Brain Pathology ISSN 1015-6305
MEETINGS PROCEEDINGS
Third International Workshop for Glycosylation Defects in Muscular Dystrophies, 18–19 April 2013, Charlotte, USA Anthony Blaeser1; Susan Sparks2; Susan C. Brown3; Kevin Campbell4; Qi Lu1 1 McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center and 2 Clinical Genetics/Department of Pediatrics, Levine Children’s Hospital, Carolinas Medical Center, Carolinas Healthcare System, Charlotte, NC. 3 Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK. 4 Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA.
Keywords dystroglycan, dystroglycanopathy, fukutin related protein, glycosylation, muscular dystrophy, therapy. Corresponding author: Anthony Blaeser, PhD, Carolinas Healthcare System, 1000 Blythe Blvd, Charlotte, NC 28203 (Email: anthony.blaeser@ carolinashealthcare.org) Received 17 December 2013 Accepted 18 December 2013 Published Online Article Accepted 8 January 2014 doi:10.1111/bpa.12118
INTRODUCTION The Third International Workshop for Glycosylation Defects in Muscular Dystrophies took place on 18–19 April 2013. It was held at the Omni Charlotte in Charlotte, NC, USA and hosted by the McColl-Lockwood Laboratory for Muscular Dystrophy Research, the Carolinas Medical Center and the Carolinas Education Institute. The event was sponsored by the Carolinas Healthcare Foundation, the Muscular Dystrophy Association (MDA), funds raised by “Jeans, Genes and Geniuses” organized by Jane and Luther Lockwood, as well as generous support by the McColl and Lockwood families. Since its founding in 2008, the International Workshop for Glycosylation Defects in Muscular Dystrophies has been a platform for bringing together top scientists and clinicians from around the world to discuss current research in the area of dystroglycanopathies [forms of muscular dystrophy associated with the hypoglycosylation of alpha-dystroglycan (α-DG) ] as well as providing a setting to facilitate collaborations. For this workshop, 22 scientists and clinicians were brought in from the US, UK and Japan for a total of 23 talks spread over 2 days. Over the past 2 years, significant progress has been made in the dystroglycanopathy field. Such advances include a greater knowledge of the mechanisms involved in the glycosylation of α-DG, the identification of additional genes that, when carrying mutations, are involved in the disease process and the development of new animal models and their use in developing new targets for treatment. New data have become available for adeno-associated virus (AAV)-
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mediated gene therapy in FKRP (fukutin-related protein) deficient animal models and compound library screening has taken place in several labs resulting in multiple hits with the potential for upregulating the functional glycosylation of α-DG and other proteins. As a testament to the progress being made, this year’s workshop devoted an entire session to discussing clinical treatments, management and endpoints. The 2013 workshop focused on the following topics: mechanisms involved in α-DG glycosylation and newly developed animal models for examining pathways and disease targets; AAV therapy and drug discovery, including possible mechanisms and targets for treating dystroglycanopathies; genetic diagnosis, clinical management and endpoint markers, including current data from clinical trials and methods for assessing treatments. The audience included members of Carolinas Medical Center, Children’s National Medical Center D.C., Genzyme Corporation, BioMarin Pharmaceuticals, Pfizer Corporation and University of Massachusetts Medical School.
BACKGROUND Dystroglycan is a key component of the dystrophin glycoprotein complex (DGC). It is encoded by a single gene (Dag1), the transcript of which is post-translationally cleaved into two subunits, alpha and beta. α-DG is heavily glycosylated and the sugar moieties in the central mucin-like region play an essential role in the binding to various extracellular matrix components such as laminin, perlecan and agrin. Alterations in the glycosylation of α-DG result Brain Pathology 24 (2014) 280–284 © 2014 International Society of Neuropathology
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in a subset of muscular dystrophies referred to as dystroglycanopathies (14). The diseases vary in severity from the severe Walker– Warburg syndrome (WWS) and muscle–eye–brain (MEB) disease, which display structural brain involvement in addition to muscular dystrophy, to the relatively mild forms of limb–girdle muscular dystrophy (LGMD) such as LGMD2I with little or no central nervous system (CNS) involvement (5, 13). The dystroglycanopathies have so far been associated with mutations in up to 16 putative or demonstrated glycosyltransferases, which include LARGE, fukutin, FKRP, POMT1, POMT2 and POMGnT1, each of which result in the aberrant glycosylation of α-DG (2, 4, 11, 12, 23). However, there are still a large proportion of patients with reduced α-DG glycosylation that lack a defined genetic diagnosis. At the current time, there is no effective therapy available for dystroglycanopathies. A number of animal models have been described previously, but no experimental therapy has been reported. The pathway leading to the functional glycosylation of α-DG is not clearly understood, and reagents and methods for identifying diversity of glycosylated α-DG remain a challenge. Research in all these areas has made significant progress during the last 2 years as presented during the meeting. At the same time, clinic preparation has geared up for future clinic trials with several lines of experimental therapies emerging from the ongoing translational research.
