A POGLUT 1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss
2016; Springer Nature; Volume: 8; Issue: 11 Linguagem: Inglês
10.15252/emmm.201505815
ISSN1757-4684
AutoresEmilia Servián‐Morilla, Hideyuki Takeuchi, Tom V. Lee, Jordi Clarimón, Fabiola Mavillard, Estela Área-Gómez, Eloy Rivas, José Luis Nieto-González, Maria Rivero, Macarena Cabrera‐Serrano, Leonardo Gómez‐Sánchez, José A. Martínez‐López, Beatriz Estrada, Celedonio Márquez, Yolanda Blanco Morgado, Xavier Suárez‐Calvet, Guillermo Pita, Anne Bigot, Eduard Gallardo, Rafael Fernández‐Chacón, Michio Hirano, Robert S. Haltiwanger, Hamed Jafar‐Nejad, Carmen Paradas,
Tópico(s)Ubiquitin and proteasome pathways
ResumoResearch Article10 October 2016Open Access Source DataTransparent process A POGLUT1 mutation causes a muscular dystrophy with reduced Notch signaling and satellite cell loss Emilia Servián-Morilla Emilia Servián-Morilla Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Search for more papers by this author Hideyuki Takeuchi Hideyuki Takeuchi Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Tom V Lee Tom V Lee Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Jordi Clarimon Jordi Clarimon Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Memory Unit, Department of Neurology and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Fabiola Mavillard Fabiola Mavillard Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Estela Area-Gómez Estela Area-Gómez Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Eloy Rivas Eloy Rivas Department of Pathology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Jose L Nieto-González Jose L Nieto-González Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Maria C Rivero Maria C Rivero Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Macarena Cabrera-Serrano Macarena Cabrera-Serrano Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Search for more papers by this author Leonardo Gómez-Sánchez Leonardo Gómez-Sánchez Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Jose A Martínez-López Jose A Martínez-López Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Beatriz Estrada Beatriz Estrada Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo Olavide, Sevilla, Spain Search for more papers by this author Celedonio Márquez Celedonio Márquez Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Yolanda Morgado Yolanda Morgado Department of Neurology, Hospital U. Valme, Sevilla, Spain Search for more papers by this author Xavier Suárez-Calvet Xavier Suárez-Calvet Laboratori de Malalties Neuromusculars, Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Guillermo Pita Guillermo Pita Human Genotyping Unit-CeGen, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Anne Bigot Anne Bigot UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, Paris, France Search for more papers by this author Eduard Gallardo Eduard Gallardo Laboratori de Malalties Neuromusculars, Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Rafael Fernández-Chacón Rafael Fernández-Chacón Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Michio Hirano Michio Hirano Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Robert S Haltiwanger Robert S Haltiwanger Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Hamed Jafar-Nejad Hamed Jafar-Nejad Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Carmen Paradas Corresponding Author Carmen Paradas [email protected] orcid.org/0000-0002-6917-2236 Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Emilia Servián-Morilla Emilia Servián-Morilla Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Search for more papers by this author Hideyuki Takeuchi Hideyuki Takeuchi Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Tom V Lee Tom V Lee Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Jordi Clarimon Jordi Clarimon Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Memory Unit, Department of Neurology and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain Search for more papers by this author Fabiola Mavillard Fabiola Mavillard Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Estela Area-Gómez Estela Area-Gómez Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Eloy Rivas Eloy Rivas Department of Pathology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Jose L Nieto-González Jose L Nieto-González Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Maria C Rivero Maria C Rivero Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Macarena Cabrera-Serrano Macarena Cabrera-Serrano Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Search for more papers by this author Leonardo Gómez-Sánchez Leonardo Gómez-Sánchez Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Jose A Martínez-López Jose A Martínez-López Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Beatriz Estrada Beatriz Estrada Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo Olavide, Sevilla, Spain Search for more papers by this author Celedonio Márquez Celedonio Márquez Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Yolanda Morgado Yolanda Morgado Department of Neurology, Hospital U. Valme, Sevilla, Spain Search for more papers by this author Xavier Suárez-Calvet Xavier Suárez-Calvet Laboratori de Malalties Neuromusculars, Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Guillermo