Histopathological scores, including the amount of amylase-resistant polysaccharide (PSSM2-QH 1.4 ± 0.6, PSSM1-QH 2.1 ± 0.3, control-QH 0 ± 0, p < 0.001), and glycogen concentrations (PSSM2-QH 129 ± 62, PSSM1-QH 175 ± 9, control-QH 80 ± 27 mmol/kg, p < 0.0001) were intermediate in PSSM2-QH with significant differences among groups. In PSSM2-QH, abnormal polysaccharide had a less filamentous ultrastructure than PSSM1-QH and phosphorylase and phosphofructokinase activities were normal. Seventeen of 30 PSSM2-QH with available pedigrees descended from one of three stallions within four generations. Of the 29 predicted high or moderate impact genetic variants identified in candidate genes, none were present in only PSSM2-QH and absent in control-QH. Analyses of PSSM2-QH and PSSM1-QH were performed on shipped samples, controls on frozen samples. PSSM2-QH is a novel glycogen storage disorder that is not the result of a mutation in genes currently known to cause muscle glycogenoses in other species.

Profiling of genetic markers useful for breeding decision in Selle Francais horse

Genetic disorders are recognised as hereditary diseases with the most significant economic impact on horse breeding, causing important foal losses, costs of treatments of horses, and maintenance of the mare during the pregnancy. The Selle Francais horses are recognized in many countries and are showing great results in equestrian sports around the world (dressage, show jumping and eventing). The study aimed to detect the presence of three mutant alleles associated with inherited diseases including Fragile Foal Syndrome (FFS), Cerebellar Abiotrophy (CA), Polysaccharide Storage Myopathy (PSSM1) and variant impacting gait type in DMRT3. This trait is important for breeding decision in Selle Francais horses and sheds new light on genetic potential and risks on this breed. The genotyping was performed on 91 Selle Francais horses using PCR-RFLP (for POLD1; GYS1 and DMRT3 genes) and PCR-ACRS (TOE1 gene) methods. The presented report indicated the presence of mutant allele A casual for PSSM1 and allele T associated with FFS syndrome occurrence, in 4% and 6% of analysed horses, respectively. Regarding CA, the present survey did not register any cases of this genetic disorder in Selle Francais horses. Our results show also that about 1% of all the Sell Francais horses studied carry the A allele of DMRT3 gene. The present findings have provided data for these fulness of monitoring genetic diseases and gait type in the investigated breed to avoid losses of offspring.

Absence of myofibrillar myopathy in Quarter Horses with a histopathological diagnosis of type 2 polysaccharide storage myopathy and lack of association with commercial genetic tests.

Genetic tests for variants in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 or P3b; rs1142918816) and MYOZ3(P4; rs1142544043) genes are offered commercially to diagnose myofibrillar myopathy (MFM) and type 2 polysaccharide storage myopathy (PSSM2) in Quarter Horses (QH). To determine if PSSM2-QH has histopathological features of MFM. To compare genotype and allele frequencies of variants P2, P3, P4 between control-QH and PSSM2-QH diagnosed by histopathology. Retrospective cross-sectional. The study includes a total of 229 healthy control-QH, 163 PSSM2-QH GYS1 mutation negative. Desmin stains of gluteal/semimembranosus muscle were evaluated. Purported disease alleles P2, P3a, P3b, P4 were genotyped by pyrosequencing. Genotype, allele frequency and total number of variant alleles or loci were compared between phenotypes using additive/genotypic and dominant models and quantitative effects evaluated by multivariable logistic regression. Histopathological features of MFM were absent in all QH. A P variant allele at any locus was not associated (P>.05) with a histopathological diagnosis of PSSM2 and one or more P variants were common in control-QH (57%) and PSSM2-QH (61%). Allele frequencies (control/PSSM2) were: 0.24/0.21 (P2), 0.07/0.12 (P3a), 0.07/0.11 (P3b) and 0.06/0.08 (P4). P3a and P3b loci were not independent (r2 =0.894); and not associated with PSSM2 histopathology comparing the haplotype of both P3a and P3b variants to other haplotypes. A receiver operator curve did not accurately predict the PSSM2 phenotype (AUC=0.67, 95% CI 0.62-0.72), and there was no difference in the total number of variant loci or total variant allele count between control-QH and PSSM2-QH. P3a and P3b were not in complete linkage disequilibrium. The P2, P3 and P4 variants in genes associated with human MFM were not associated with PSSM2 in 392 QH. Their use would improperly diagnose PSSM2/MFM in 57% of healthy QH and fail to diagnose PSSM2 in 40% of QH with histopathological evidence of PSSM2.

