Genomics of muscle disorders

Eventual sequencing of the GBE1 gene coding sequences resulted in the discovery of a mutation in exon 1 that results in a premature stop codon (Ward et al., 2004). GBED was then demonstrated to be an autosomal recessive disease currently known only to affect Quarter Horse and Paint Horse breeds. Glycogen is a required energy source in the rapidly growing fetus and neonate and is synthesized by glycogen synthase, which creates straight chains of glucose with alpha 1,4-glycosidic linkages, and by glycogen branching enzyme, which creates a branched structure through alpha 1,6-linkages. It is likely that the GBE1 mutation and lack of GBE enzymatic activity results in cardiac and skeletal muscle, liver, and brain being unable to store and mobilize glycogen to maintain normal glucose homeostasis.

Carrier frequency estimates of 7.1% and 8.3% in the Paint and Quarter Horse breeds, respectively, have been reported (Wagner et al., 2006), which, along with the tracing of pedigrees of carriers back to founders of the breed in the early 1900s, would indicate that many GBED foals are, or should be, born every year. However, clinically, the number of GBED foals born alive appears less than expected. After the discovery of the GBE1 mutation, genotyping of foals that were aborted or stillborn due to unknown causes demonstrated that up to 5% of such cases were the result of homozygosity for the GBE1 mutation (Render et al., 1999; Valberg et al., 2001; Wagner et al., 2006). Thus, abortion may be the primary clinical presentation for GBED. Within Quarter Horse performance horse types, Western Pleasure horses have the highest prevalence of carriers of GBED, with 28% of horses being heterozygous (Tryon et al., 2009).

Polysaccharide Storage Myopathy Type 1

In 1932, Carlstrom recognized that episodes of rhabdomyolysis in Draft Horses were associated with increased glycogen storage; however, this condition was not yet recognized as a specific disease (Carlstrom, 1932). Polysaccharide storage myopathy (PSSM) in Quarter Horses, also characterized by increased muscle glycogen concentration, abnormal polysaccharide accumulation, and rhabdomyolysis, was first described in 1992 (Valberg et al., 1992). Subsequently, a similar combination of findings was also recognized in Belgian and Percheron horses (Valentine et al., 1997; Firshman et al., 2005).

PSSM in any breed of horse has been diagnosed primarily by the histological demonstration of periodic acid Schiff’s (PAS) positive inclusions of abnormal polysaccharide in as few as one or two fibers, to as many as 30% of the Type II skeletal muscle fibers in a fresh-frozen biopsy (McCue, Ribiero, & Valberg, 2006). Controversy existed as to whether diagnostic criteria for abnormal-appearing polysaccharide should be restricted to amylase-resistant polysaccharide or should also include increased amylase-sensitive glycogen (Figure 11.2). Abnormal polysaccharide has been described in the cardiac muscle of a severely affected Quarter Horse and a Belgian horse; however, it is not considered a typical feature of the disease (Valentine et al., 1997; Annandale et al., 2004). No histological abnormalities have been found in the liver or adipose tissue of affected horses.

Figure 11.2 PSSM Types 1 and 2 distinguished by PAS staining of fresh-frozen muscle biopsy sections with and without amylase pre-incubation. Note abnormal dark-staining PAS-positive material. This material in Type 1 PSSM has a more aggregated and crystalline appearance throughout the sarcoplasm (2A), while in Type 2 PSSM this material is located more under the sarcolemma (1A). The PAS-positive material is typically removed by amylase digestion in Type 2 PSSM (1B), but not Type 1 PSSM (2B).


Environmental factors clearly play a role in the clinical phenotype of horses with PSSM. Providing dietary fat as an alternative energy substrate to carbohydrate, limiting the daily consumption of starch and sugar, and gradually increasing daily exercise typically decrease muscle stiffness and eliminate episodes of ER in Quarter Horses with PSSM (Firshman et al., 2003; Ribiero et al., 2004). Phenotypic variability is also readily apparent between breeds, with PSSM Quarter Horses having a higher frequency of ER but less likely to have gait abnormalities than Draft Horses; however, Draft Horses are more likely to have muscle atrophy than Quarter Horses. Warmbloods with PSSM frequently present with a gait abnormality and lack of impulsion with infrequent episodes of rhabdomyolysis. Thus, Draft Horses and Quarter Horses have similar histopathological features of a glycogenosis but differences in clinical signs.