Pita Guillermo Pita Human Genotyping Unit-CeGen, Spanish National Cancer Research Centre, Madrid, Spain Search for more papers by this author Anne Bigot Anne Bigot UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, Paris, France Search for more papers by this author Eduard Gallardo Eduard Gallardo Laboratori de Malalties Neuromusculars, Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Rafael Fernández-Chacón Rafael Fernández-Chacón Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Michio Hirano Michio Hirano Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Robert S Haltiwanger Robert S Haltiwanger Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA Search for more papers by this author Hamed Jafar-Nejad Hamed Jafar-Nejad Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Carmen Paradas Corresponding Author Carmen Paradas [email protected] orcid.org/0000-0002-6917-2236 Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Author Information Emilia Servián-Morilla1,2,‡, Hideyuki Takeuchi3,15,‡, Tom V Lee4,‡, Jordi Clarimon2,5, Fabiola Mavillard2,6, Estela Area-Gómez7, Eloy Rivas8, Jose L Nieto-González2,6, Maria C Rivero2,6, Macarena Cabrera-Serrano1,2, Leonardo Gómez-Sánchez2,6, Jose A Martínez-López2,6, Beatriz Estrada9, Celedonio Márquez1, Yolanda Morgado10, Xavier Suárez-Calvet11,12, Guillermo Pita13, Anne Bigot14, Eduard Gallardo11,12, Rafael Fernández-Chacón2,6, Michio Hirano7, Robert S Haltiwanger3,15, Hamed Jafar-Nejad4 and Carmen Paradas *,1,2,7 1Neuromuscular Disorders Unit, Department of Neurology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 3Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA 4Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 5Memory Unit, Department of Neurology and Sant Pau Biomedical Research Institute, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain 6Department of Medical Physiology and Biophysics, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 7Department of Neurology, Columbia University Medical Center, New York, NY, USA 8Department of Pathology, Instituto de Biomedicina de Sevilla, Hospital U. Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 9Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo Olavide, Sevilla, Spain 10Department of Neurology, Hospital U. Valme, Sevilla, Spain 11Laboratori de Malalties Neuromusculars, Institut de Recerca de HSCSP, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain 12Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER), Barcelona, Spain 13Human Genotyping Unit-CeGen, Spanish National Cancer Research Centre, Madrid, Spain 14UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, Sorbonne Universités, Paris, France 15Present address: Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +34 955923045; Fax: +34 955013536; E-mail: [email protected] EMBO Mol Med (2016)8:1289-1309https://doi.org/10.15252/emmm.201505815 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limb-girdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces O-glucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific α-dystroglycan hypoglycosylation not present in patients' fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells. Synopsis A protein O-glucosyltransferase 1 (POGLUT1) homozygous D233E mutation underlies a novel autosomal recessive muscular dystrophy, wherein altered Notch signaling affects muscle regeneration and, as a consequence, α-dystroglycan glycosylation. POGLUT1 D233E exhibits decreased enzymatic activity toward Notch EGF repeats. POGLUT1 D233E leads to Notch activity downregulation, which affects muscle regeneration due to satellite cell (SC) loss of quiescence, depletion of PAX7+ cells, and premature and enhanced differentiation. Reduced Notch signaling accelerates muscle differentiation and disrupts the progressive and coordinated process of α-dystroglycan glycosylation during differentiation, and hence, mild α-dystroglycan hypoglycosylation is observed in skeletal muscle from POGLUT1 D233E patients. Defective regeneration, combined with α-dystroglycan hypoglycosylation, likely results in skeletal muscle degeneration and finally gives rise to muscular dystrophy. Introduction Cell surface glycans are diverse in structure and function and play critical roles in many biological processes including infection, cancer, and development (2009). Broadly speaking, glycans affect the function of proteins to which they are linked by modulating their structure and/or by serving as a direct recognition signal for other proteins (Varki & Sharon, 2009). Glycans are involved in protein folding, stability, and trafficking and regulate the activity of important signaling pathways such as Notch, WNT, BMP, and Hedgehog (Christian, 2000; Yan & Lin, 2009; Jafar-Nejad et al, 2010; Takeuchi & Haltiwanger, 2014). Not surprisingly, aberrant glycosylation leads to a variety of human diseases, the list of which is growing (Freeze et al, 2014). Protein glycosylation can directly regulate signaling events. A clear example of this is the regulation of the evolutionarily conserved Notch signaling pathway, which is an intercellular communication mechanism widely used during animal development (Hori et al, 2013). The extracellular domain of the Notch receptor is modified with O-fucose and