- Email: [email protected]
International workshop: Glycosylation defects in muscular dystrophies – Enhancing glycosylation to fight muscle diseases, 15–16 May, 2008, Charlotte, USA
Neuromuscular Disorders 18 (2008) 1002–1004
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Workshop report
International workshop: Glycosylation defects in muscular dystrophies – Enhancing glycosylation to fight muscle diseases, 15–16 May, 2008, Charlotte, USA Yiumo Michael Chan a,*, Susan Brown b, Qi Lu a a b
McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC 28231, USA Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Campus, UK
1. Introduction
3. Report summary
The first international workshop on glycosylation defects in muscular dystrophies (IWGDMD) took place on May 15 and 16 in Charlotte, North Carolina, USA. The meeting was organized by the McColl–Lockwood Laboratory for Muscular Dystrophy Research at the Carolinas Medical Center (CMC) and assembled 15 clinicians and scientists from US, Europe and Japan. In addition, the workshop was joined by participants from Carolinas Medical Center, University of North Carolina at Charlotte, University of North Carolina at Chapel Hill, Wake Forest University and Virginia Tech. The goals of the workshop are (1) to examine the current status of research in a new class of muscular dystrophies that are primarily associated with glycosylation defects, (2) to facilitate international collaborations in developing treatment plans.
3.1. Clinical and laboratory diagnosis
2. Background Glycosylation is a common post-translational modification event for proteins to maintain essential cellular functions. Recently, a new class of genes has been implicated in different types of muscular dystrophies [1]. Despite the variations in the clinical phenotypes, they are often associated with abnormal glycosylation of a-dystroglycan in muscle, therefore commonly referred as ‘‘dystroglycanopathies” [2–4]. As of today, mutations in six genes have been identified in patients with different dystroglycanopathies; these are fukutin-related protein (FKRP), fukutin, POMT1, POMT2, POMGnT1 and LARGE [5–10]. All these genes encode known or putative glycosyltransferases that are thought to be involved in the O-mannosylation (a special type of O-linked glycosylation) of a-dystroglycan [11]. It has been suggested that aberrant glycosylation of a-dystroglycan disrupts its binding to several proteins in the extracellular matrix including laminin, agrin and perlecan, resulting in muscle weakness and cell death [12–14]. However, the exact disease mechanism is not fully understood and currently, there is no effective treatment available.
* Corresponding author. Tel.: +1 704 355 6427; fax: +1 704 355 1679. E-mail address: [email protected] (Y.M. Chan).
Herbert Bonkovsky, Vice President of Research in CMC, delivered the opening remarks for the conference and welcomed the delegates to Charlotte and Carolinas Medical Center. The first session, chaired by Benjamin Brooks, Director of the Carolinas neuromuscular/ALS-MDA Center at CMC, provided an overview of current clinical diagnosis and management of the diseases. Susan Sparks discussed the challenge in determining genotype– phenotype correlation in limb girdle muscular dystrophy type 2I (LGMD2I) due to the highly variable clinical spectrums associated with FKRP mutations. Although there seems to be a general correlation between the amount of residual glycosylation of a-dystroglycan and severity, exceptions were observed in some patients. To better define and characterize LGMD2I, microarray technology was used to identify molecular networks that are specifically altered in LGMD2I but not in other types of muscular dystrophies. This approach has the potential to allow identification of critical downstream elements that can be targeted for therapies in the future. Pascale Guicheney also reiterated that the spectrum of clinical phenotypes associated with the dystroglycanopathies is broader than expected. Noticeably, some patients have mild phenotypes despite the severe reduction of a-dystroglycan glycosylation. It was proposed that a combined approach using genetics, immunohistochemistry and measurement of glycosyltransferase activities (namely POMT and POMGnT1) in immortalized lymphoblastoid cell lines from patients is helpful in the identification and characterization of dystroglycanopathies. Ichizo Nishino concluded the morning session with his work on Fukuyama congenital muscular dystrophy (FCMD), which is caused by mutations in the fukutin gene. He pointed out that the frequency of each subtype of a-dystroglycanopathies varies among different ethnic groups, with FCMD being the most predominant form of congenital muscular dystrophy in Japan. Interestingly, despite extensive genetic analysis, there are still patients without mutations in the six known genes responsible for dystroglycanopathies, indicating the existence of yet-to-be-identified genes.