REPORT SUMMARY Glycosylation The workshop began with opening remarks by Bernard Brigonnet, Vice President of Research, Carolinas Healthcare System. The first session was chaired by James Ervasti from the University of Minnesota Department of Biochemistry, Molecular Biology and Biophysics. To start the session Dr. James Ervasti shared his view of the more complex nature of α-DG glycosylation in muscle and the difficulties associated with determining the status of so-called functional glycosylation, a critical step if we are to understand the function of each of the so-called “glycosylation” genes in the glycosylation process. Sample preparation, sample buffer composition and pH, denaturation temperature and duration, as well as protease inhibiters could all affect the size and signal intensity of the species of α-DG glycosylation detected by IIH6 (an antibody commonly used to identify the glycosylation status of α-DG) and laminin-binding assays. Dr. Ervasti suggested that greater care should be taken to standardize blotting procedures for detecting α-DG and data interpretation. This also raised the need to develop new reagents for possibly more reliable determination of α-DG glycosylation status (see below). Work from Dr. Ervasti’s lab also suggested a possible interaction between dystroglycan and other membrane-associated proteins such as caveolin, which might play a role for maintaining muscle fiber stability (10). To address the difficulty in detecting dystroglycan reliably, Dr. Glenn Morris presented his lab’s efforts (with support from LGMD2I Fund) to generate specific monoclonal antibodies for functionally glycosylated α- and β-DG. Dr. Morris reported initial evidence that clones of such antibodies have been created. However, further selection and characterization are required. Using a zebra fish model system, Dr. Tamao Endo illustrated his studies showing that POMT1/2 play an essential role in the normal
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development of zebra fish through the O-mannosylation of α-DG. Whilst the glycosylation of α-DG is important for maintaining muscle stability, the regulation of this process remains largely unknown. Dr. Endo reported the importance of human natural killer-1 sulfotransferase (HNK-1ST) in modulating function of α-DG. Using a coexpression system in retinoic acid-treated S91 melanoma cells, Dr. Endo showed that expression of HNK-1ST mediated the incorporation of sulfate into α-DG. This is associated with suppressed expression and reduced ligand-binding activity of α-DG in the presence and absence of LARGE overexpression. The results suggest that sulfotransferase activity of HNK-1ST may be one of the mechanisms modulating α-DG glycosylation though the precise pathways are not understood. Dr. Susan Brown has followed up her earlier study of developing an FKRP knockdownneoY307N mutant mouse model. The original model proved to be embryonic and perinatal lethal. By crossbreeding with a strain of mouse expressing Cre recombinase under the SOX-1 promoter, Dr. Brown reported the creation of a new line with the restoration of functional α-DG glycosylation in the CNS but not skeletal muscle, thus producing viable mice with clear dystrophic phenotype. This model will now be more useful for mechanistic and therapeutic studies. Dr. Anthony Blaeser presented further data in FKRP mouse model characterization conducted in McColl-Lockwood Lab. Through crossbreeding of original strains of FKRP mutants with P448L, L276I and E310X nonsense mutation, a set of mouse models with wide range of disease severity has been created with dystrophic phenotypes ranging from very mild, limb–girdle muscular dystrophy 2I, to severe Walker–Warburg syndrome and muscle–eye–brain disease with brain and eye defects (3, 8). These animal models will play a crucial role in understanding the mechanisms of α-DG glycosylation as well as in developing experimental therapies. Dystroglycan phosphorylation was suggested as a new therapeutic target for dystroglycanopathies by Dr. Steve Winder. Dr. Winder proposed that the tyrosine phosphorylation of β-DG plays a crucial role in the loss of DGC from the membrane and might represent a therapeutic target. Using mouse myoblasts, Dr. Winder was able to demonstrate an increase in non-phosphorylated dystroglycan. Also, in sapje (dystrophin-deficient) zebra fish, the inhibition of tyrosine phosphorylation was able to rescue the dystrophic phenotype. Dr. Huaiyu Hu presented new data suggesting a role for glycosyltranferases in neural development. Using POMGnT1 knockout mice, Dr. Hu demonstrated that the hypoglycosylation of α-DG resulted in the disruption of basement membrane assembly during neural tissue development, leading to reduced levels of key basement membrane components and the weakening of the basement membrane. This could be the mechanism involved in the alteration of neuronal migration. To explore regulatory pathways of α-DG glycosylation, Dr. Minoru Fukuda applied the siRNA library screening technique and identified Fer kinase as a key regulator of laminin-binding glycan expression on breast and prostate cancer cells (22). Dr. Fukuda demonstrated that knockdown of Fer kinase increased transcription levels of β3GnT1 and LARGE. However, expression of Fert2, (a homolog of Fer) in mouse C2C12 myoblast cells, downregulated β3GntT1 expression but not LARGE. The knockdown of Fert2 was able to upregulate laminin-binding glycan expression and 281
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myotube formation, suggesting a novel role of Fer kinase in glycosylation regulation. Dr. Kevin Campbell presented data of LARGE function in the glycosylation of α-DG. Dr. Campbell demonstrated the bifunctionality of LARGE with both xylosyltransferase and glucoronyltransferase activities (9). These activities play an important role in the laminin-binding efficiency of glycosylated α-DG.
AAV THERAPY/DRUG DISCOVERY The second session was chaired by Qi Long Lu from the Carolinas Medical Center. Dr. Xiao Xiao began the session by discussing the use of AAV9 vectors for the delivery of FKRP to L276I-FKRP mutant mice, which exhibit a mild disease phenotype with late onset. It was apparent that the expression of a human FKRP transgene restored glycosylation of α-DG and ameliorated the dystrophic pathology in both skeletal and cardiac muscles. Dr. Qi Long Lu further demonstrated the effectiveness of AAV9-FKRP therapy in FKRP-P448Lneo mutant mice, which present moderate dystrophic phenotype with disease onset before 4 weeks of age. Dr. Lu showed that AAV-FKRP treatment was able to restore the functional glycosylation of α-DG in all skeletal and heart muscles. This led to a significant improvement in muscle pathology, strength and serum markers. No detectable toxicity was observed, suggesting the feasibility of gene therapy as an effective treatment for FKRP-related muscular dystrophies. In line with the effort to move FKRP gene therapy using AAV vectors toward clinic trials, Dr. John Gray introduced clinical trials using AAV8-mediated gene therapy in Hemophilia B patients. Preliminary results from this study proved to be encouraging with patients maintaining expression at 1%–6% normal levels after a period of 1.5 years. However, cell-mediated immunity to AAV8 remains a concern with some of the high-dose cohort patients showing elevated levels of AAV8 capsid-specific T-cells. Dr. Tatsushi Toda discussed the use of antisense oligonucleotides targeting various splice acceptor and enhancer sites to prevent pathogenic exon-trapping in Fukuyama congenital muscular dystrophy (FCMD) mouse models and the cells of patients with FCMD. The disease is largely the result of retrotransposal insertion in the 3′ UTR region of the fukutin gene, leading to the altered splicing and the addition of a new sequence in the 3′ end of the transcript with loss of function. Dr. Toda designed specific antisense oligonucleotides targeting splicing acceptor and enhancer sites for restoration of normal fukutin protein and reported success in doing so by using a cocktail of antisense oligonucleotides in both human cells and in the mouse model. Dr. Toda also presented data supporting the hypothesis that FKRP is also involved in the post-phosphoryl modification of α-DG. In addition, Dr. Toda reported two distinct fukutin conditional knockout (cKO) mice and AAV9-fukutin therapy. They proposed that myofiber-selective expression of fukutin can also be an effective therapeutic strategy. Using cell-based assays on patient-derived cells, Dr. Anne Bang has developed a 384-well based assay for identifying lead compounds to treat LGMD2I. A screening of 6000 pharmacologically active compounds resulted in the identification of nine compounds that increase laminin binding in FKRP-deficient patient cells. Dr. Bang has also developed induced pluripotent stem cells (iPSC) from dystroglycanopathy patient-derived tissue. This will allow Dr. Bang to further differentiate the iPSC into disease-relevant cell 282