O-glucose glycans (Moloney et al, 2000). The structures of these glycans change in a tissue-specific and developmentally regulated manner (Johnston et al, 1997), and altering Notch glycosylation dramatically affects its activity (Stanley & Okajima, 2010; Takeuchi & Haltiwanger, 2010). One of the cell types whose regulation by Notch signaling has been intensely studied in recent years is the muscle-specific adult stem cell called the satellite cell (SC) (Mourikis & Tajbakhsh, 2014). SCs reside between the basal lamina and sarcolemma of myofibers (Mauro, 1961) and are the primary contributors to skeletal muscle growth and repair (Collins et al, 2005). Despite continuous regeneration, pools of SC are maintained in healthy muscle. This is accomplished through asymmetric cell divisions of SC, which give rise both to self-renewing SC and to committed myogenic progenitors that differentiate (Kuang et al, 2007; Sacco et al, 2008). In this process, the Notch pathway is key for maintaining quiescence in SC and for homing of SC-derived myoblasts (Bjornson et al, 2012; Brohl et al, 2012). A recent report indicates that mice in which Notch signaling is specifically blocked in SCs exhibit a decrease in the number of SCs and histological features of muscular dystrophy even upon normal daily activity (Lin et al, 2013), suggesting that Notch-mediated maintenance of an active SC pool is essential for repairing muscle damage caused by regular activity and for maintaining healthy muscle. Muscular dystrophies are inherited disorders characterized by progressive weakness due to skeletal muscle degeneration, due to mutations in a growing list of responsible genes (Chandrasekharan & Martin, 2010; Rahimov & Kunkel, 2013). However, to date, no primary molecular defects in SC or Notch pathway components have been identified in human muscular dystrophies. Mutations in several genes disrupt various aspects of the dystrophin-associated glycoprotein complex, which links the cytoskeleton to the extracellular matrix in muscle (Chandrasekharan & Martin, 2010). A key component of this complex is dystroglycan (Ervasti et al, 1990), a transmembrane protein essential for normal basement membrane development and muscle maintenance. Dystroglycan comprises a transmembrane β-subunit non-covalently linked to an extracellular α-subunit containing a mucin-like domain, which is modified with numerous O-linked glycans (Barresi & Campbell, 2006). The β-subunit is linked to the actin cytoskeleton, and the O-linked glycans on the α-subunit are critical for its binding capacity to extracellular matrix proteins such as laminin and agrin (Ervasti & Campbell, 1993; Michele et al, 2002). Mutations in dystroglycan itself result in the primary dystroglycanopathies (Henry & Campbell, 1998; Cote et al, 1999; Hara et al, 2011; Willer et al, 2014; Riemersma et al, 2015; Kanagawa et al, 2016). Secondary dystroglycanopathies are caused by interruption of α-dystroglycan–ligand interactions due to mutations in a growing list of genes [currently eighteen (Bonnemann et al, 2014)] encoding glycosyltransferases and accessory proteins responsible for α-dystroglycan's extensive posttranslational modifications (Muntoni et al, 2011; Inamori et al, 2012; Yoshida-Moriguchi et al, 2013). These include protein O-mannosyltransferase 1 (POMT1) and POMT2, which add O-linked mannose to α-dystroglycan (Manya et al, 2004; Willer et al, 2004), and like-acetylglucosaminyltransferase (LARGE), which is responsible for the addition of repeating xylose-glucuronic acid units on the O-mannosyl glycans on α-dystroglycan (Inamori et al, 2012). The dystroglycan gene (DAG1) is expressed in SCs (Cohn et al, 2002), but glycosylated α-dystroglycan is not required by myogenic cells during proliferation (Awano et al, 2015), a notion supported by the observation that trace amounts of glycosylated α-dystroglycan are found in C2C12 and human primary myoblasts during proliferation. Cultures of freshly isolated muscle fibers suggest that glycosylation of α-dystroglycan is needed for proliferation of SCs in vivo, but it is not necessary when SCs are removed from their niche (Ross et al, 2012). The transition to larger glycans occurs rapidly after differentiation is induced, and a direct correlation exists between LARGE-dependent extension of O-glycans on α-dystroglycan and its function as an extracellular matrix receptor (Goddeeris et al, 2013). Despite extensive studies regarding the genetics of dystroglycanopathies, the responsible gene is still unknown in a significant number of patients; thus, mutations in additional genes will likely be implicated in this group of diseases. Here, we report a homozygous missense mutation (D233E) in the protein O-glucosyltransferase 1 gene, POGLUT1, in four siblings with autosomal recessive limb-girdle muscular dystrophy. POGLUT1 is the sole enzyme that directly adds O-glucose to a distinct serine residue of epidermal growth factor-like (EGF) repeats containing a CXSX(P/A)C consensus sequence (Rana et al, 2011), many of which are found in Notch extracellular domains (Fernandez-Valdivia et al, 2011; Rana et al, 2011). The D233E mutation dramatically reduces the O-glucosyltransferase activity of POGLUT1, leading to impaired Notch signaling and a dramatic decrease