Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle.

Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.

Brain magnetic resonance imaging in the DE50-MD dog model of Duchenne muscular dystrophy reveals regional reductions in cerebral gray matter.

BACKGROUND: Duchenne muscular dystrophy is a X-linked disease characterized by severe and progressive muscle weakness, alongside cognitive impairment and a range of neurobehavioral disorders secondary to brain dystrophin deficiency. Duchenne muscular dystrophy patients have reduced cerebral gray matter and altered white matter ultrastructure (detected by magnetic resonance imaging) compared to age-matched controls. METHODS: We studied the DE50-MD canine model of Duchenne muscular dystrophy, which is deficient in full length brain dystrophin (Dp427) isoforms and has a neurocognitive phenotype. Eight DE50-MD and 6 age-matched littermate wild type male dogs underwent serial brain magnetic resonance imaging from 14 to 33 months of age. RESULTS: Reduced regional gray matter was detected in DE50-MD dogs compared with wildtype, including the piriform lobe, hippocampus and cingulate gyrus. Lateral ventricle volume was larger in DE50-MD dogs. Differences did not progress over time. White matter volume did not differ between DE50-MD and wildtype dogs. There was no difference in brain nor cranial vault volume between DE50-MD and wildtype dogs. CONCLUSION: Dystrophin deficiency in the canine brain results in structural changes that likely contribute to the neurocognitive phenotype.

Genetic characterisation of the Connemara pony and the Warmblood horse using a within-breed clustering approach.

BACKGROUND: The Connemara pony (CP) is an Irish breed that has experienced varied selection by breeders over the last fifty years, with objectives ranging from the traditional hardy pony to an agile athlete. We compared these ponies with well-studied Warmblood (WB) horses, which are also selectively bred for athletic performance but with a much larger census population. Using genome-wide single nucleotide polymorphism (SNP) and whole-genome sequencing data from 116 WB (94 UK WB and 22 European WB) and 36 CP (33 UK CP and 3 US CP), we studied the genomic diversity, inbreeding and population structure of these breeds. RESULTS: The k-means clustering approach divided both the CP and WB populations into four genetic groups, among which the CP genetic group 1 (C1) associated with non-registered CP, C4 with US CP, WB genetic group 1 (W1) with Holsteiners, and W3 with Anglo European and British WB. Maximum and mean linkage disequilibrium (LD) varied significantly between the two breeds (mean from 0.077 to 0.130 for CP and from 0.016 to 0.370 for WB), but the rate of LD decay was generally slower in CP than WB. The LD block size distribution peaked at 225 kb for all genetic groups, with most of the LD blocks not exceeding 1 Mb. The top 0.5% harmonic mean pairwise fixation index (FST) values identified ontology terms related to cancer risk when the four CP genetic groups were compared. The four CP genetic groups were less inbred than the WB genetic groups, but C2, C3 and C4 had a lower proportion of shorter runs of homozygosity (ROH) (74 to 76% < 4 Mb) than the four WB genetic groups (80 to 85% < 4 Mb), indicating more recent inbreeding. The CP and WB genetic groups had a similar ratio of effective number of breeders (Neb) to effective population size (Ne). CONCLUSIONS: Distinct genetic groups of individuals were revealed within each breed, and in WB these genetic groups reflected population substructure better than studbook or country of origin. Ontology terms associated with immune and inflammatory responses were identified from the signatures of selection between CP genetic groups, and while CP were less inbred than WB, the evidence pointed to a greater degree of recent inbreeding. The ratio of Neb to Ne was similar in CP and WB, indicating the influence of popular sires is similar in CP and WB.

Recognising the potential of large animals for modelling neuromuscular junction physiology and disease.

The aetiology and pathophysiology of many diseases of the motor unit remain poorly understood and the role of the neuromuscular junction (NMJ) in this group of disorders is particularly overlooked, especially in humans, when these diseases are comparatively rare. However, elucidating the development, function and degeneration of the NMJ is essential to uncover its contribution to neuromuscular disorders, and to explore potential therapeutic avenues to treat these devastating diseases. Until now, an understanding of the role of the NMJ in disease pathogenesis has been hindered by inherent differences between rodent and human NMJs: stark contrasts in body size and corresponding differences in associated axon length underpin some of the translational issues in animal models of neuromuscular disease. Comparative studies in large mammalian models, including examination of naturally occurring, highly prevalent animal diseases and evaluation of their treatment, might provide more relevant insights into the pathogenesis and therapy of equivalent human diseases. This review argues that large animal models offer great potential to enhance our understanding of the neuromuscular system in health and disease, and in particular, when dealing with diseases for which nerve length dependency might underly the pathogenesis.