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types including myogenic cells and myofibers, cardiomyocytes, and neural cells. This could be highly valuable for drug screening as effect of any potential compound may well be cell-specific. Dr. Xiaohua Wu reported his finding on drug screening of a larger library with about 420 000 small compounds in Chinese hamster ovary (CHO) cells with glycosylation levels and laminin binding as a readout. Several dozen compounds were identified that significantly increase the expression of functionally glycosylated α-DG. However, the enhanced expression of α-DG appears to be cell-type dependant, suggesting cell-specific regulatory mechanisms may underlie functional glycosylation. Dr. Wu emphasized the importance of cell-type specificity of drug response and the need to establish more relevant in vitro screen system for further drug development. Another strategy for drug screening was adopted by Dr. Lou Kunkel. Dr. Kunkel screened a drug library in dystroglycan-null zebra fish, in an attempt to find candidate compounds capable of alleviating the dystrophic phenotype. Preliminary screening resulted in 11 candidates that prevent muscle pathology in short-term treatments. Of these compounds, one was able to dramatically increase the survival of dystroglycannull zebra fish and reverse the pathology. This is interesting as it implies that factors other than functional glycosylated α-DG are able to compensate for the lack of dystroglycan. Dr. Kunkel is further testing the candidate drug in different cell and animal model systems. Currently, immunohistochemistry and western blot are the most widely used methods of detecting functionally glycosylated α-DG. However, Dr. Francesco Muntoni demonstrated the use of flow cytometry for the detection of IIH6-reactive functionally glycosylated α-DG in patients-derived fibroblasts. The results show that the levels of glycosylated α-DG in fibroblasts are comparable to that in patient-derived skeletal muscle cells, supporting the view that examination of fibroblasts in conjunction with flow cytometry could be of diagnostic value for dystroglycanopathies. Dr. Muntoni also applied flow cytometry for drug screening with a library of about 1000 compounds and reported several hits with increase in α-DG glycosylation. Dr. Muntoni suggested the use of flow cytometry as a platform for conducting high throughput assays.
CLINICAL MANAGEMENT/ENDPOINTS The final session was chaired by Susan Sparks from the Carolinas Medical Center. Dr. Francesco Muntoni returned to discuss the identification of a number of novel genes, mutations in which are associated with a dystroglycanopathy phenotype. These included at least six other genes in the last 2 years and include:—B3GALnT2, a glycosyltransferase located in the endoplasmic reticulum, mutations in which were found to underlie WWS and MEB (18); GDP-mannose pyrophosphorylase B (GMPPB), mutations in which were identified in patients with varying severities of disease from MEB to LGMD (7); isoprenoid synthase domain containing (ISPD) gene, mutations in which were shown in individuals with severe WWS (16, 20) and subsequently milder LGMD (19); dolichol synthesis (DPM1 and DPM2), mutations in which were associated with a dystroglycanopathy-type congenital muscular dystrophy (1, 21); B3GNT1, mutations in which were found to underlie WWS (6, 17); GTDC2, mutations in which were identified
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in patients with WWS (15); and finally SGK196, mutations in which were associated with congenital muscular dystrophy (24). Dr. Susan Sparks reported on a study of expression profiling in patients by looking at the expression of various growth factors and cytokines in LGMD patients with FKRP mutations. Dr. Sparks identified 373 transcripts, which are differentially expressed compared with normal controls. The results showed that FKRP patients contain more downregulated transcripts than typically seen in other dystrophies. Of note was the downregulation of genes involved in the TGF-B and IGF-II signaling pathways, which are generally upregulated in other dystrophies. Identifying biomarkers specific for FKRP-dystroglycanopathies and disease progression remains to be investigated, but crucial for clinic evaluation of any experimental therapy. Dr. Madhuri Hegde addressed the issue of identifying disease subtypes through the use of next-generation sequencing and whole exomes. She reviewed the current technology, utilization and limitations. Utilizing patient-derived data, including sequencing and phenotypes, known and unknown variants can be catalogued using a newly established website. This site can then be used to examine relevant genes associated with a patient’s clinical presentation. This allows enhanced evaluation for clinical phenotypes. Tina Duong continued the discussion of evaluating clinical outcome measures in patients with LGMD. Examining various conditions, based on strength and functional tests, baseline measures were established for a variety of LGMD subtypes. When compared with other LGMD subtypes, patients with FKRP showed lower forced vital capacity and sparing of grip strength. Additionally, 6-minute walking test (MWT) values seem to be more highly correlated to lower extremity strength than respiratory function. Results from this pilot study validate previously published works on FKRP phenotype using a much smaller cohort and would help in future experimental designs and outcomes selection. Dr. Volker Straub assessed LGMD patients with FKRP of different clinical severities with similar evaluation methods as those described by Tina Duong. He also included muscle magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) showing a correlation between fat infiltration and muscle function. Dr. Straub suggests that quantitative MRI is better suited to measure disease progression than physical assessments. Furthermore, Dr. Straub presented the global FKRP registry websites (https:// www.fkrp-registry.org) to identify potential patients eligible for clinical trials as well as providing a better understanding of the prevalence and distribution of those affected by FKRP mutations. The final talk of the session was given by Dr. Claudia Mitchell regarding various research supported by the LGMD2I Research Fund. These include research in developing patient iPS cells, antibodies to α-DG, various FKRP animal models and drug screening as well as various experimental therapies. The Fund is also involved in the formation of various websites and clinical trials as well as discussing social media avenues to encourage patient involvement in research.
WORKSHOP ORGANIZERS The workshop was organized with the help of Dr. Qi Long Lu, Dr. Anthony Blaeser, Dr. Kevin Campbell, Dr. Susan Sparks, Jeannie Maggio, Cameron Davis, Caren Anderson and Ashley Katkin.