in the number of SCs in adult muscles. Transgenic experiments in Drosophila indicate that D233E impairs the ability of human POGLUT1 to rescue the muscle phenotype caused by the loss of fly Poglut1 activity. Patient muscles show α-dystroglycan hypoglycosylation and decreased binding to laminin, but normal binding to agrin and normal basement membrane structure. Moreover, unlike other dystroglycanopathies, patient fibroblasts exhibit normal α-dystroglycan glycosylation and laminin binding. Together, our findings indicate that exhaustion of the SC pool plays a primary role in this novel form of muscular dystrophy. Results Clinical and radiological findings A consanguineous family from southern Spain comprises 17 individuals spanning three generations (Fig 1A). Four out of five siblings from generation II presented a phenotype consistent with a limb-girdle muscular dystrophy. Specifically, the patients exhibited muscle weakness predominantly in the proximal lower limbs, with onset during the third decade. The disease course was progressive, leading to scapular winging and wheelchair confinement. For more extended clinical data regarding this family, see the Appendix Information, Appendix Fig S1, and Appendix Tables S1 and S2. Serum creatine kinase level was normal in three patients and mildly elevated in one (Appendix Table S1). Muscle biopsies from all four affected siblings revealed histological features ranging from very mild myopathic changes to classic dystrophic pathology (Fig 1A). Proteins typically affected in myopathies displayed normal expression in muscle, except for a reduction in α-dystroglycan (Appendix Fig S2). Muscle magnetic resonance imaging (MRI) of the legs revealed a striking pattern of muscle involvement (Fig 1C), with early fatty replacement of internal regions of thigh muscles that spared external areas. This "from inside-to-outside" mode of fatty degeneration progressed over the years and did not match the distribution patterns typically associated with other forms of muscular dystrophies (Appendix Information and Appendix Figs S3 and S4). Figure 1. POGLUT1 missense mutation in a family with a limb-girdle muscular dystrophy The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers, and double line denotes a consanguineous marriage. The pictures show scapular winging, which is a consistent clinical sign in affected individuals. Hematoxylin and eosin staining (H&E) of skeletal muscle from patient II.1 shows histological features of moderate-to-severe dystrophic pattern. Scale bar, 50 μm. T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartments, with relative sparing of the rectus femoris, sartorius, and gracilis muscles until late stages (4, 10, and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5–9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posterior muscle (14) in the lower leg. Download figure Download PowerPoint Expression and functional modification of α-dystroglycan in patients Given the key role played by aberrant α-dystroglycan glycosylation and function in a subset of muscular dystrophies and because of the observed decrease in α-dystroglycan levels in patient muscles, we examined the glycosylation status and ligand-binding ability of α-dystroglycan in our patients. Immunofluorescence staining of frozen cross sections from skeletal muscle biopsy with an antibody against glycosylated α-dystroglycan [IIH6 (Ervasti & Campbell, 1991)] revealed a variable reduction in the glycosylated form of α-dystroglycan at the sarcolemma in patients, while antibodies against α-dystroglycan core protein, β-dystroglycan, and laminin α2 showed normal staining (Fig 2A and Appendix Fig S5A). In agreement with this observation, Western blots showed a reduction in α-dystroglycan glycosylation in patient muscle, accompanied by a mild decrease in the molecular weight of glycosylated α-dystroglycan compared with controls. To examine whether decreased α-dystroglycan glycosylation affected binding to ligands, we performed a ligand overlay assay. As shown in Fig 2B, the laminin-binding activity was diminished in muscle. However, the agrin-binding activity to the patients' muscle extracts showed no difference compared with controls (Fig 2B). Moreover, in skin fibroblasts from patients, the level of both functional α-dystroglycan glycosylation, examined by Western blot and flow cytometry (Stevens et al, 2013b), and its ability to bind laminin (Fig 2B and Appendix Fig S5B) were normal, unlike known secondary dystroglycanopathies, which usually result in decreased functional α-dystroglycan glycosylation in both muscle and the skin fibroblasts (Willer et al, 2012; Carss et al, 2013; Stevens et al, 2013a). Finally, although basement membrane defects are commonly observed in dystroglycanopathies (Yamamoto et al, 1997; Goddeeris et al, 2013), transmission electron microscopy showed normal muscle ultrastructure in patients, with no alterations in basement membrane compaction (Fig 2C and Appendix Fig S5C). Figure 2. A muscle-specific decrease in α-dystroglycan glycosylation and laminin binding upon POGLUT1 mutation Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG), and laminin-α2 is similar to control (scale bar, 100 μm). Western blots and ligand overlay (O/L) of
Referência(s)