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. 2008 Dec;18(12):942-52.
doi: 10.1016/j.nmd.2008.08.002. Epub 2008 Nov 5.

Muscular dystrophy associated with alpha-dystroglycan deficiency in Sphynx and Devon Rex cats

Paul T Martin  1 , G Diane Shelton, Peter J Dickinson, Beverly K Sturges, Rui Xu, Richard A LeCouteur, Ling T Guo, Robert A Grahn, Harriet P Lo, Kathryn N North, Richard Malik, Eva Engvall, Leslie A Lyons
Affiliations

Muscular dystrophy associated with alpha-dystroglycan deficiency in Sphynx and Devon Rex cats

Paul T Martin et al. Neuromuscul Disord. 2008 Dec.

Abstract

Recent studies have identified a number of forms of muscular dystrophy, termed dystroglycanopathies, which are associated with loss of natively glycosylated alpha-dystroglycan. Here we identify a new animal model for this class of disorders in Sphynx and Devon Rex cats. Affected cats displayed a slowly progressive myopathy with clinical and histologic hallmarks of muscular dystrophy including skeletal muscle weakness with no involvement of peripheral nerves or CNS. Skeletal muscles had myopathic features and reduced expression of alpha-dystroglycan, while beta-dystroglycan, sarcoglycans, and dystrophin were expressed at normal levels. In the Sphynx cat, analysis of laminin and lectin binding capacity demonstrated no loss in overall glycosylation or ligand binding for the alpha-dystroglycan protein, only a loss of protein expression. A reduction in laminin-alpha2 expression in the basal lamina surrounding skeletal myofibers was also observed. Sequence analysis of translated regions of the feline dystroglycan gene (DAG1) in affected cats did not identify a causative mutation, and levels of DAG1 mRNA determined by real-time QRT-PCR did not differ significantly from normal controls. Reduction in the levels of glycosylated alpha-dystroglycan by immunoblot was also identified in an affected Devon Rex cat. These data suggest that muscular dystrophy in Sphynx and Devon Rex cats results from a deficiency in alpha-dystroglycan protein expression, and as such may represent a new type of dystroglycanopathy where expression, but not glycosylation, is affected.