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LIST OF SPEAKERS (i) Anne Bang, Sanford-Burnham Medical Research Institute (ii) Anthony Blaeser, Carolinas Medical Center (iii) Susan Brown, Royal Veterinary College, University of London (iv) Kevin Campbell, University of Iowa (v) Tina Duong, Children’s National Medical Center (vi) Tamao Endo, Tokyo Metropolitan Institute of Gerontology (vii) James Ervasti, University of Minnesota (viii) Minoru Fukuda, Sanford-Burnham Medical Research Institute (ix) John Gray, St. Jude Children’s Research Hospital (x) Madhuri Hegde, Emory University School of Medicine (xi) Huaiyu Hu, SUNY Upstate Medical University (xii) Louis Kunkel, Boston Children’s Hospital (xiii) Qi Long Lu, Carolinas Medical Center (xiv) Claudia Mitchell, LGMD2i Research Fund (xv) Glenn Morris, Robert Jones & Agnes Hunt Orthopaedic Hospital (xvi) Francesco Muntoni, University College London (xvii) Susan Sparks, Levine Children’s Hospital (xviii) Volker Straub, Newcastle University (xix) Tatsushi Toda, Kobe University (xx) Steve Winder, University of Sheffield (xxi) Xiaohua Wu, Carolinas Medical Center (xxii) Xiao Xiao, University of North Carolina at Chapel Hill
WORKSHOP WEBSITE A website for the Third International Workshop for Glycosylation Defects can be accessed at http://www.carolinashealthcare.org/ md-international-workshop.
ACKNOWLEDGEMENTS Generous financial support for the workshop was provided by “Jeans, Genes and Geniuses” charity event organized by Jane and Luther Lockwood, the McColl and Lockwood families, the Carolinas Healthcare Foundation and the Muscular Dystrophy Association (MDA). Special thanks would also be given to the following people for their help: Cameron Davis, Ashley Katkin and Susan Rucho from CEI for planning the event; Jane Howard for making all of the travel arrangements; Frederick Jones for arranging our audio visual needs; Rebekah Law from the Omni Charlotte for all of her help organizing the event; and Malia Rodgers for her assistance in setting up the workshop website.
REFERENCES 1. Barone R, Aiello C, Race V, Morava E, Foulquier F, Riemersma M et al (2012) DPM2-CDG: a muscular dystrophy-dystroglycanopathy syndrome with severe epilepsy. Ann Neurol 72:550–558. 2. Beltran-Valero de Bernabe D, Currier S, Steinbrecher A, Celli J, van Beusekom E, van der Zwaag B et al (2002) Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker–Warburg syndrome. Am J Hum Genet 71:1033–1043.
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3. Blaeser A, Keramaris E, Chan YM, Sparks S, Cowley D, Xiao X, Lu QL (2013) Mouse models of fukutin-related protein mutations show a wide range of disease phenotypes. Hum Genet 132:923–934. 4. Brockington M, Blake DJ, Prandini P, Brown SC, Torelli S, Benson MA et al (2001) Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 69:1198–1209. 5. Brown SC, Torelli S, Brockington M, Yuva Y, Jimenez C, Feng L et al (2004) Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 164:727–737. 6. Buysse K, Riemersma M, Powell G, van Reeuwijk J, Chitayat D, Roscioli T et al (2013) Missense mutations in beta-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker–Warburg syndrome. Hum Mol Genet 22:1746–1754. 7. Carss KJ, Stevens E, Foley AR, Cirak S, Riemersma M, Torelli S et al (2013) Mutations in GDP-mannose pyrophosphorylase B cause congenital and limb–girdle muscular dystrophies associated with hypoglycosylation of alpha-dystroglycan. Am J Hum Genet 93:29–41. 8. Chan YM, Keramaris-Vrantsis E, Lidov HG, Norton JH, Zinchenko N, Gruber HE et al (2010) Fukutin-related protein is essential for mouse muscle, brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies. Hum Mol Genet 19:3995–4006. 9. Inamori K, Hara Y, Willer T, Anderson ME, Zhu Z, Yoshida-Moriguchi T, Campbell KP (2013) Xylosyl- and glucuronyltransferase functions of LARGE in alpha-dystroglycan modification are conserved in LARGE2. Glycobiology 23:295–302. 10. Johnson EK, Li B, Yoon JH, Flanigan KM, Martin PT, Ervasti J, Montanaro F et al (2013) Identification of new dystroglycan complexes in skeletal muscle. PLoS ONE 8:e73224. 11. Lefeber DJ, Schonberger J, Morava E, Guillard M, Huyben KM, Verrijp K et al (2009) Deficiency of Dol-P-Man synthase subunit DPM3 bridges the congenital disorders of glycosylation with the dystroglycanopathies. Am J Hum Genet 85:76–86. 12. Longman C, Brockington M, Torelli S, Jimenez-Mallebrera C, Kennedy C, Khalil N et al (2003) Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet 12:2853–2861.
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13. Mendell JR, Boue DR, Martin PT (2006) The congenital muscular dystrophies: recent advances and molecular insights. Pediatr Dev Pathol 9:427–443. 14. Muntoni F (2004) Journey into muscular dystrophies caused by abnormal glycosylation. Acta Myol 23:79–84. 15. Ogawa M, Nakamura N, Nakayama Y, Kurosaka A, Manya H, Kanagawa M et al (2013) GTDC2 modifies O-mannosylated alpha-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody. Biochem Biophys Res Commun 440:88–93. 16. Roscioli T, Kamsteeg EJ, Buysse K, Maystadt I, van Reeuwijk J, van den Elzen C et al (2012) Mutations in ISPD cause Walker–Warburg syndrome and defective glycosylation of alpha-dystroglycan. Nat Genet 44:581–585. 17. Shaheen R, Faqeih E, Ansari S, Alkuraya FS (2013) A truncating mutation in B3GNT1 causes severe Walker–Warburg syndrome. Neurogenetics 14:243–245. 18. Stevens E, Carss KJ, Cirak S, Foley AR, Torelli S, Willer T et al (2013) Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of alpha-dystroglycan. Am J Hum Genet 92:354–365. 19. Tasca G, Moro F, Aiello C, Cassandrini D, Fiorillo C, Bertini E et al (2013) Limb–girdle muscular dystrophy with alphadystroglycan deficiency and mutations in the ISPD gene. Neurology 80:963–965. 20. Willer T, Lee H, Lommel M, Yoshida-Moriguchi T, de Bernabe DB, Venzke D et al (2012) ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker–Warburg syndrome. Nat Genet 44:575–580. 21. Yang AC, Ng BG, Moore SA, Rush J, Waechter CJ, Raymond KM et al (2013) Congenital disorder of glycosylation due to DPM1 mutations presenting with dystroglycanopathy-type congenital muscular dystrophy. Mol Genet Metab 110:345–351. 22. Yoneyama T, Angata K, Bao X, Courtneidge S, Chanda SK, Fukuda M (2012) Fer kinase regulates cell migration through alpha-dystroglycan glycosylation. Mol Biol Cell 23:771–780. 23. Yoshida A, Kobayashi K, Manya H, Taniguchi K, Kano H, Mizuno M et al (2001) Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 1:717–724. 24. Yoshida-Moriguchi T, Willer T, Anderson ME, Venzke D, Whyte T, Muntoni F et al (2013) SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function. Science 341:896–899.