An Investigation into Equine Nutrition Knowledge and Educational Needs of Equine Veterinarians.

This study investigated equine nutrition knowledge and educational needs of licensed veterinarians in the United States who were exclusively or predominately equine practitioners. It found veterinarians regard their peers as an important resource of nutritional knowledge, ranking ahead of all other sources except a PhD equine nutritionist. Interestingly, only 21% of veterinarians felt good about their knowledge level in equine nutrition after graduating from veterinary school. Although veterinarians in this study reported equine nutrition to be an area of weakness, 75% had not pursued continuing education in the field of nutrition within the last year. Additionally, they devoted only 65 minutes per year on average to improving their knowledge of equine nutrition, yet the majority (82.2%) had been providing nutritional advice to clients. This study revealed that time spent practicing veterinary medicine increases (p < .001) a veterinarian's self-perceived knowledge level of equine nutrition, shifting from just below average after graduation from veterinary school to just above average at the time of this study. The majority (70%) of veterinarians in this study believe nutrition is very important in their practice philosophy, and 71% showed interest in taking online continuing education courses; thus, curriculum should be developed and offered in areas of need as identified by this study. These areas include insulin resistance, equine gastric ulcer syndrome, equine metabolic syndrome, performance horses, equine pituitary pars intermedia dysfunction, equine polysaccharide storage myopathy, and arthritis/joint pain, along with how to assess nutritional status during general wellness examinations.

Some inherited disorders in pacing horses in Turkey

This study was carried out to detect the presence of mutant alleles of polysaccharide storage myopathy (PSSM) and severe combined immunodeficiency (SCID) disorders in pacing horses raised in different regions of Turkey. Blood/hair samples from 264 (182 Indigenous, 31 Iranian, 24 Afghan, and 27 Bulgarian) pacing horses aged 4 and over were used. As a result of the study, a mutation-heterozygosis (GA) in the GYS1 gene related to PSSM disease was detected in one of the pacing horses (Bulgarian horse). No deletions were observed in the DNA-PKcs gene region in the pacing horses for SCID disease. This study has been carried out to identify the status of two inherited disorders in pacing horses in Turkey. It was determined that there are no mutant genes in indigenous pacing horses, in terms of PSSM and SCID disorders, which are the major hereditary disorders in horses.

Biodiversity and functional variability of the allele pool of horse breed populations

Polysaccharide storage myopathy (PSSM1) is a disease of excessive accumulation of glycogen in muscles, which is encoded by the enzyme glycogen synthase (GYS1). The horses with a mutation at the locus (GYS1) have substandard performance, they often experience muscle pain with intense work, get sweaty and have stiffness in movement, which is generally accompanied by a decrease in performance. The aim of our research was to study the GYS1 mutation (g.18940324 G> A) in glycogen accumulation in horses of different breeds. For the study, horses (n = 181) of ten factory and local breeds were selected, including Russian Donskaya, Bashkirskaya, Buryatskaya, Vladimirskaya, Vyatskaya, Percheronskaya, Russian heavy draft, Soviet heavy draft, Orlov trotter and Thoroughbred horse breeds. According to the results of testing, the GYS1 mutation was found in Bashkirskaya horse breed (3.2%), Buryatskaya horse breed (10%), Vyatskaya horse breed (9.5%), Soviet heavy draft horse breed (30%), Russian heavy draft horse breed (49.9%) and Percheronskaya horse breed (90%). Donskaya, Thoroughbred and Orlov trotter horse breeds showed no PSSM1 defect. Timely diagnostics and monitoring of horse breeds for the presence of genetic abnormalities that reduce their health and performance allow avoiding the accumulation of genetic burden in populations and contribute to increasing the efficiency of breeding programs.

