G.P.12.04 A missense variant in the MTM1 gene associated with X-linked myotubular myopathy in Labrador retrievers

Mutations in the MTM1 gene, encoding myotubularin, cause X-linked myotubular myopathy, a well-defined subgroup of centronuclear myopathies in humans. Five male Labrador retrievers from 3 to 4 months of age, and from three apparently unrelated litters, were evaluated for generalized weakness and muscle atrophy. All dogs tested for the known centronuclear myopathy (CNM) mutation in the PTPLA gene were negative. Cryostat sections of muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas. The subsarcolemmal and central dense areas stained darkly positive with the mitochondrial specific reactions for SDH and COX allowing histologic differentiation from Labrador retrievers with CNM. Immunoblot analysis using an anti-myotubularin antibody (Santa Cruz, Biotechnology) showed that myotubularin protein was absent in muscle extracts from affected dogs but present in muscle from a control littermate. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure and mitochondrial accumulations. Triads were infrequent with an abnormal orientation of T tubules. Immunofluorescence staining of frozen muscle sections using antibodies against T tubules (DHPRa1, Abcam) and adjacent sarcoplasmic reticulum (RYR1, Abcam) confirmed an abnormal distribution of these structures. DNA analysis of exonic sequences from the MTM1 gene in all five affected males revealed a unique variant in exon 7 causing the non-conservative missense change N155K in the linker region between the GRAM-PH and phosphatase domains of myotubularin. Two proven carrier dams from one of these kindreds were shown to be heterozygous for the mutation. Analysis of a world-wide panel of 237 unrelated and unaffected Labrador retrievers, and 59 additional control dogs from 25 breeds, failed to identify this variant, suggesting it is the pathogenic mutation responsible for the myopathy in these dogs.