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MEETINGS PROCEEDINGS
Third International Workshop for Glycosylation Defects in Muscular Dystrophies, 18–19 April 2013, Charlotte, USA Anthony Blaeser1; Susan Sparks2; Susan C. Brown3; Kevin Campbell4; Qi Lu1 1 McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center and 2 Clinical Genetics/Department of Pediatrics, Levine Children’s Hospital, Carolinas Medical Center, Carolinas Healthcare System, Charlotte, NC. 3 Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK. 4 Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA.
Keywords dystroglycan, dystroglycanopathy, fukutin related protein, glycosylation, muscular dystrophy, therapy. Corresponding author: Anthony Blaeser, PhD, Carolinas Healthcare System, 1000 Blythe Blvd, Charlotte, NC 28203 (Email: anthony.blaeser@ carolinashealthcare.org) Received 17 December 2013 Accepted 18 December 2013 Published Online Article Accepted 8 January 2014 doi:10.1111/bpa.12118
INTRODUCTION The Third International Workshop for Glycosylation Defects in Muscular Dystrophies took place on 18–19 April 2013. It was held at the Omni Charlotte in Charlotte, NC, USA and hosted by the McColl-Lockwood Laboratory for Muscular Dystrophy Research, the Carolinas Medical Center and the Carolinas Education Institute. The event was sponsored by the Carolinas Healthcare Foundation, the Muscular Dystrophy Association (MDA), funds raised by “Jeans, Genes and Geniuses” organized by Jane and Luther Lockwood, as well as generous support by the McColl and Lockwood families. Since its founding in 2008, the International Workshop for Glycosylation Defects in Muscular Dystrophies has been a platform for bringing together top scientists and clinicians from around the world to discuss current research in the area of dystroglycanopathies [forms of muscular dystrophy associated with the hypoglycosylation of alpha-dystroglycan (α-DG) ] as well as providing a setting to facilitate collaborations. For this workshop, 22 scientists and clinicians were brought in from the US, UK and Japan for a total of 23 talks spread over 2 days. Over the past 2 years, significant progress has been made in the dystroglycanopathy field. Such advances include a greater knowledge of the mechanisms involved in the glycosylation of α-DG, the identification of additional genes that, when carrying mutations, are involved in the disease process and the development of new animal models and their use in developing new targets for treatment. New data have become available for adeno-associated virus (AAV)-
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mediated gene therapy in FKRP (fukutin-related protein) deficient animal models and compound library screening has taken place in several labs resulting in multiple hits with the potential for upregulating the functional glycosylation of α-DG and other proteins. As a testament to the progress being made, this year’s workshop devoted an entire session to discussing clinical treatments, management and endpoints. The 2013 workshop focused on the following topics: mechanisms involved in α-DG glycosylation and newly developed animal models for examining pathways and disease targets; AAV therapy and drug discovery, including possible mechanisms and targets for treating dystroglycanopathies; genetic diagnosis, clinical management and endpoint markers, including current data from clinical trials and methods for assessing treatments. The audience included members of Carolinas Medical Center, Children’s National Medical Center D.C., Genzyme Corporation, BioMarin Pharmaceuticals, Pfizer Corporation and University of Massachusetts Medical School.
BACKGROUND Dystroglycan is a key component of the dystrophin glycoprotein complex (DGC). It is encoded by a single gene (Dag1), the transcript of which is post-translationally cleaved into two subunits, alpha and beta. α-DG is heavily glycosylated and the sugar moieties in the central mucin-like region play an essential role in the binding to various extracellular matrix components such as laminin, perlecan and agrin. Alterations in the glycosylation of α-DG result Brain Pathology 24 (2014) 280–284 © 2014 International Society of Neuropathology
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in a subset of muscular dystrophies referred to as dystroglycanopathies (14). The diseases vary in severity from the severe Walker– Warburg syndrome (WWS) and muscle–eye–brain (MEB) disease, which display structural brain involvement in addition to muscular dystrophy, to the relatively mild forms of limb–girdle muscular dystrophy (LGMD) such as LGMD2I with little or no central nervous system (CNS) involvement (5, 13). The dystroglycanopathies have so far been associated with mutations in up to 16 putative or demonstrated glycosyltransferases, which include LARGE, fukutin, FKRP, POMT1, POMT2 and POMGnT1, each of which result in the aberrant glycosylation of α-DG (2, 4, 11, 12, 23). However, there are still a large proportion of patients with reduced α-DG glycosylation that lack a defined genetic diagnosis. At the current time, there is no effective therapy available for dystroglycanopathies. A number of animal models have been described previously, but no experimental therapy has been reported. The pathway leading to the functional glycosylation of α-DG is not clearly understood, and reagents and methods for identifying diversity of glycosylated α-DG remain a challenge. Research in all these areas has made significant progress during the last 2 years as presented during the meeting. At the same time, clinic preparation has geared up for future clinic trials with several lines of experimental therapies emerging from the ongoing translational research.