A method to identify, dissect and stain equine neuromuscular junctions for morphological analysis.

Morphological study of the neuromuscular junction (NMJ), a specialised peripheral synapse formed between a lower motor neuron and skeletal muscle fibre, has significantly contributed to the understanding of synaptic biology and neuromuscular disease pathogenesis. Rodent NMJs are readily accessible, and research into conditions such as amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), and spinal muscular atrophy (SMA) has relied heavily on experimental work in these small mammals. However, given that nerve length dependency is an important feature of many peripheral neuropathies, these rodent models have clear shortcomings; large animal models might be preferable, but their size presents novel anatomical challenges. Overcoming these constraints to study the NMJ morphology of large mammalian distal limb muscles is of prime importance to increase cross-species translational neuromuscular research potential, particularly in the study of long motor units. In the past, NMJ phenotype analysis of large muscle bodies within the equine distal pelvic limb, such as the tibialis cranialis, or within muscles of high fibrous content, such as the soleus, has posed a distinct experimental hurdle. We optimised a technique for NMJ location and dissection from equine pelvic limb muscles. Using a quantification method validated in smaller species, we demonstrate their morphology and show that equine NMJs can be reliably dissected, stained and analysed. We reveal that the NMJs within the equine soleus have distinctly different morphologies when compared to the extensor digitorum longus and tibialis cranialis muscles. Overall, we demonstrate that equine distal pelvic limb muscles can be regionally dissected, with samples whole-mounted and their innervation patterns visualised. These methods will allow the localisation and analysis of neuromuscular junctions within the muscle bodies of large mammals to identify neuroanatomical and neuropathological features.

Cultured dissociated primary dorsal root ganglion neurons from adult horses enable study of axonal transport.

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival.

Identification and validation of genetic variants predictive of gait in standardbred horses.

Several horse breeds have been specifically selected for the ability to exhibit alternative patterns of locomotion, or gaits. A premature stop codon in the gene DMRT3 is permissive for "gaitedness" across breeds. However, this mutation is nearly fixed in both American Standardbred trotters and pacers, which perform a diagonal and lateral gait, respectively, during harness racing. This suggests that modifying alleles must influence the preferred gait at racing speeds in these populations. A genome-wide association analysis for the ability to pace was performed in 542 Standardbred horses (n = 176 pacers, n = 366 trotters) with genotype data imputed to ~74,000 single nucleotide polymorphisms (SNPs). Nineteen SNPs on nine chromosomes (ECA1, 2, 6, 9, 17, 19, 23, 25, 31) reached genome-wide significance (p < 1.44 x 10-6). Variant discovery in regions of interest was carried out via whole-genome sequencing. A set of 303 variants from 22 chromosomes with putative modifying effects on gait was genotyped in 659 Standardbreds (n = 231 pacers, n = 428 trotters) using a high-throughput assay. Random forest classification analysis resulted in an out-of-box error rate of 0.61%. A conditional inference tree algorithm containing seven SNPs predicted status as a pacer or trotter with 99.1% accuracy and subsequently performed with 99.4% accuracy in an independently sampled population of 166 Standardbreds (n = 83 pacers, n = 83 trotters). This highly accurate algorithm could be used by owners/trainers to identify Standardbred horses with the potential to race as pacers or as trotters, according to the genotype identified, prior to initiating training and would enable fine-tuning of breeding programs with designed matings. Additional work is needed to determine both the algorithm's utility in other gaited breeds and whether any of the predictive SNPs play a physiologically functional role in the tendency to pace or tag true functional alleles.

Species-specific consequences of an E40K missense mutation in superoxide dismutase 1 (SOD1).