0960-8966/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.09.007
Y.M. Chan et al. / Neuromuscular Disorders 18 (2008) 1002–1004
3.2. Pathogenesis and animal models The afternoon session devoted to the critical role of glycosylation in muscular dystrophies. Participants reviewed the use of different animal models and other essential tools to investigate the diseases. Jane Hewitt used the myodystrophy (myd) mice to study the LARGE gene family. Several studies have suggested that up-regulation of LARGE can be used as a potential therapy to compensate for the loss of other glycosyltransferases in dystroglycanopathies. The role and function of LARGE2, a separate but similar gene to LARGE, was discussed. In addition, zebrafish was explored as a useful alternative model for the diseases since it appeared that the glycosylation pathway for a-dystroglycan is highly conserved in this organism. Genri Kawahara discussed the development of a zebrafish model for LGMD2I. When FKRP expression was knocked down with morpholinos, zebrafish displayed abnormal formation of muscle, heart and head, similar to human patients. Given that several of the mouse models for dystroglycanopathies are embryonic lethal, the use of zebrafish as an alternative model clearly provides a major benefit and advantage. Susan Brown then presented a new exciting mouse model for FKRP-related muscular dystrophies. A FKRP mutant line of mice was generated and developed a phenotype reminiscent of muscle-eye-brain disease. These mice display a severe reduction of a-dystroglycan offering new and valuable insights into the function of FKRP. One of the key issues will be to validate and develop these mice into a useful model that can be used to test potential therapeutic compounds in the future. Tatsushi Toda discussed his work on fukutin and FCMD. By generating different lines of fukutin mutant mice, he suggested that a small amount of functional glycosylated a-dystroglycan might be sufficient to prevent disease progress. To develop effective therapies, a major consideration is the level of the functional dystroglycan and glycosyltransferase in muscle required for normal function. Another key question is how to document the putative enzymatic activities for fukutin and FKRP. Surprisingly, Dr. Toda demonstrated an unexpected role of fukutin in interacting with POMGnT1 and regulating its glycosyltransferase activity. Kevin Campbell further emphasized the importance of dystroglycan in the disease process. He reported the identity of specific conserved residues that are important for functional dystroglycan glycosylation by LARGE. The basic mechanism underlying this subset of muscular dystrophies seems to be hypoglycosylation of a-dystroglycan, resulting in disrupting interaction with its ligand, namely, laminin in the extracellular matrix. The selection of appropriate promoters for generating mouse models was mentioned in the presentation since the use of different promoters might affect the expression of the genes they regulate, thereby changing the outcomes and interpretations. During the discussion, the issue regarding the glycoepitopes recognized by the two most commonly used antibodies against a-dystroglycan (IIH6C4 and VIA4-1) was raised. The characterization of these epitopes will be essential for understanding dystroglycan glycosylation and the nature of the aberrantly glycosylated dystroglycan. Derek Blake has investigated the role of FKRP in LGMD2I from a different perspective. Using a heterologous cell expression system, his group found that many of the disease-causing FKRP mutations caused the proteins to misfold in specific manners, leading to their retention in the endoplasmic reticulum. Similar to the mislocalization of CFTR mutant protein documented in cystic fibrosis, the folding, processing and trafficking of FKRP might contribute to the pathogenesis of muscular dystrophies. The possibility of identifying proteins that can correct the folding and trafficking of mutant FKRP was discussed. One of the approaches discussed was to utilize mass spectrometry.
1003
Tamao Endo provided an extensive review of the complexity of the O-mannosylation pathway with respect to a-dystroglycan. To gain insight into the regulation of dystroglycan glycosylation, the specific amino acids on a-dystroglycan recognized by POMT1 and 2 were determined. It also appeared that POMT1 and POMT2 form an active enzyme complex essential for glycosyltransferase activity. The ability to assay this activity will be an important part of work focused on identifying additional O-mannosylated proteins and their functional role in muscular dystrophies. The evening was concluded with welcome addresses by James McDeavitt, Senior Vice President in CMC and Mr. Hugh McColl, Jr., the founding donor of the Carolinas Muscular Dystrophy Research Endowment. 