REPORT SUMMARY Glycosylation The workshop began with opening remarks by Bernard Brigonnet, Vice President of Research, Carolinas Healthcare System. The first session was chaired by James Ervasti from the University of Minnesota Department of Biochemistry, Molecular Biology and Biophysics. To start the session Dr. James Ervasti shared his view of the more complex nature of α-DG glycosylation in muscle and the difficulties associated with determining the status of so-called functional glycosylation, a critical step if we are to understand the function of each of the so-called “glycosylation” genes in the glycosylation process. Sample preparation, sample buffer composition and pH, denaturation temperature and duration, as well as protease inhibiters could all affect the size and signal intensity of the species of α-DG glycosylation detected by IIH6 (an antibody commonly used to identify the glycosylation status of α-DG) and laminin-binding assays. Dr. Ervasti suggested that greater care should be taken to standardize blotting procedures for detecting α-DG and data interpretation. This also raised the need to develop new reagents for possibly more reliable determination of α-DG glycosylation status (see below). Work from Dr. Ervasti’s lab also suggested a possible interaction between dystroglycan and other membrane-associated proteins such as caveolin, which might play a role for maintaining muscle fiber stability (10). To address the difficulty in detecting dystroglycan reliably, Dr. Glenn Morris presented his lab’s efforts (with support from LGMD2I Fund) to generate specific monoclonal antibodies for functionally glycosylated α- and β-DG. Dr. Morris reported initial evidence that clones of such antibodies have been created. However, further selection and characterization are required. Using a zebra fish model system, Dr. Tamao Endo illustrated his studies showing that POMT1/2 play an essential role in the normal
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development of zebra fish through the O-mannosylation of α-DG. Whilst the glycosylation of α-DG is important for maintaining muscle stability, the regulation of this process remains largely unknown. Dr. Endo reported the importance of human natural killer-1 sulfotransferase (HNK-1ST) in modulating function of α-DG. Using a coexpression system in retinoic acid-treated S91 melanoma cells, Dr. Endo showed that expression of HNK-1ST mediated the incorporation of sulfate into α-DG. This is associated with suppressed expression and reduced ligand-binding activity of α-DG in the presence and absence of LARGE overexpression. The results suggest that sulfotransferase activity of HNK-1ST may be one of the mechanisms modulating α-DG glycosylation though the precise pathways are not understood. Dr. Susan Brown has followed up her earlier study of developing an FKRP knockdownneoY307N mutant mouse model. The original model proved to be embryonic and perinatal lethal. By crossbreeding with a strain of mouse expressing Cre recombinase under the SOX-1 promoter, Dr. Brown reported the creation of a new line with the restoration of functional α-DG glycosylation in the CNS but not skeletal muscle, thus producing viable mice with clear dystrophic phenotype. This model will now be more useful for mechanistic and therapeutic studies. Dr. Anthony Blaeser presented further data in FKRP mouse model characterization conducted in McColl-Lockwood Lab. Through crossbreeding of original strains of FKRP mutants with P448L, L276I and E310X nonsense mutation, a set of mouse models with wide range of disease severity has been created with dystrophic phenotypes ranging from very mild, limb–girdle muscular dystrophy 2I, to severe Walker–Warburg syndrome and muscle–eye–brain disease with brain and eye defects (3, 8). These animal models will play a crucial role in understanding the mechanisms of α-DG glycosylation as well as in developing experimental therapies. Dystroglycan phosphorylation was suggested as a new therapeutic target for dystroglycanopathies by Dr. Steve Winder. Dr. Winder proposed that the tyrosine phosphorylation of β-DG plays a crucial role in the loss of DGC from the membrane and might represent a therapeutic target. Using mouse myoblasts, Dr. Winder was able to demonstrate an increase in non-phosphorylated dystroglycan. Also, in sapje (dystrophin-deficient) zebra fish, the inhibition of tyrosine phosphorylation was able to rescue the dystrophic phenotype. Dr. Huaiyu Hu presented new data suggesting a role for glycosyltranferases in neural development. Using POMGnT1 knockout mice, Dr. Hu demonstrated that the hypoglycosylation of α-DG resulted in the disruption of basement membrane assembly during neural tissue development, leading to reduced levels of key basement membrane components and the weakening of the basement membrane. This could be the mechanism involved in the alteration of neuronal migration. To explore regulatory pathways of α-DG glycosylation, Dr. Minoru Fukuda applied the siRNA library screening technique and identified Fer kinase as a key regulator of laminin-binding glycan expression on breast and prostate cancer cells (22). Dr. Fukuda demonstrated that knockdown of Fer kinase increased transcription levels of β3GnT1 and LARGE. However, expression of Fert2, (a homolog of Fer) in mouse C2C12 myoblast cells, downregulated β3GntT1 expression but not LARGE. The knockdown of Fert2 was able to upregulate laminin-binding glycan expression and 281
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myotube formation, suggesting a novel role of Fer kinase in glycosylation regulation. Dr. Kevin Campbell presented data of LARGE function in the glycosylation of α-DG. Dr. Campbell demonstrated the bifunctionality of LARGE with both xylosyltransferase and glucoronyltransferase activities (9). These activities play an important role in the laminin-binding efficiency of glycosylated α-DG.
AAV THERAPY/DRUG DISCOVERY The second session was chaired by Qi Long Lu from the Carolinas Medical Center. Dr. Xiao Xiao began the session by discussing the use of AAV9 vectors for the delivery of FKRP to L276I-FKRP mutant mice, which exhibit a mild disease phenotype with late onset. It was apparent that the expression of a human FKRP transgene restored glycosylation of α-DG and ameliorated the dystrophic pathology in both skeletal and cardiac muscles. Dr. Qi Long Lu further demonstrated the effectiveness of AAV9-FKRP therapy in FKRP-P448Lneo mutant mice, which present moderate dystrophic phenotype with disease onset before 4 weeks of age. Dr. Lu showed that AAV-FKRP treatment was able to restore the functional glycosylation of α-DG in all skeletal and heart muscles. This led to a significant improvement in muscle pathology, strength and serum markers. No detectable toxicity was observed, suggesting the feasibility of gene therapy as an effective treatment for FKRP-related muscular dystrophies. In line with the effort to move FKRP gene therapy using AAV vectors toward clinic trials, Dr. John Gray introduced clinical trials using AAV8-mediated gene therapy in Hemophilia B patients. Preliminary results from this study proved to be encouraging with patients maintaining expression at 1%–6% normal levels after a period of 1.5 years. However, cell-mediated immunity to AAV8 remains a concern with some of the high-dose cohort patients showing elevated levels of AAV8 capsid-specific T-cells. Dr. Tatsushi Toda discussed the use of antisense oligonucleotides targeting various splice acceptor and enhancer sites to prevent pathogenic exon-trapping in Fukuyama congenital muscular dystrophy (FCMD) mouse models and the cells of patients with FCMD. The disease is largely the result of retrotransposal insertion in the 3′ UTR region of the fukutin gene, leading to the altered splicing and the addition of a new sequence in the 3′ end of the transcript with loss of function. Dr. Toda designed specific antisense oligonucleotides targeting splicing acceptor and enhancer sites for restoration of normal fukutin protein and reported success in doing so by using a cocktail of antisense oligonucleotides in both human cells and in the mouse model. Dr. Toda also presented data supporting the hypothesis that FKRP is also involved in the post-phosphoryl modification of α-DG. In addition, Dr. Toda reported two distinct fukutin conditional knockout (cKO) mice and AAV9-fukutin therapy. They proposed that myofiber-selective expression of fukutin can also be an effective therapeutic strategy. Using cell-based assays on patient-derived cells, Dr. Anne Bang has developed a 384-well based assay for identifying lead compounds to treat LGMD2I. A screening of 6000 pharmacologically active compounds resulted in the identification of nine compounds that increase laminin binding in FKRP-deficient patient cells. Dr. Bang has also developed induced pluripotent stem cells (iPSC) from dystroglycanopathy patient-derived tissue. This will allow Dr. Bang to further differentiate the iPSC into disease-relevant cell 282