A glutamic acid to lysine (E40K) residue substitution in superoxide dismutase 1 (SOD1) is associated with canine degenerative myelopathy: the only naturally occurring large animal model of amyotrophic lateral sclerosis (ALS). The E40 residue is highly conserved across mammals, except the horse, which naturally carries the (dog mutant) K40 residue. Here we hypothesized that in vitro expression of mutant dog SOD1 would recapitulate features of human ALS (ie, SOD1 protein aggregation, reduced cell viability, perturbations in mitochondrial morphology and membrane potential, reduced ATP production, and increased superoxide ion levels); further, we hypothesized that an equivalent equine SOD1 variant would share similar perturbations in vitro, thereby explain horses' susceptibility to certain neurodegenerative diseases. As in human ALS, expression of mutant dog SOD1 was associated with statistically significant increased aggregate formation, raised superoxide levels (ROS), and altered mitochondrial morphology (increased branching (form factor)), when compared to wild-type dog SOD1-expressing cells. Similar deficits were not detected in cells expressing the equivalent horse SOD1 variant. Our data helps explain the ALS-associated cellular phenotype of dogs expressing the mutant SOD1 protein and reveals that species-specific sequence conservation does not necessarily predict pathogenicity. The work improves understanding of the etiopathogenesis of canine degenerative myelopathy.

Assessing pathological changes within the nucleus ambiguus of horses with recurrent laryngeal neuropathy: An extreme, length-dependent axonopathy.

INTRODUCTION: Equine recurrent laryngeal neuropathy (RLN) is a naturally occurring model of length-dependent axonopathy characterized by asymmetrical degeneration of recurrent laryngeal nerve axons (RLn). Distal RLn degeneration is marked, but it is unclear whether degeneration extends to include cell bodies (consistent with a neuronopathy). METHODS: With examiners blinded to RLN severity, brainstem location, and side, we examined correlations between RLN severity (assessed using left distal RLn myelinated axon count) and histopathological features (including chromatolysis and glial responses) in the nucleus ambiguus cell bodies, and myelinated axon count of the right distal RLn of 16 horses. RESULTS: RLN severity was not associated with RLn cell body number (P > .05), or degeneration. A positive correlation between the left and right distal RLn myelinated axon counts was identified (R2 = 0.57, P < .05). DISCUSSION: We confirm that RLN, a length-dependent distal axonopathy, occurs in the absence of detectable neuronopathy.

Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Energy turnover in mammalian skeletal muscle in contractions mimicking locomotion: effects of stimulus pattern on work, impulse and energetic cost and efficiency.

Active muscle performs various mechanical functions during locomotion: work output during shortening, work absorption when resisting (but not preventing) lengthening, and impulse (force-time integral) whenever there is active force. The energetic costs of these functions are important components in the energy budget during locomotion. We investigated how the pattern of stimulation and movement affects the mechanics and energetics of muscle fibre bundles isolated from wild rabbits (Oryctolagus cuniculus). The fibres were from muscles consisting of mainly fast-twitch, type 2 fibres. Fibre length was held constant (isometric) or a sinusoidal pattern of movement was imposed at a frequency similar to the stride frequency of running wild rabbits. Duty cycle (stimulation duration×movement frequency) and phase (timing of stimulation relative to movement) were varied. Work and impulse were measured as well as energy produced as heat. The sum of net work (work output-work input) and heat was taken as a measure of energetic cost. Maximum work output was produced with a long duty cycle and stimulation starting slightly before shortening, and was produced quite efficiently. However, efficiency was even higher with other stimulation patterns that produced less work. The highest impulse (considerably higher than isometric impulse) was produced when stimulation started while the muscle fibres were being lengthened. High impulse was produced very economically because of the low cost of producing force during lengthening. Thus, locomotion demanding high work, high impulse or economical work output or impulse requires a distinct pattern of stimulation and movement.

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems.

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes.

Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy.

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog's phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5' donor splice site of intron 50 in the CKCS breed.

Ultrasonography detects early laryngeal muscle atrophy in an equine neurectomy model.

INTRODUCTION: A unilateral neurectomy model was used to study the relationship between histologic and ultrasonographic tissue characteristics during muscle atrophy over time. METHODS: This investigation was an in vivo experimental study in an equine model (n = 28). Mean pixel intensity of ultrasonographic images was measured, a muscle appearance grade was assigned weekly, and muscles were harvested from 4 to 32 weeks. Minimum fiber diameter, fiber density per unit area, percent collagen, percent fat, and fiber type profile were measured from muscle cryosections and correlated with the ultrasonographic parameters. RESULTS: A significant relationship was identified between collagen content, minimum fiber diameter, and ultrasonographic muscle appearance by as early as 8 weeks. There was no apparent association between fat content of muscle and the ultrasonographic appearance of atrophy before 28 weeks. CONCLUSIONS: Early muscle atrophy before fatty infiltration is detectable with ultrasound. The effect of muscle collagen content on echointensity may be mediated by reduced fiber diameter.