Considerable evidence indicated that a form of PSSM in Quarter Horses was highly heritable (Valberg et al., 1996). A genome-wide scanning approach was therefore considered possible, and at that time increasingly feasible. A study with 105 microsatellite markers spread across the genome enabled the identification of a region on ECA10 associated with PSSM in a population of 48 PSSM Quarter Horses descended from a founder stallion and 48 controls (McCue et al., 2008a). Following fine-mapping with local microsatellites and validation in an unrelated population, the skeletal muscle glycogen synthase gene (GYS1) was clearly the most plausible positional candidate gene. Sequence analysis of the entire coding region, as well as the 5′ and 3′ UTRs of the GYS1 gene, revealed a single polymorphism in exon 6 that changes the normal arginine (R) codon (CGT) to a histidine (H) codon (CAT). This polymorphism is at amino acid residue 309 of the 737 amino acid equine GS protein. Almost the entire amino acid sequence of exon 6 is conserved in sequenced vertebrate GYS1 genes, suggesting both an essential role in glycogen synthase function and the plausibility that this polymorphism is the causative PSSM mutation. Segregation of the H allele consistent with autosomal dominant inheritance was confirmed in a Quarter Horse herd developed as a controlled breeding trial (McCue, 2008a). The activity of the glycogen synthase enzyme was found to be increased both without and with the allosteric activator glucose 6 phosphate, indicating this is a gain of function mutation (McCue, 2008a). Subsequently, it has become clear that there is a reduced penetrance of the H allele in herds across the United States, consistent with the known environmental influences on disease expression. In any event, discovery of this disease involving a well-known gene, for which no naturally occurring gain of function mutations had previously been found, demonstrates the power of genetic studies in horses to define new mechanisms of muscle disease. It is interesting that two of the mutations found to date in Quarter Horses (GBED and PSSM1) involve the synthesis of glycogen.

Glycogen synthase catalyzes the rate-limiting step in muscle glycogen synthesis and is under stringent covalent and allosteric regulation (Pederson et al., 2000). Several possibilities exist as to how an Arg309His substitution would result in gain of muscle GS function and excessive glycogen accumulation. Perhaps the best possibility is that the mutant enzyme is resistant to negative regulation, or more sensitive to positive regulation, due either to altered phosphorylation/dephosphorylation by a multitude of protein kinases and phosphatases, altered affinity for its substrates (UDP-glucose and glycogen), or altered allosteric effects resulting from glucose 6-phosphate binding (Figure 11.3). At this time we know only that maximal GS activity assayed by the incorporation of 14C UDP-Glucose into glycogen is significantly higher in PSSM than in control muscle homogenates in both the presence and the absence of a maximally activating concentration of glucose 6-phosphate (McCue et al., 2008a).

Figure 11.3 Regulation of glycogen synthase activity by glucose 6 phosphate and phosphorylation. Inserts represent the kinetics of synthase activity in relation to its UDP-glucose substrate in each state. The solid line is the phosphorylated enzyme, and the dashed line is the dephosphorylated enzyme. Adapted from Pederson et al. (2000).


A number of studies have described physiological and biochemical effects of the mutation in Quarter Horses. For example, during maximal exercise tests, Quarter Horses with PSSM deplete 26% of the resting glycogen concentration and accumulate twice as much muscle lactate as control horses (Valberg et al., 1999). Thus, excessive glycogen accumulation in horses with PSSM is not due to an inability to utilize glycogen, and these horses actually have a higher rate of glycolysis than normal horses with maximal exertion. Nevertheless, horses with PSSM do have exercise intolerance and a cellular energy deficit with submaximal exercise (Valberg, Townsend, & Mickelson, 1998; Valberg et al., 1999). When ATP levels in muscle cannot be effectively restored by metabolic pathways, the myokinase reaction increasingly is used to produce ATP and AMP from ADP. AMP is then increasingly degraded to IMP by AMP deaminase. During submaximal exercise tests designed to mimic low workload in normal horses, lactate, ATP, ADP, and AMP concentrations are not significantly different in normal and PSSM horses; however, IMP concentrations are significantly elevated in horses with PSSM (Annandale, Valberg, & Essen Gustavsson, 2005). Premature degradation of adenine nucleotides to IMP in PSSM horses may be caused by abnormal regulation of the flux of substrates into aerobic metabolism.