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types including myogenic cells and myofibers, cardiomyocytes, and neural cells. This could be highly valuable for drug screening as effect of any potential compound may well be cell-specific. Dr. Xiaohua Wu reported his finding on drug screening of a larger library with about 420 000 small compounds in Chinese hamster ovary (CHO) cells with glycosylation levels and laminin binding as a readout. Several dozen compounds were identified that significantly increase the expression of functionally glycosylated α-DG. However, the enhanced expression of α-DG appears to be cell-type dependant, suggesting cell-specific regulatory mechanisms may underlie functional glycosylation. Dr. Wu emphasized the importance of cell-type specificity of drug response and the need to establish more relevant in vitro screen system for further drug development. Another strategy for drug screening was adopted by Dr. Lou Kunkel. Dr. Kunkel screened a drug library in dystroglycan-null zebra fish, in an attempt to find candidate compounds capable of alleviating the dystrophic phenotype. Preliminary screening resulted in 11 candidates that prevent muscle pathology in short-term treatments. Of these compounds, one was able to dramatically increase the survival of dystroglycannull zebra fish and reverse the pathology. This is interesting as it implies that factors other than functional glycosylated α-DG are able to compensate for the lack of dystroglycan. Dr. Kunkel is further testing the candidate drug in different cell and animal model systems. Currently, immunohistochemistry and western blot are the most widely used methods of detecting functionally glycosylated α-DG. However, Dr. Francesco Muntoni demonstrated the use of flow cytometry for the detection of IIH6-reactive functionally glycosylated α-DG in patients-derived fibroblasts. The results show that the levels of glycosylated α-DG in fibroblasts are comparable to that in patient-derived skeletal muscle cells, supporting the view that examination of fibroblasts in conjunction with flow cytometry could be of diagnostic value for dystroglycanopathies. Dr. Muntoni also applied flow cytometry for drug screening with a library of about 1000 compounds and reported several hits with increase in α-DG glycosylation. Dr. Muntoni suggested the use of flow cytometry as a platform for conducting high throughput assays.
CLINICAL MANAGEMENT/ENDPOINTS The final session was chaired by Susan Sparks from the Carolinas Medical Center. Dr. Francesco Muntoni returned to discuss the identification of a number of novel genes, mutations in which are associated with a dystroglycanopathy phenotype. These included at least six other genes in the last 2 years and include:—B3GALnT2, a glycosyltransferase located in the endoplasmic reticulum, mutations in which were found to underlie WWS and MEB (18); GDP-mannose pyrophosphorylase B (GMPPB), mutations in which were identified in patients with varying severities of disease from MEB to LGMD (7); isoprenoid synthase domain containing (ISPD) gene, mutations in which were shown in individuals with severe WWS (16, 20) and subsequently milder LGMD (19); dolichol synthesis (DPM1 and DPM2), mutations in which were associated with a dystroglycanopathy-type congenital muscular dystrophy (1, 21); B3GNT1, mutations in which were found to underlie WWS (6, 17); GTDC2, mutations in which were identified
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in patients with WWS (15); and finally SGK196, mutations in which were associated with congenital muscular dystrophy (24). Dr. Susan Sparks reported on a study of expression profiling in patients by looking at the expression of various growth factors and cytokines in LGMD patients with FKRP mutations. Dr. Sparks identified 373 transcripts, which are differentially expressed compared with normal controls. The results showed that FKRP patients contain more downregulated transcripts than typically seen in other dystrophies. Of note was the downregulation of genes involved in the TGF-B and IGF-II signaling pathways, which are generally upregulated in other dystrophies. Identifying biomarkers specific for FKRP-dystroglycanopathies and disease progression remains to be investigated, but crucial for clinic evaluation of any experimental therapy. Dr. Madhuri Hegde addressed the issue of identifying disease subtypes through the use of next-generation sequencing and whole exomes. She reviewed the current technology, utilization and limitations. Utilizing patient-derived data, including sequencing and phenotypes, known and unknown variants can be catalogued using a newly established website. This site can then be used to examine relevant genes associated with a patient’s clinical presentation. This allows enhanced evaluation for clinical phenotypes. Tina Duong continued the discussion of evaluating clinical outcome measures in patients with LGMD. Examining various conditions, based on strength and functional tests, baseline measures were established for a variety of LGMD subtypes. When compared with other LGMD subtypes, patients with FKRP showed lower forced vital capacity and sparing of grip strength. Additionally, 6-minute walking test (MWT) values seem to be more highly correlated to lower extremity strength than respiratory function. Results from this pilot study validate previously published works on FKRP phenotype using a much smaller cohort and would help in future experimental designs and outcomes selection. Dr. Volker Straub assessed LGMD patients with FKRP of different clinical severities with similar evaluation methods as those described by Tina Duong. He also included muscle magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) showing a correlation between fat infiltration and muscle function. Dr. Straub suggests that quantitative MRI is better suited to measure disease progression than physical assessments. Furthermore, Dr. Straub presented the global FKRP registry websites (https:// www.fkrp-registry.org) to identify potential patients eligible for clinical trials as well as providing a better understanding of the prevalence and distribution of those affected by FKRP mutations. The final talk of the session was given by Dr. Claudia Mitchell regarding various research supported by the LGMD2I Research Fund. These include research in developing patient iPS cells, antibodies to α-DG, various FKRP animal models and drug screening as well as various experimental therapies. The Fund is also involved in the formation of various websites and clinical trials as well as discussing social media avenues to encourage patient involvement in research.
WORKSHOP ORGANIZERS The workshop was organized with the help of Dr. Qi Long Lu, Dr. Anthony Blaeser, Dr. Kevin Campbell, Dr. Susan Sparks, Jeannie Maggio, Cameron Davis, Caren Anderson and Ashley Katkin.