Heritability of Recurrent Exertional Rhabdomyolysis in Standardbred and Thoroughbred Racehorses Derived From SNP Genotyping Data.

Recurrent exertional rhabdomyolysis (RER) in Thoroughbred and Standardbred racehorses is characterized by episodes of muscle rigidity and cell damage that often recur upon strenuous exercise. The objective was to evaluate the importance of genetic factors in RER by obtaining an unbiased estimate of heritability in cohorts of unrelated Thoroughbred and Standardbred racehorses. Four hundred ninety-one Thoroughbred and 196 Standardbred racehorses were genotyped with the 54K or 74K SNP genotyping arrays. Heritability was calculated from genome-wide SNP data with a mixed linear and Bayesian model, utilizing the standard genetic relationship matrix (GRM). Both the mixed linear and Bayesian models estimated heritability of RER in Thoroughbreds to be approximately 0.34 and in Standardbred racehorses to be approximately 0.45 after adjusting for disease prevalence and sex. To account for potential differences in the genetic architecture of the underlying causal variants, heritability estimates were adjusted based on linkage disequilibrium weighted kinship matrix, minor allele frequency and variant effect size, yielding heritability estimates that ranged between 0.41-0.46 (Thoroughbreds) and 0.39-0.49 (Standardbreds). In conclusion, between 34-46% and 39-49% of the variance in RER susceptibility in Thoroughbred and Standardbred racehorses, respectively, can be explained by the SNPs present on these 2 genotyping arrays, indicating that RER is moderately heritable. These data provide further rationale for the investigation of genetic mutations associated with RER susceptibility.

Genome-wide analysis reveals selection for important traits in domestic horse breeds.

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.

A standardised approach to quantifying activity in domestic dogs.

Objective assessment of activity via accelerometry can provide valuable insights into dog health and welfare. Common activity metrics involve using acceleration cut-points to group data into intensity categories and reporting the time spent in each category. Lack of consistency and transparency in cut-point derivation makes it difficult to compare findings between studies. We present an alternative metric for use in dogs: the acceleration threshold (as a fraction of standard gravity, 1 g = 9.81 m/s2) above which the animal's X most active minutes are accumulated (MXACC) over a 24-hour period. We report M2ACC, M30ACC and M60ACC data from a colony of healthy beagles (n = 6) aged 3-13 months. To ensure that reference values are applicable across a wider dog population, we incorporated labelled data from beagles and volunteer pet dogs (n = 16) of a variety of ages and breeds. The dogs' normal activity patterns were recorded at 200 Hz for 24 hours using collar-based Axivity-AX3 accelerometers. We calculated acceleration vector magnitude and MXACC metrics. Using labelled data from both beagles and pet dogs, we characterize the range of acceleration outputs exhibited enabling meaningful interpretation of MXACC. These metrics will help standardize measurement of canine activity and serve as outcome measures for veterinary and translational research.

Characterisation of phenotypic patterns in equine exercise-associated myopathies.

BACKGROUND: Equine exercise-associated myopathies are prevalent, clinically heterogeneous, generally idiopathic disorders characterised by episodes of myofibre damage that occur in association with exercise. Episodes are intermittent and vary within and between affected horses and across breeds. The aetiopathogenesis is often unclear; there might be multiple causes. Poor phenotypic characterisation hinders genetic and other disease analyses. OBJECTIVES: The aim of this study was to characterise phenotypic patterns across exercise-associated myopathies in horses. STUDY DESIGN: Historical cross-sectional study, with subsequent masked case-control validation study. METHODS: Historical clinical and histological features from muscle samples (n = 109) were used for k-means clustering and validated using principal components analysis and hierarchical clustering. For further validation, a blinded histological study (69 horses) was conducted comparing two phenotypic groups with selected controls and horses with histopathological features characterised by myofibrillar disruption. RESULTS: We identified two distinct broad phenotypes: a non-classic exercise-associated myopathy syndrome (EAMS) subtype was associated with practitioner-described signs of apparent muscle pain (p < 0.001), reluctance to move (10.85, p = 0.001), abnormal gait (p < 0.001), ataxia (p = 0.001) and paresis (p = 0.001); while a non-specific classic RER subtype was not uniquely associated with any particular variables. No histological differences were identified between subtypes in the validation study, and no identifying histopathological features for other equine myopathies identified in either subtype. MAIN LIMITATIONS: Lack of an independent validation population; small sample size of smaller identified subtypes; lack of positive control myofibrillar myopathy cases; case descriptions derived from multiple independent and unblinded practitioners. CONCLUSIONS: This is the first study using computational clustering methods to identify phenotypic patterns in equine exercise-associated myopathies, and suggests that differences in patterns of presenting clinical signs support multiple disease subtypes, with EAMS a novel subtype not previously described. Routine muscle histopathology was not helpful in sub-categorising the phenotypes in our population.