Another approach to define the cellular responses involved in PSSM utilized a microarray containing ∼750 oligonucleotide probes to measure expression of a subset of genes from the skeletal muscle of PSSM (GYS1 mutation heterozygous and homozygotes) and control Normand Cob horses. The analysis revealed 129 genes significantly modulated, with 16 genes up-regulated over 1.5-fold and 37 genes down-regulated over 1.5-fold. Data mining showed that protein synthesis, apoptosis, cellular movement, growth, and proliferation were the main cellular functions significantly associated with the modulated genes. The authors concluded that PSSM mitochondrial dysfunction, glycogenesis inhibition, and chronic hypoxia may contribute to the PSSM disease process (Barrey et al., 2009).

A number of recent studies have identified the GYS1 mutation in a large number of breeds in both North America and Europe (Herszberg et al., 2008; Stanley et al., 2009; Baird et al., 2010; McCue et al., 2010). Many Draft breeds originating in continental Europe are at particular risk, especially breeds that are related to the original Belgian breed. The GYS1 genotype distribution in Quarter Horses and Paint Horses is similar at 6–7% heterozygous or homozygous for the H allele. However, approximately 33% of Belgians and more than 50% of Percherons are either heterozygous or homozygous for the H allele. Assuming a dominant inheritance, this means that approximately 7% of all Quarter Horses and Paint horses, 33% of all Belgians, and 50% of all Percherons are actually genetically susceptible to PSSM. The prevalence of the GYS1 mutation is very low in athletic light breeds such as Arabians, Thoroughbreds, and Standardbreds and relatively low in horses diagnosed with polysaccharide storage myopathy on the basis of amylase-sensitive accumulation of glycogen (McCue et al., 2008b).

We now know that there are likely multiple forms of PSSM that can be present to varying extents in different breeds, with potentially different penetrance and different clinical signs due to different genetic backgrounds. Phenotypic variability in the clinical presentation among horses with PSSM is also explained, at least in some instances, by a modifying gene or genes. Horses in several of the Type 1 PSSM Quarter Horse families used in the original microsatellite genome scan had a severe and occasionally fatal PSSM phenotype (McCue et al., 2008a). That same genetic association analysis was able to identify microsatellite markers on another segment of ECA10 as being associated with the severe PSSM phenotype in these families, but not in other, less severely affected families (McCue et al., 2009b). The ECA10 segment coincided with the location of the RYR1 gene, in which the known equine RYR1 mutation (Aleman et al., 2004) was also found to segregate in this family. Retrospective analysis of muscle biopsy submissions and a controlled exercise trial demonstrated that horses with both the GYS1 and RYR1 mutations had a more severe clinical phenotype than horses with the GYS1 mutation alone (McCue et al., 2009b).

Polysaccharide Storage Myopathy Type 2

Approximately 20% of the Quarter Horses, 11% of Belgians, and 80% of Warmbloods with abnormal polysaccharide in muscle biopsies and clinical signs of PSSM examined thus far do not carry the GYS1 H allele. Although it is possible that some horses receive a false-positive diagnosis of PSSM based on histopathology and clinical signs, it is far more likely that a population of horses with PSSM have separate, distinct glycogenosis(es) resulting from different molecular and cellular causes (McCue et al., 2008a; McCue et al., 2008b). This possibility has been examined in some detail in Quarter Horses. To distinguish the two forms of PSSM, we have proposed that the term “Type 1 PSSM” be used for horses that possess the GYS1 mutation and “Type 2 PSSM” be used to distinguish the group of horses with abnormal polysaccharide in muscle biopsies that do not have the GYS1 mutation (McCue et al., 2008b).

PSSM Types 1 and 2 can be histochemically and clinically distinguished by appropriate analyses (McCue et al., 2008b). The abnormal polysaccharide in Type 1 PSSM is often amylase-resistant and coarse granular in appearance and is typically located in the cytoplasm, whereas in Type 2 PSSM it is often amylase-sensitive, fine granular/ homogeneous in appearance, and located under the sarcolemma (Figure 11.2). Both forms of PSSM occur in young horses, but a high percentage of horses with Type 2 PSSM are younger than 1 year old. In contrast, the mean age of GYS1-positive horses with PSSM was greater than GYS1-negative PSSM, and less than 10% of Type 1 horses with PSSM were younger than 1 year old. Lastly, horses with Type 2 PSSM more commonly presented with an obscure or undiagnosed gait abnormality compared to horses with Type 1 PSSM.