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LIST OF SPEAKERS (i) Anne Bang, Sanford-Burnham Medical Research Institute (ii) Anthony Blaeser, Carolinas Medical Center (iii) Susan Brown, Royal Veterinary College, University of London (iv) Kevin Campbell, University of Iowa (v) Tina Duong, Children’s National Medical Center (vi) Tamao Endo, Tokyo Metropolitan Institute of Gerontology (vii) James Ervasti, University of Minnesota (viii) Minoru Fukuda, Sanford-Burnham Medical Research Institute (ix) John Gray, St. Jude Children’s Research Hospital (x) Madhuri Hegde, Emory University School of Medicine (xi) Huaiyu Hu, SUNY Upstate Medical University (xii) Louis Kunkel, Boston Children’s Hospital (xiii) Qi Long Lu, Carolinas Medical Center (xiv) Claudia Mitchell, LGMD2i Research Fund (xv) Glenn Morris, Robert Jones & Agnes Hunt Orthopaedic Hospital (xvi) Francesco Muntoni, University College London (xvii) Susan Sparks, Levine Children’s Hospital (xviii) Volker Straub, Newcastle University (xix) Tatsushi Toda, Kobe University (xx) Steve Winder, University of Sheffield (xxi) Xiaohua Wu, Carolinas Medical Center (xxii) Xiao Xiao, University of North Carolina at Chapel Hill
WORKSHOP WEBSITE A website for the Third International Workshop for Glycosylation Defects can be accessed at http://www.carolinashealthcare.org/ md-international-workshop.
ACKNOWLEDGEMENTS Generous financial support for the workshop was provided by “Jeans, Genes and Geniuses” charity event organized by Jane and Luther Lockwood, the McColl and Lockwood families, the Carolinas Healthcare Foundation and the Muscular Dystrophy Association (MDA). Special thanks would also be given to the following people for their help: Cameron Davis, Ashley Katkin and Susan Rucho from CEI for planning the event; Jane Howard for making all of the travel arrangements; Frederick Jones for arranging our audio visual needs; Rebekah Law from the Omni Charlotte for all of her help organizing the event; and Malia Rodgers for her assistance in setting up the workshop website.
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3. Blaeser A, Keramaris E, Chan YM, Sparks S, Cowley D, Xiao X, Lu QL (2013) Mouse models of fukutin-related protein mutations show a wide range of disease phenotypes. Hum Genet 132:923–934. 4. Brockington M, Blake DJ, Prandini P, Brown SC, Torelli S, Benson MA et al (2001) Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 69:1198–1209. 5. Brown SC, Torelli S, Brockington M, Yuva Y, Jimenez C, Feng L et al (2004) Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 164:727–737. 6. Buysse K, Riemersma M, Powell G, van Reeuwijk J, Chitayat D, Roscioli T et al (2013) Missense mutations in beta-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker–Warburg syndrome. Hum Mol Genet 22:1746–1754. 7. Carss KJ, Stevens E, Foley AR, Cirak S, Riemersma M, Torelli S et al (2013) Mutations in GDP-mannose pyrophosphorylase B cause congenital and limb–girdle muscular dystrophies associated with hypoglycosylation of alpha-dystroglycan. Am J Hum Genet 93:29–41. 8. Chan YM, Keramaris-Vrantsis E, Lidov HG, Norton JH, Zinchenko N, Gruber HE et al (2010) Fukutin-related protein is essential for mouse muscle, brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies. Hum Mol Genet 19:3995–4006. 9. Inamori K, Hara Y, Willer T, Anderson ME, Zhu Z, Yoshida-Moriguchi T, Campbell KP (2013) Xylosyl- and glucuronyltransferase functions of LARGE in alpha-dystroglycan modification are conserved in LARGE2. Glycobiology 23:295–302. 10. Johnson EK, Li B, Yoon JH, Flanigan KM, Martin PT, Ervasti J, Montanaro F et al (2013) Identification of new dystroglycan complexes in skeletal muscle. PLoS ONE 8:e73224. 11. Lefeber DJ, Schonberger J, Morava E, Guillard M, Huyben KM, Verrijp K et al (2009) Deficiency of Dol-P-Man synthase subunit DPM3 bridges the congenital disorders of glycosylation with the dystroglycanopathies. Am J Hum Genet 85:76–86. 12. Longman C, Brockington M, Torelli S, Jimenez-Mallebrera C, Kennedy C, Khalil N et al (2003) Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan. Hum Mol Genet 12:2853–2861.
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13. Mendell JR, Boue DR, Martin PT (2006) The congenital muscular dystrophies: recent advances and molecular insights. Pediatr Dev Pathol 9:427–443. 14. Muntoni F (2004) Journey into muscular dystrophies caused by abnormal glycosylation. Acta Myol 23:79–84. 15. Ogawa M, Nakamura N, Nakayama Y, Kurosaka A, Manya H, Kanagawa M et al (2013) GTDC2 modifies O-mannosylated alpha-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody. Biochem Biophys Res Commun 440:88–93. 16. Roscioli T, Kamsteeg EJ, Buysse K, Maystadt I, van Reeuwijk J, van den Elzen C et al (2012) Mutations in ISPD cause Walker–Warburg syndrome and defective glycosylation of alpha-dystroglycan. Nat Genet 44:581–585. 17. Shaheen R, Faqeih E, Ansari S, Alkuraya FS (2013) A truncating mutation in B3GNT1 causes severe Walker–Warburg syndrome. Neurogenetics 14:243–245. 18. Stevens E, Carss KJ, Cirak S, Foley AR, Torelli S, Willer T et al (2013) Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of alpha-dystroglycan. Am J Hum Genet 92:354–365. 19. Tasca G, Moro F, Aiello C, Cassandrini D, Fiorillo C, Bertini E et al (2013) Limb–girdle muscular dystrophy with alphadystroglycan deficiency and mutations in the ISPD gene. Neurology 80:963–965. 20. Willer T, Lee H, Lommel M, Yoshida-Moriguchi T, de Bernabe DB, Venzke D et al (2012) ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker–Warburg syndrome. Nat Genet 44:575–580. 21. Yang AC, Ng BG, Moore SA, Rush J, Waechter CJ, Raymond KM et al (2013) Congenital disorder of glycosylation due to DPM1 mutations presenting with dystroglycanopathy-type congenital muscular dystrophy. Mol Genet Metab 110:345–351. 22. Yoneyama T, Angata K, Bao X, Courtneidge S, Chanda SK, Fukuda M (2012) Fer kinase regulates cell migration through alpha-dystroglycan glycosylation. Mol Biol Cell 23:771–780. 23. Yoshida A, Kobayashi K, Manya H, Taniguchi K, Kano H, Mizuno M et al (2001) Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 1:717–724. 24. Yoshida-Moriguchi T, Willer T, Anderson ME, Venzke D, Whyte T, Muntoni F et al (2013) SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function. Science 341:896–899.
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