MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers

Abstract

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14-26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

(A) A 4-mo-old male Labrador Retriever (dog I.a) affected with XLMTM illustrating the generalized muscle atrophy and characteristic kyphosis and ventroflexion of the neck indicative of generalized muscle weakness. (B) Pedigrees and genetic status of affected Labrador Retrievers from Saskatchewan, Canada. In the five litters depicted, only males were affected, supporting an X-linked recessive mode of inheritance. Three apparently unrelated probands (arrows) were eventually linked to two identifiable kindreds (I and II). All affected males (■) are related through carrier females (circles with red dots). Males marked with an asterisk were all genetically confirmed to be hemizygous for the MTM1 c.465C>A mutation, and females with red dots are confirmed heterozygous carriers. “CNM clear” indicates dogs whose DNA tested negative for the PTLPA SINE insertion mutation that causes canine CNM. Numbers within or below male or female symbols indicate the number of additional pups of a particular gender and affection status as inferred from their owners' testimony. Not shown are two unaffected male and two unaffected female littermates of proband I.e and unknown additional unaffected littermates of dams I.b and I.c.

Fig. 2.

Skeletal muscle histology in an affected male, I.e (A, C, and E), and unaffected carrier female, I.f (B, D, and F), whose muscle exhibited no detectable abnormal findings. H&E-stained fresh frozen muscle biopsy sections from the triceps (A and B) reveal extensive fiber size variation with numerous small, centrally nucleated myofibers resembling fetal myotubes characteristic of XLMTM, in the affected male (A). The vastus lateralis muscles (C–F) consistently exhibited similar but less extreme pathology. Numerous “necklace” fibers were highlighted with the oxidative stain NADH-TR (E and F). (Scale bar: 100 μm.)

Fig. 3.

(A) Indirect immunofluorescence analysis of biopsies from the vastus lateralis muscle of two male Labrador Retrievers affected with X-linked myotubular myopathy (dogs II.d and I.e in Fig. 1B) and an unaffected carrier female (I.f in Fig. 1B) whose muscle histology and immunohistochemistry were indistinguishable from normal. Biopsy specimens from affected males revealed abnormal distribution of the SR and T tubule markers, ryanodine receptors (RYR1) and dihydropyridine receptors (DHPRα1), in numerous myofibers that exhibited subsarcolemmal and internal localizations; this pattern was not seen in myofibers from the unaffected littermate (I.f). (B) Indirect immunofluorescence analysis of the triceps muscle from dog I.e (Fig. 1B) showing apparent colocalization of a triad marker (DHPRα1 antibody) and myotubularin (R2867 C-terminal antibody) in abnormal distributions consistent with occurrence of necklace fibers. Staining of muscle from carrier I.f (bottom row) is indistinguishable from that in control biopsies. All panels are the same magnification (scale bar: 50 μm) except the magnified images of I.e in row 5 (scale bar: 10 μm).

Fig. 4.

Ultrastructural studies of a Labrador Retriever (I.e in Fig. 1B) with XLMTM (A–F) and an unaffected female carrier littermate (I.f in Fig 1B) (G) showing centrally located nuclei (A), myofibrillar disarray (B–E) with or without mitochondrial accumulation, and whirled membranous structures (C–E). Triads were infrequent and in disarray in the affected dog (F, arrow and inset showing higher-power view) compared with the unaffected littermate (G, arrow and inset showing higher-power view). (Scale bars: 3.6 μm for A, 2.2 μm for B and G, 3.0 μm for C, and 1.8 μ for E and insets.)

Fig. 5.

(A) Schematic diagram of mammalian myotubularin indicating the phosphoinositide-binding “GRAM-PH “domain, the catalytically active “phosphatase” domain, the “coiled-coil” domain likely responsible for binding other myotubularin-related proteins, and the C-terminal “PDZ-binding” domain. Exon 7 (indicated) encodes residues in a short linker between the GRAM-PH and phosphatase domains. (B) DNA sequence of a portion of MTM1 exon 7 from an unaffected control Labrador Retriever (Control), affected males (II.d and I.e), and a carrier female (I.b). An MTM1 c.465C>A transversion (in red box) was found to be hemizygous in all affected males and heterozygous in the obligate female carriers. (C) The affected amino acid, p.N155, is located directly at the interface between the beta pleated sheet-containing GRAM-PH (Upper Left) and the alpha-helical phosphatase (Lower Right) domains. (D) Amino acid alignment of MTM1 orthologs demonstrate that this region is highly conserved among mammals and p.N155 (in red box) is conserved in all vertebrates examined as far as amphibians (Xenopus). (E) Amino acid lineup of human myotubularin (MTM1) with the seven catalytically active human MTMRs demonstrates that p.N155 (red box) is not conserved in myotubularin-related proteins.

Fig. 6.

Abnormal expression of mutated myotubularin protein in vivo and in vitro. Expression of normal and mutated myotubularin in affected skeletal muscles (A and B) and COS-1 cells (C–I). Immunoblot analysis of myotubularin in striated muscles reveals loss of correctly sized (65 kDa) immunoreactive protein in affected dogs. (A) Skeletal muscle lysates from affected males (I.e and I.d in Fig. 1B) and an unaffected heterozygous female Labrador Retriever (I.f), reacted with goat polyclonal sc-14781 raised against the N terminus of myotubularin (N-20). (B) Canine quadriceps (Quad) and heart muscle lysates reacted with R2827 (labeled Ab2827) antisera, raised against myotubularin C terminus, confirm loss of detectable myotubularin in affected (I.e) tissues. C, normal control canine tissues. Parallel staining of WT and Mtm1 KO mouse skeletal muscles confirms specificity of the antiserum (Right). An antibody against 43 kDa β-actin was used as a loading control in A and B. Typical fluorescent images of COS-1 cells transfected with WT canine GFP-myotubularin (C) or p.N155K GFP-myotubularin (F) reveal unusual punctate localization of mutated protein. Double-label immunofluorescence for SUG1, the 26S ATPase subunit of proteasomes (D and G) reveals colocalization of mutated N155K GFP-myotubularin (H), but not the WT protein (E). Nuclei are stained blue by DAPI. (I) Quantitation of the percentage of cells transfected with each construct exhibiting varying numbers of aggregated punctate “dots” per cell. Two independent plasmid preparations were tested for each construct, and numbers represent 500 cells per construct over several independent transfection experiments.