F.L. Robinson1; A.P. Misizin1; D.P. O'Brien2; G.S. Johnson2; J.E. Dixon1; G.D. Shelton1
Three male Manchester terrier littermates were presented at 2 months of age for weakness and failure to thrive. The dam and sire were clinically normal. The dam was previously bred to a different male with 2 male puppies showing similar clinical signs. Muscle biopsy specimens from all three puppies showed centrally placed nuclei resembling fetal myotubes, or a central clear zone, within most type 1 fibers. Cryostat sections reacted for oxidative enzymes showed attenuated activity at the periphery of the fibers, and dense oxidative staining at the center of the fibers, which lacked myosin ATPase activity. Ultrastructural analysis correlated well with the histochemical findings including myofilaments forming a compact peripheral rim around a central nucleus, or around a central zone, containing mitochondria, glycogen, or dilated membranous profiles. In humans, recessive mutations in the myotubularin (MTM1) gene cause X-linked myotubular myopathy. MTM1 is a member of a family of phosphoinositide 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. The canine MTM1 gene also resides on the X chromosome. Accordingly, MTM1 was investigated as a possible gene responsible for the described myotubular myopathy in Manchester Terriers. MTM1 protein was analyzed in affected and normal dog muscle. MTM1 protein in detergent extracts was immunoprecipitated with an anti-MTM1 antibody (Santa Cruz Biotechnology) and protein-A agarose, then analyzed by SDS-PAGE and immunoblotting using an anti-MTM1 antibody. MTM1 protein was not detected in the muscle extracts from affected dogs. MTM1 mRNA analysis of muscle was performed by RT-PCR. Exon 1 containing mRNA was not detected in affected dogs. In conclusion, myotubular myopathy in Manchester Terrier dogs is clinically, histologically, ultrastructurally, and biochemically similar to that of human X-linked myotubular myopathy. The absence of MTM1 protein and exon 1 containing mRNA in muscle tissue of affected dogs suggests that loss of function mutations in this PI 3-phosphatase may be the causative genetic defect. Preliminary data suggests a mutation in exon 1 of the MTM1 gene.
Originally presented at the World Muscle Society Meeting, Giardini-Naxos, Taormina, Sicily, October 2007.