144 Lack of correspondence between commercial genetic test variants for type 2 polysaccharide storage myopathy and a histopathologic diagnosis in Quarter Horses

A diagnosis of type 2 polysaccharide storage myopathy (PSSM2) in Quarter Horse-related breeds (QH) is currently based on the absence of the glycogen synthase 1 ( GYS1) mutation causing type 1 PSSM (PSSM1) and presence of aggregates polysaccharide in myofibers. Recently, genetic tests for PSSM2 have been offered by a commercial company based on variants in the genes myotilin (termed P2), filamin C (P3) and myozenin 3 (P4). The company claims these variants are associated with PSSM2 as phenotyped by clinical signs without regard to muscle histopathology. The purpose of this study was to compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323 and P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Quarter Horses diagnosed with PSSM2 by muscle histopathology and healthy Quarter Horse controls with normal muscle histology (periodic acid Schiff's stain). DNA was isolated from hair bulb, buffy coat, whole blood or muscle tissue samples. Primers were designed and genotyping performed using pyrosequencing. Genotypes at each locus (homozygous reference vs. heterozygous, homozygous alternate) were compared between PSSM2 (n = 54, 7.6 ± 4.2 y) and control horses (n = 121, 9.8 ± 5.7 y) using a Fisher's exact test ( P ≤ 0.05). There was no significant association between any P locus and a histopathological diagnosis of PSSM2. Minor allele frequencies (cases/controls) were: 20%/27% (P2), 13%/10% (P3a), 11%/9% (P3b)/and 11%/10% (P4). The P2 variant was present in 48% of control and 33% of PSSM2 Quarter Horses. In conclusion, the P2, P3a, P3b and P4 commercial test variants appear to be relatively common variants in Quarter Horses not associated with a diagnosis of PSSM2 established by muscle biopsy.

Comparison of gluteus medius muscle activity in Haflinger and Noriker horses with polysaccharide storage myopathy.

Type 1 polysaccharide storage myopathy caused by genetic mutation in the glycogen synthase 1 gene is present in many breeds including the Noriker and Haflinger horses. In humans, EMG has already been used to document changes in the muscle activity patterns of patients affected by human glycogen storage disorders. Therefore, the aim of the present study was to describe gluteus muscle activity with surface electromyography (sEMG) in Haflinger and Noriker horses with known GYS1 mutation status during walk and trot. Thirty‐two horses (11 Haflinger and 21 Noriker horses) with homozygous non‐affected (GG), heterozygous affected (GA) and homozygous affected (AA) status of GYS1 mutation without overt clinical signs of any myopathy were selected for the current study. Using surface electromyography gluteus medius muscle activity at walk and at trot was measured, and muscle activity was described in relation to the maximum observed value at the same sensor and the same gait. In order to further describe the signals in detail comprising both frequencies and amplitudes, the crossings through the baseline and the 25, 50 and 75 percentile lines were determined. The result of the relative muscle activity did not show a consistent difference between affected and non‐affected horses. Genetically affected (GA and AA) horses showed significantly less density of muscle activity for both gaits and horse breeds except for the crossings per second at the baseline and 75 percentile at walk in the Haflinger horses and 75 percentile at trot in the Noriker horses. The medians of all calculated density values were significantly lower in the GA Haflingers compared to the GG Haflingers (p = 0.012) and also in the AA Norikers compared to the GG Norikers (p = 0.011). Results indicate that the GYS1 mutation reduces the number of functional muscle fibres detected by sEMG measurements even in the absence of overt clinical signs.

Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis.

