Allele copy number and underlying pathology are associated with subclinical severity in equine type 1 polysaccharide storage myopathy (PSSM1)

Abstract

Equine type 1 polysaccharide storage myopathy (PSSM1), a common glycogenosis associated with an R309H founder mutation in the glycogen synthase 1 gene (GYS1), shares pathological features with several human myopathies. In common with related human disorders, the pathogenesis remains unclear in particular, the marked phenotypic variability between affected animals. Given that affected animals accumulate glycogen and alpha-crystalline polysaccharide within their muscles, it is possible that physical disruption associated with the presence of this material could exacerbate the phenotype. The aim of this study was to compare the histopathological changes in horses with PSSM1, and specifically, to investigate the hypothesis that the severity of underlying pathology, (e.g. vacuolation and inclusion formation) would (1) be higher in homozygotes than heterozygotes and (2) correlate with clinical severity. Resting and post-exercise plasma creatine kinase (CK) and aspartate aminotransferase (AST) enzyme activity measurements and muscle pathology were assessed in matched cohorts of PSSM1 homozygotes, heterozygotes or control horses. Median (interquartile range (IR)) resting CK activities were 364 (332-764) U/L for homozygotes, 301 (222-377) U/L for heterozygotes and 260 (216-320) U/L for controls, and mean (+/- SD) AST activity for homozygotes were 502 (+/116) U/L, for heterozygotes, 357 (+/-92) U/L and for controls, 311 (+/-64) U/L and were significantly different between groups (P = 0.04 and P = 0.01 respectively). Resting plasma AST activity was significantly associated with the severity of subsarcolemmal vacuolation (rho = 0.816; P = 0.01) and cytoplasmic inclusions (rho = 0.766; P = 0.01). There were fewer type 2× and more type 2a muscle fibres in PSSM1-affected horses. Our results indicate that PSSM1 has incomplete dominance. Furthermore, the association between plasma muscle enzyme activity and severity of underlying pathology suggests that physical disruption of myofibres may contribute to the myopathic phenotype. This work provides insight into PSSM1 pathogenesis and has implications for related human glycogenoses.

Figure 1. The effect of GYS1 genotype on skeletal muscle histopathology.

a–d: Boxplots illustrating the percentage of muscle fibres containing (a) internalised nuclei, (b) subsarcolemmal vacuoles, (c) cytoplasmic inclusions and (d) diastase resistant inclusions in horses homozygous (HH) and heterozygous (HR) for GYS1 mutation and controls (RR). * Denotes significant differences between individual groups (p<0.05), ** (p<0.01), following post- hoc analysis. e–h: Representative images of the pathology (arrows) identified (e) internalized nuclei, (f) subsarcolemmal vacuoles, (g) cytoplasmic inclusions and (h) diastase resistant inclusions (e–g: haematoxylin and eosin; h: Periodic acid Schiff following diastase digestion). Bar = 50 µm.

Figure 2. Dystrophin localisation.

Representative images of dystrophin immunohistochemistry (brown staining) in skeletal muscle counterstained with haematoxylin and eosin from (A) a control horse and (B) a horse heterozygous for the GYS1 mutation. Note the normal dystrophin expression and localisation in the region of the subsarcolemmal vacuoles (arrows). Bar = 50 µm.

Figure 3. Muscle fibre type distributions.

Boxplots illustrating the muscle fibre type composition of horses homozygous (HH) (n = 4), heterozygous (HR) (n = 8) and control horses (RR) (n = 6). * Denotes significant differences between individual groups (p<0.05) following post- hoc analysis.

Figure 4. Resting muscle enzyme activities.

Box plots illustrating the resting (a) CK activity and (b) AST activity for each GYS1 genotype (n = 8) (HH = homozygotes, HR = heterozygote, RR = control) * Denotes significant differences between individual groups (p<0.05) following post- hoc analysis.

Figure 5. Pre- and post exercise muscle enzyme activites.

Graph illustrating the pre and post exercise (a) CK activity and (b) AST activity for each GYS1 genotype (n = 4) (HH = homozygotes, HR = heterozygote, RR = control).

Figure 6. The correlation between muscle enzyme activity and muscle histopathology.

Scattergraphs illustrating the correlation between the Log¹⁰ resting CK activity (A) and Log¹⁰ AST activity (B) and the percentage of fibres containing subsarcolemmal vacuoles. (HH = triangles, HR = stars, RR = circles) (n = 18).