CONTEXTE: Les myopathies induites à l'exercice demeurent fréquentes, hétérogènes cliniquement et représentent des désordres idiopathiques caractérisés par des épisodes de dommages myofibrillaires en lien avec l'exercice. Les épisodes sont intermittents et varient à la fois chez le même cheval, entre chevaux et entre les différentes races. L'étiopathogénie demeure obscure et pourrait être multifactorielle. La pauvre caractérisation phénotypique des myopathies ne simplifie pas les analyses génétiques ni celles d'autres maladies. OBJECTIFS: Le but de cette étude est de caractériser les patrons phénotypiques en lien avec les myopathies induites à l'exercice chez le cheval. TYPE D'ÉTUDE: Étude transversale historique et étude subséquente de validation de cas témoins aveugle. MÉTHODES: Les facteurs clés cliniques et histologiques provenant d'échantillons de muscles (n = 109) ont été utilisés pour l'algorithme de K­moyennes et validés par le biais d'analyse des composantes principales et de classification hiérarchique. Pour validation additionnelle, une étude histologique à l'aveugle (69 chevaux) a été faite comparant les deux groupes phénotypiques avec des contrôles sélectionnés et des chevaux avec éléments histopathologiques caractérisés par de la discontinuité myofibrillaire. RÉSULTATS: Deux phénotypes distincts ont été identifiés: un premier sous­type de syndrome de myopathie induite à l'exercice non­classique (EAMS) associé à de la douleur musculaire telle que décrite par le praticien suivant le cheval (χ2 (df=1,n=109) = 19.33, p < 0.001), difficulté à se déplacer (χ2 (df=1,n=109) = 10.85, p = 0.001), démarche anormale (χ2 (df=1,n=109) = 34.61, p < 0.001), ataxie (χ2 (df=1,n=109) = 10.88, p = 0.001) et parésie (χ2 (df=1,n=109) = 10.88, p = 0.001); alors qu'un sous­type RER classique non­spécifique n'était associé à aucune variable en particulier. Aucune différente histologique n'a été identifié entre les sous­types dans l'étude de validation et aucune caractéristique histopathologique d'autres myopathies équines n'a été identifiées dans les différents sous­types. LIMITES PRINCIPALES: Aucune population indépendante pour validation; petite taille d'échantillon pour les sous­types peu nombreux identifiés; aucun cas contrôles positifs de myopathie fibrillaire; description des cas provenant de multiples praticiens indépendants et non­aveugles. CONCLUSION: Cette étude est la première utilisant des méthodes de regroupement informatique pour identifier des patrons phénotypiques de myopathies équines induites à l'exercice et suggère que des différences existent dans les patrons de signes cliniques en faveur de multiples sous­types de maladie, incluant EAMS qui représente un nouveau sous­type non décrit jusqu'à maintenant. L'histopathologie musculaire de routine n'a pas permis de sous­catégoriser les phénotypes dans cette population.

When Size Really Matters: The Eccentricities of Dystrophin Transcription and the Hazards of Quantifying mRNA from Very Long Genes.

At 2.3 megabases in length, the dystrophin gene is enormous: transcription of a single mRNA requires approximately 16 h. Principally expressed in skeletal muscle, the dystrophin protein product protects the muscle sarcolemma against contraction-induced injury, and dystrophin deficiency results in the fatal muscle-wasting disease, Duchenne muscular dystrophy. This gene is thus of key clinical interest, and therapeutic strategies aimed at eliciting dystrophin restoration require quantitative analysis of its expression. Approaches for quantifying dystrophin at the protein level are well-established, however study at the mRNA level warrants closer scrutiny: measured expression values differ in a sequence-dependent fashion, with significant consequences for data interpretation. In this manuscript, we discuss these nuances of expression and present evidence to support a transcriptional model whereby the long transcription time is coupled to a short mature mRNA half-life, with dystrophin transcripts being predominantly nascent as a consequence. We explore the effects of such a model on cellular transcriptional dynamics and then discuss key implications for the study of dystrophin gene expression, focusing on both conventional (qPCR) and next-gen (RNAseq) approaches.