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Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

Böhm J, Vasli N, Maurer M, Cowling BS, Cowling B, Shelton GD, Kress W, Toussaint A, Prokic I, Schara U, Anderson TJ, Weis J, Tiret L, Laporte J - PLoS Genet. (2013)

Bottom Line: In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy.Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes.Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD.

View Article: PubMed Central - PubMed

Affiliation: IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Illkirch, France.

ABSTRACT
Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

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Common ultrastructural and membrane defects in affected human and dog.(A–D) Electron microscopic analysis of a patient biopsy showing a centralized nucleus surrounded by organelles and mild sarcomeric disarray (arrow, A), accumulations of membranes and vacuoles containing whorled membranes (arrow, B), autophagic vacuoles containing myelin-like material (arrow, C), and widened tubules at the triads (arrow, D). The inset shows normal triads in an age-matched biopsy. (E–H) Ultrastructural analysis of a IMGD dog biopsy revealed central nuclei surrounded by mitochondrial accumulations (E), membranous whorls (arrow, F), deep membrane invaginations (arrowhead, F), lipofuscin granules (arrow, G), and abnormal triads (arrow, H). The inserted picture shows a normal triad in an age-matched canine control.
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pgen-1003430-g005: Common ultrastructural and membrane defects in affected human and dog.(A–D) Electron microscopic analysis of a patient biopsy showing a centralized nucleus surrounded by organelles and mild sarcomeric disarray (arrow, A), accumulations of membranes and vacuoles containing whorled membranes (arrow, B), autophagic vacuoles containing myelin-like material (arrow, C), and widened tubules at the triads (arrow, D). The inset shows normal triads in an age-matched biopsy. (E–H) Ultrastructural analysis of a IMGD dog biopsy revealed central nuclei surrounded by mitochondrial accumulations (E), membranous whorls (arrow, F), deep membrane invaginations (arrowhead, F), lipofuscin granules (arrow, G), and abnormal triads (arrow, H). The inserted picture shows a normal triad in an age-matched canine control.

Mentions: To uncover the pathological defects underlying this highly progressive form of centronuclear myopathy and to validate the canine model, we analyzed human and dog muscle biopsies by electron microscopy. Ultrastructural analysis of the human muscle biopsy revealed centralized nuclei surrounded by an area devoid of myofibrils and containing glycogen granules and other organelles (Figure 5A, Figure S2), as commonly seen in MTM1, DNM2 and BIN1-related CNM. Myofibrillar disintegration with occasional Z-band streaming (arrow, Figure 5A) was seen in the adjacent sarcomeres. Triad structures were found to be aberrant and we observed frequent enlarged structures, most probably originating from the sarcoplasmic reticulum (arrow, Figure 5D). We also noted other membrane alterations, including accumulations of membranes and tubules, vacuoles containing whorled membranes (arrow, Figure 5B), as well as a high number of myelin-like membranous structures suggestive of autophagosomes (arrow, Figure 5C). Likewise, ultrastructural analysis of muscle biopsies from an affected Great Dane dog showed nuclear internalization, mitochondrial accumulations around the internalized nuclei and myofibrillar disarray (Figure 5E, Figure S3). We furthermore found membranous whorls (arrow, Figure 5F) as reported for the X-linked CNM Labrador retriever model with MTM1 mutation [31], deep membrane invaginations (arrowhead, Figure 5F), lipofuscin granules (arrow, Figure 5G), and abnormal triads in almost all fibers (arrow, Figure 5H). Sarcolemmal invaginations contained basement membranes and often pointed towards centralized nuclei. Taken together and considering the histological analysis described above, histopathology of IMGD dogs and human patients appear strikingly similar, emphasizing common alterations of membrane structures.


Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

Böhm J, Vasli N, Maurer M, Cowling BS, Cowling B, Shelton GD, Kress W, Toussaint A, Prokic I, Schara U, Anderson TJ, Weis J, Tiret L, Laporte J - PLoS Genet. (2013)

Common ultrastructural and membrane defects in affected human and dog.(A–D) Electron microscopic analysis of a patient biopsy showing a centralized nucleus surrounded by organelles and mild sarcomeric disarray (arrow, A), accumulations of membranes and vacuoles containing whorled membranes (arrow, B), autophagic vacuoles containing myelin-like material (arrow, C), and widened tubules at the triads (arrow, D). The inset shows normal triads in an age-matched biopsy. (E–H) Ultrastructural analysis of a IMGD dog biopsy revealed central nuclei surrounded by mitochondrial accumulations (E), membranous whorls (arrow, F), deep membrane invaginations (arrowhead, F), lipofuscin granules (arrow, G), and abnormal triads (arrow, H). The inserted picture shows a normal triad in an age-matched canine control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675003&req=5

pgen-1003430-g005: Common ultrastructural and membrane defects in affected human and dog.(A–D) Electron microscopic analysis of a patient biopsy showing a centralized nucleus surrounded by organelles and mild sarcomeric disarray (arrow, A), accumulations of membranes and vacuoles containing whorled membranes (arrow, B), autophagic vacuoles containing myelin-like material (arrow, C), and widened tubules at the triads (arrow, D). The inset shows normal triads in an age-matched biopsy. (E–H) Ultrastructural analysis of a IMGD dog biopsy revealed central nuclei surrounded by mitochondrial accumulations (E), membranous whorls (arrow, F), deep membrane invaginations (arrowhead, F), lipofuscin granules (arrow, G), and abnormal triads (arrow, H). The inserted picture shows a normal triad in an age-matched canine control.
Mentions: To uncover the pathological defects underlying this highly progressive form of centronuclear myopathy and to validate the canine model, we analyzed human and dog muscle biopsies by electron microscopy. Ultrastructural analysis of the human muscle biopsy revealed centralized nuclei surrounded by an area devoid of myofibrils and containing glycogen granules and other organelles (Figure 5A, Figure S2), as commonly seen in MTM1, DNM2 and BIN1-related CNM. Myofibrillar disintegration with occasional Z-band streaming (arrow, Figure 5A) was seen in the adjacent sarcomeres. Triad structures were found to be aberrant and we observed frequent enlarged structures, most probably originating from the sarcoplasmic reticulum (arrow, Figure 5D). We also noted other membrane alterations, including accumulations of membranes and tubules, vacuoles containing whorled membranes (arrow, Figure 5B), as well as a high number of myelin-like membranous structures suggestive of autophagosomes (arrow, Figure 5C). Likewise, ultrastructural analysis of muscle biopsies from an affected Great Dane dog showed nuclear internalization, mitochondrial accumulations around the internalized nuclei and myofibrillar disarray (Figure 5E, Figure S3). We furthermore found membranous whorls (arrow, Figure 5F) as reported for the X-linked CNM Labrador retriever model with MTM1 mutation [31], deep membrane invaginations (arrowhead, Figure 5F), lipofuscin granules (arrow, Figure 5G), and abnormal triads in almost all fibers (arrow, Figure 5H). Sarcolemmal invaginations contained basement membranes and often pointed towards centralized nuclei. Taken together and considering the histological analysis described above, histopathology of IMGD dogs and human patients appear strikingly similar, emphasizing common alterations of membrane structures.

Bottom Line: In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy.Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes.Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD.

View Article: PubMed Central - PubMed

Affiliation: IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), Illkirch, France.

ABSTRACT
Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

Show MeSH
Related in: MedlinePlus