BackgroundCommercial genetic tests for type 2 polysaccharide storage myopathy (PSSM2) and myofibrillar myopathy (MFM) have not been validated by peer‐review, and formal regulation of veterinary genetic testing is lacking.ObjectivesTo compare genotype and allele frequencies of commercial test variants (P variants) in MYOT (P2; rs1138656462), FLNC (P3a; rs1139799323), FLNC (P3b; rs1142918816) and MYOZ3 (P4; rs1142544043) between Warmblood (WB) and Arabian (AR) horses diagnosed with PSSM2/MFM by muscle histopathology, and phenotyped breed‐matched controls. To quantify variant frequency in public repositories of ancient and modern horse breeds.Study designCross sectional using archived clinical material and publicly available data.MethodsWe studied 54 control‐WB, 68 PSSM2/MFM‐WB, 30 control‐AR, 30 PSSM2/MFM‐AR and 205 public genotypes. Variants were genotyped by pyrosequencing archived DNA. Genotype and allele frequency, and number of variant alleles or loci were compared within breed between controls, PSSM2/MFM combined and MFM or PSSM2 horses considered separately using additive/genotypic and dominant models (Fisher's exact tests). Variant frequencies in modern, early domestic and Przewalski horses were determined from a public data repository.ResultsThere was no significant association between any P locus and a histopathological diagnosis of PSSM2/MFM, and no difference between control and myopathic horses in total loci with alternative alleles, or total alternate alleles when PSSM2/MFM was considered combined or separately as PSSM2 or MFM. For all tests, sensitivity was <0.33. Allele frequencies in WB (controls/cases) were: 8%/15% (P2), 5%/6% (P3a/b) and 9%/13% (P4); in AR, frequencies were: 12%/17% (P2), 2%/2% (P3a/b) and 7%/12% (P4). All P variants were present in early domestic (400‐ to 5500‐year‐old) horses and P2 present in the Przewalski.ConclusionsBecause of the lack of significant association between a histopathological diagnosis of PSSM2 or MFM and the commercial genetic test variants P2, P3 and P4 in WB and AR, we cannot recommend the use of these variant genotypes for selection and breeding, prepurchase examination or diagnosis of a myopathy.

P.73Correlation of gluteus medius muscle activities at walk and trot with myopathy changes on biopsies of the exact same locations in horses without clinical signs of type 1 polysaccharide storage myopathy

In the native Austrian breed of Haflinger horses, abnormal polysaccharide accumulations and signs of myopathy in skeletal muscle are commonly caused by type 1 polysaccharide storage myopathy (T1PSSM), based on the genetic mutation of the glycogen synthase 1 (GYS1) gene. In the absence of clinical signs, mild histological myopathy changes in muscle fibres on biopsy as well as changes in the gluteus muscle activity pattern could be documented in Haflinger horses. Surface electromyography (sEMG) of the gluteus medius muscle during walk and trot as well as superficial muscle biopsies in the exact same location taken immediately after sEMG measurements are available of 13 Haflinger horses with homozygous non-affected status (GG, n=5) and heterozygous affected status (GA, n=6) of the GYS1 gene. Thus the aim of the present study is to investigate the correlation between sEMG activity parameters and histology scores for myopathy, to elucidate which histology changes are potentially useful to quantify the functional effect. Parameters used for sEMG signal analysis comprising frequency and amplitude information were the crossings per second (C/s) of the signal through the baseline and the 25, 50, and 75 percentile lines of the sEMG signal at the same location during the same gait. Of the biopsies of the same horses at the same location taken after the sEMG measurement myopathy changes were scored for anguloid atrophy, necrosis or macrophages, central nuclei, and fiber size variation, and numbers of fibers with characteristic cPAS inclusions were expressed as percentage of total fibers counted. There was a significant negative correlation between C/s parameters and myopathy scores over all horses; however, this was not significant within the GA horses alone for any of the myopathy characteristics documented. This may indicate that the functional relevance of the histology parameters in these clinically not affected horses is more of a qualitative than of a quantitative nature.

P.68Breed specifics of muscle function changes in the Austrian Haflinger and Austrian Noriker horses with polysaccharide storage myopathy (PSSM)

P.68Breed specifics of muscle function changes in the Austrian Haflinger and Austrian Noriker horses with polysaccharide storage myopathy (PSSM)