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Figures

Figure 1
Figure 1. Sphynx cats with muscular dystrophy showing postures consistent with generalized muscle weakness
(A) dorsal protrusion of the scapulae, (B) “chipmunk” or “squirreling” posture, and (C) ventroflexion of the head and neck.
Figure 2
Figure 2. Muscle biopsies from dystrophic Sphynx cats show only mild myopathic changes
Pathological changes include variability in myofiber size (all figures) and a type 1 fiber predominance (C). A: H&E; B: Modified Gomori trichrome; C: NADH dehydrogenase; D: Periodic acid-Schiff. Bar = 50 μm for all figures.
Figure 3
Figure 3. Immunofluorescence staining of frozen muscle biopsy sections from a dystrophic Sphynx cat showed decreased staining for laminin α-2 and increased staining for utrophin compared to a control cat
Staining intensity with monoclonal antibodies against the rod (DYS1) and carboxy terminus (DYS2) of dystrophin, β-sarcoglycan (β-SG), β-dystroglycan (β-DG), and spectrin in the Sphynx cat (S) was similar to control (C) muscle. Staining for laminin α-2 (Lama2) was decreased while staining for utrophin (UTR) was increased in the dystrophic Sphynx cat compared to control muscle. Bar = 50 μm.
Figure 4
Figure 4. Compared to staining in normal cats, staining for natively glycosylated α-dystroglycan was decreased
Immunofluorescence staining of fresh frozen muscle biopsy sections from a dystrophic Sphynx cat, and control (mixed breed and normal Devon Rex) cats was compared with antibodies against natively glycosylated α-dystroglycan (IIH6 anti-carbohydrate antibody). Bar = 50 μm.
Figure 5
Figure 5. Protein expression of natively glycosylated α-dystroglycan is reduced in Sphynx and Devon Rex cats
A. Muscle protein lysate from a Sphynx cat (S) and a normal (N) cat were compared by immunoblotting. Levels of dystrophin (DYS), laminin α2 (Lamα2), β dystroglycan (βDG), α-δ sarcoglycan (α-δSG), desmin (DES), and dystrobrevin (DB) were similar in both samples, while the amount of native α-dystroglycan (αDG, recognized by the IIH6 anti-carbohydrate antibody) was reduced and the level of utrophin (UTR) was increased. Some increase in β sarcoglycan may also have occurred, although this signal was very weak on immunoblots. Tentative molecular weights are 140 kDa and 120 kDa for α- DG, 43 kDa for β-DG, 50-55 kDa for desmin, 55 kDa for α- SG, 43 kDa for β-SG, 35 and 90 kDa for γ-SG, 50 kDa for δ-SG, 350 kDa for laminin-α2, 350-400 kDa for dystrophin and utrophin, and 80-100 kDa and 35-50 kDa for dystrobrevin. B. Muscle proteins from the affected Devon Rex (D) and control (C) cats were compared by immunoblot analysis. By densitometry, amounts of native α-DG (recognized by the VIA4-1 antibody) was reduced approximately 29% in the Devon Rex cat compared to control. This antibody also shows a high level of cross-reactivity with myosin heavy chain. By comparison, densitometry showed only a small decrease in levels of laminin-α2. Coomassie blue staining of actin demonstrates relative loading of skeletal muscle protein.
Figure 6
Figure 6. Relative expression of α-dystroglycan to β-dystroglycan and to total cell protein was reduced in Sphynx cat skeletal muscle
Different amounts of total muscle cell lysates were loaded to compare α- and β-dystroglycan expression by immunoblotting. An antibody that recognizes natively glycosylated α-dystroglycan (IIH6) and one that recognizes α-dystroglycan polypeptide (DG3) were used, and laminin-1 binding to α-dystroglycan was visualized by laminin overlay. Levels of α-dystroglycan, both by IIH6 and DG3 immunoblotting, were lower in Sphynx cat skeletal muscle than in normal controls (C), as was laminin binding to α dystroglycan, when comparing equivalent amounts of loaded protein (20ug). Increasing the amount of Sphynx protein lysate loaded increased IIH6 blotting and laminin binding to levels beyond wild type. α-dystroglycan in normal cat skeletal muscle migrates about 20kDa lower than α-dystroglycan in mouse skeletal muscle.
Figure 7
Figure 7. Lectin precipitation profile of α dystroglycan did not differ between Sphynx and normal cat skeletal muscle
Skeletal muscle protein lysates were normalized to allow for precipitation of equivalent amounts of α-dystroglycan between control (C) and Sphynx (S) samples. Lysates were then subjected to lectin precipitation followed by immunoblotting for native (glycosylated) α-dystroglycan (IIH6) or β-dystroglycan. Laminin overlays were done on precipitated protein to visualize laminin binding to α-dystroglycan. ConA, PNA, and WGA precipitated α-dystroglycan well. VVA-B4 showed intermediate levels, while LCA and MAA showed poor precipitation. The profile of α-dystroglycan binding to lectins, and also of laminin binding of precipitated α-dystroglycan protein, was unchanged between Sphynx and normal cat muscle.
Figure 8
Figure 8. Expression of DAG1 mRNA in muscle biopsy specimens did not differ between dystrophic Sphynx cats and controls
The top and bottom edge of the box plot represent the upper and lower quartile respectively. The line within the box represents the median. The tails extend to the farthest point that is within 1.5 interquartile ranges of the quartiles. DAG1 expression levels varied markedly between individual muscle samples, although DAG1 mRNA was present in all affected Sphynx muscle samples and no statistical difference in expression level was seen between control (5 control cats: 3 samples from each cat) and two Sphynx (3 samples from each Sphynx) cats (p=0.57).
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