The similarity in clinical presentation between the two forms of PSSM should not be unexpected as it is consistent with findings in other species. There are 11 known inherited glycogenoses affecting skeletal muscle in humans that can cause rhabdomyolysis, pain with exertion, muscle cramping, and myoglobinuria or fixed, progressive weakness (DiMauro & Lamperti, 2001; DiMauro, Hayes, & Tsujino, 2004). These clinical characteristics are shared by GYS1-positive and GYS1-negative horses with PSSM. A more thorough investigation of pedigrees, clinical history, clinical findings, and muscle biochemistry should help determine if there is a familial basis for GYS1-negative PSSM and what are the major distinguishing clinical and biochemical characteristics. Whole genome association studies with the EquineSNP50 BeadChip are also currently underway to identify significantly associated SNPs with Type 2 PSSM in Quarter Horses.

Recurrent Exertional Rhabdomyolysis

While ER is observed in many breeds of horse, the condition of recurrent exertional rhabdomyolysis (RER) is likely the most common form of tying-up and the most important muscular disorder of Thoroughbred horses (Valberg et al., 1992). About 5–10% of all Thoroughbreds in the United States and the United Kingdom develop ER at some point during a racing season, up to 75% of trainers have at least one horse with RER, and recurrence is so frequent in 17% of the affected horses that they do not race again that season (MacLeay et al., 1999a; McGowan, Fordham, & Christley, 2002). An increased prevalence of RER in young nervous fillies has been consistently identified. RER is clearly distinct from sporadic forms of ER that occur in otherwise healthy athletic horses due to overexertion, electrolyte depletion, or a dietary excess of carbohydrates. When horses with RER become riding horses later in life, they often have subtle signs of sore muscles, which, in our experience, results from RER.

Efforts have been made to determine if specific cellular or molecular defects can be associated with RER, and to explain the clinical findings of muscle cramping and rhabdomyolysis. Muscle histopathology in RER Thoroughbreds is distinguished only by the characteristic but nonspecific finding of increased numbers of central nuclei in horses with active clinical disease (Lentz et al., 1999). However, an in vitro alteration in the regulation of muscle contractility in RER horses has been demonstrated with intact electrically stimulated tendon-to-tendon muscle fiber bundles, dissected from surgically removed external intercostal muscles (Lentz et al., 1999). These preparations demonstrated a significantly increased sensitivity of RER muscles to the development of potassium-, caffeine- or halothane-induced contractures; all three types of treatment stimulate the release of calcium from the sarcoplasmic reticulum via the calcium release channel (Figure 11.4). In addition, a significantly increased rate of relaxation during a twitch was noted in the RER muscles (Figure 11.4). The fact that altered muscle contractility in RER could be recapitulated in vitro suggests an intrinsic muscle defect. This hypothesis was further supported by the observation that muscle cells grown in cell culture as myotubes displayed higher myoplasmic Ca2+concentrations in response to caffeine (Lentz et al., 2002). It has also been demonstrated that the increased contracture sensitivity of RER muscles is not due to an enhanced Ca2+sensitivity of the contractile apparatus (Mlekoday et al., 2001), and there was no support for altered activity of the sarcoplasmic reticulum Ca2+release channel or Ca2+-ATPase (Ward et al., 2000). An approach that uses a vector to force expression of MyoD and transform equine fibroblasts into myogenic cells that fuse to form myotubes in cell culture offers an exciting alternative approach to defining the possible RER muscle defects at a cellular level (Fernandez-Fuente et al., 2008).

Figure 11.4 Abnormal sensitivity to in vitro contracture of muscle fiber bundles from RER-susceptible Thoroughbreds: (left) Representative twitch force records of muscle bundles from normal and RER susceptible Thoroughbreds. Note the faster return of the force to baseline in the RER muscle indicating a faster rate of relaxation. (right) Contracture force versus caffeine concentration for muscle bundles from normal and RER susceptible Thoroughbreds. Note the significantly greater contracture forces produced at lower caffeine concentrations in RER muscle, compared to normal, indicating an increased sensitivity to contracture. Adapted from Lentz et al. (1999).


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Jul 9, 2017 | Posted by in EQUINE MEDICINE | Comments Off on Genomics of muscle disorders

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