Bayesian Networks in Equine Genetic Counseling

Evaluation of the individualized risks of occurrence of genetic disorders in horses can be challenging and necessitates adequate methods. One of these methods is the Bayes network (BN). The objective of this study is to create a BN and test its utility in evaluating risk occurrence of type 1 polysaccharide storage myopathy (PSSM1) as an example. Current knowledge on the hereditary mode of transmission of PSSM1 and on the effects of breed, diet, exercise regimen, and age on the disease was retrieved from the scientific literature. Uncertainty on this background information was converted into prior beta distributions. Utility values of different potential mating were based on recommendations toward preventing the production of affected animals and reducing the frequency of asymptomatic carriers. The network suggested, for example, it is impossible to observe parents free of PSSM1 clinical signs if their offspring present PSSM1 clinical signs. Expected utilities for the alternatives “breed” or “not to breed”, two individuals free of PSSM1 clinical signs, are equivalent if an arbitrary score of 50 is given to each copy of the deleterious allele. It is higher for the alternative “breed” if a score of 100 is given to a mating leading to a heterozygous offspring. Results of the sensitivity analyses agree with intuitive expectations about the usefulness of genetic test in determining the genetic status of an individual. Limited current knowledge about the risk factors of PSSM1 may affect the results but the network may be easily updated with incoming new information.

Muscle glycogen concentrations and response to diet and exercise regimes in Warmblood horses with type 2 Polysaccharide Storage Myopathy.

Type 1 polysaccharide storage myopathy (PSSM1) is a glycogen storage disorder of known cause whereas the basis for type 2 PSSM (PSSM2) is unknown. The same diet and exercise regime prescribed for PSSM1 is recommended for PSSM2; however, the benefit of these recommendations for PSSM2 is undocumented. The objectives of this study were to determine traits of PSSM2 Warmblood horses (WB), determine the changes in exercise responses that occur with a recommended low-starch/fat-supplemented diet and exercise regime, and determine if glycogen concentrations correspond to the severity of signs. Owners of PSSM2 WB (2008–2016), completed a retrospective questionnaire regarding their horse. Glycogen concentrations were analyzed in skeletal muscle of PSSM2 WB (n = 36) obtained prior to recommendations and in control WB with no evident myopathy (n = 23). Chi-square, Fisher’s exact, McNemar’s tests with Bonferroni correction and Mann Whitney testing were utilized. Abnormal exercise responses reported by owners, began at approximately 6 years of age and included a decline in performance, a reluctance to collect and reluctance to go forward in over 50% of horses. With the recommended diet and exercise regime, 80% of PSSM2 WB owners reported an overall improvement with significant decreases in the proportion of horses showing a decline in performance and rhabdomyolysis. However, 53% of PSSM2 WB were still not advancing as expected with reluctance to go forward and collect persisting in approximately one third of horses. Median muscle glycogen concentrations did not differ between PSSM2 WB and WB with no evident myopathy. PSSM2 WB with the highest glycogen concentrations were significantly more likely to show a decline in performance than those with lower glycogen concentrations. In conclusion, diet and exercise recommendations ideal for PSSM1 improve but do not eliminate the decline in performance and reluctance to go forward under saddle characteristic of PSSM2.

Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

Estimation of the Allele Frequency of Type 1 Polysaccharide Storage Myopathy and Malignant Hyperthermia in Quarter Horses in Brazil

Type 1 polysaccharide storage myopathy (PSSM1) and malignant hyperthermia (MH) are autosomal dominant genetic diseases caused by a point mutation in equine GYS1 and RYR1, respectively. Although the prevalence of PSSM1 and MH have been described in other countries as causes of myopathy in horses, there are no studies evaluating the allele frequency of those mutations in Brazil. The aim of the present study was to determine the allele frequency of the mutated alleles causing susceptibility for PSSM1 and MH mutations in Quarter Horses (QHs) in Brazil. Deoxyribonucleic acid was purified from blood samples of 741 QHs used in five competitive disciplines (i.e., barrel racing, cutting, halter, racing, and reining). The PCR was optimized to identify the mutations using primers designed from the sequence of equine GYS1 and RYR1. The allele frequencies and the prevalence of PSSM1 were 0.034 and 6.7%, respectively. The highest allele frequency for the GYS1 mutated gene was identified in halter horses (0.163), and the gene mutation was not observed in racing animals. The mutation in the RYR1 was not identified in any of the sampled horses. The PSSM1 allele frequency observed in the present study indicates the relevance of the disease in the differential diagnosis of myopathies in horses. In addition, the absence of the RYR1 causative mutation suggests little importance of MH as a genetic muscle disorder in the Brazilian QHs.