3.3. Experimental therapies The second day’s session focused on potential treatments for dystroglycanopathies. Speakers reviewed and discussed different treatment strategies. Key areas of emphasis included how to effectively disseminate current knowledge/technology into the development of applicable therapies. John Vissing discussed the potential benefits of endurance training for LGMD2I patients. It was suggested that aerobic training, such as cycling, is safe and provides some benefit to patients. The potential benefit of other types of exercises, for example, strengthen training is also being investigated. Discussions highlighted the possibility of combination therapies using both drugs and physical exercise for muscular dystrophies in the future. In addition, Dr. Vissing reported that the L276I FKRP mutation has a very high prevalence in Denmark, suggesting a founder effect for this particular mutation. Paul Martin provided evidence that overexpression of cytotoxic T cell GalNAc transferase (Galgt2) has therapeutic values in several animal models of muscular dystrophies, including DMD, CMD and sarcoglycan-deficient LGMD. The possibility of GalgT2 being a suitable molecular target for therapy in dystroglycanopathies was discussed but it was pointed out that initial efforts must be addressed to increase efficiency and level of expression. Drug screening strategy to identify compounds that can up-regulate GalgT2 activity in human was also examined. Xiao Xiao discussed the various adeno-associated virus (AAV)based vectors and approaches to improve the efficiency of gene transfer and to reduce the immune response associated with gene therapy. Dr. Xiao’s recently studies have demonstrated successful systematic delivery of (1) a miniature dystrophin gene to DMD mouse and dog models, (2) a d-sarcoglycan gene to a LGMD2F hamster model, (3) an agrin gene to a congenital muscular dystrophy mouse model. These vectors are being further developed for systemic delivery of FKRP gene. In addition, they can be used to deliver small hairpin interference RNA post-natally in mice to knock down FKRP expression to generate LGMD2I phenotypes for functional studies. Ellen Welch took a different approach with regard to developing a potential treatment for muscular dystrophies. The read-through strategy allows translational machinery in cells to ignore premature stop codons due to nonsense mutations, leading to production of near-normal protein. Currently, clinical trials for a subset of DMD patients with nonsense mutations are being conducted using read-through compounds developed by PTC Therapeutics. This strategy clearly has the potential to be used to treat dystroglycanopathies caused by nonsense mutations. The key question to be addressed is whether the read-through compounds can result in sufficient amounts of glycosyltransferases to restore normal muscle function. In addition, considerable efforts have been made to identify small molecules that can up-regulate specific protein targets known to be involved in muscular dystrophies.
1004
Y.M. Chan et al. / Neuromuscular Disorders 18 (2008) 1002–1004
Qi Lu provided a detailed overview of the diseases and other potential treatments currently pursued in the laboratory. It has become apparent that an important approach will be to artificially increase a-dystroglycan glycosylation. A drug screening system has been established to search for small molecules with the potential to enhance glycosylation of the targeted a-dystroglycan. The laboratory is also collaborating with PTC Therapeutics to determine the potential value of candidate read-through drugs for LGMD2I. To conclude the workshop, Dr. Lu stated that ‘‘It is clear that wider collaborations among the limited number of laboratories engaged in the study of dystroglycanopathies are important to further our understanding of the disease mechanisms and development of animal models and effective treatments’. 4. The workshop organizers Yiumo Michael Chan, PhD, Carolinas Medical Center, US, Jeannie Maggio, Carolinas Medical Center, US, Qi Lu, MD, PhD, Carolinas Medical Center, US, Herbert Bonkovsky, MD, Carolinas Medical Center, US, Derek Blake, PhD, Cardiff University, UK, Francesco Muntoni, MD, Imperial College, UK.
McColl and Lockwood families as well as Carolinas HealthCare Foundation. The laboratory focuses on the translational research for limb girdle muscular dystrophy type 2I. Acknowledgements This workshop was made possible by the financial support of the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation and the Muscular Dystrophy Association, USA. Special thanks are due to Mr. and Mrs. Luther Lockwood for their substantial support of this workshop through their successful charity event, ‘‘Jeans, Genes and Geniuses”. In additional, the Organizing Committee thank Jeannie Maggio for working meticulously on every aspect of the meeting. Gracious support was also provided by Dr. Lu, Dr. Blake and Dr. Muntoni. The Organizing Committee extend appreciation to Libby Anderson, Ashley Ginn and Denise Moseley at the Marketing Department for website design and marketing materials and Frederick Jones and Trey Maggio for audio-visual assistance. References
5. List of participants 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Derek Blake, PhD, Cardiff University, UK Susan Brown, PhD, Imperial College, UK Kevin Campbell, PhD, University of Iowa, USA Tamao Endo, PhD, TMIG, Japan Pascale Guicheney, PhD, INSERM, France Jane Hewitt, PhD, University of Nottingham, UK Genri Kawahara, PhD, Children’s Hospital Boston, USA Qi Lu, MD, PhD, Carolinas Medical Center, USA Paul Martin, PhD, Ohio State University, USA Ichizo Nishino, MD, PhD, NCNP, Japan Susan Sparks, MD, PhD, Children’s National Medical Center, USA Tatsushi Toda, MD, PhD, Osaka University, Japan John Vissing, MD, DMSci, University of Copenhagen, Denmark Ellen Welch, PhD, PTC Therapeutics, USA Xiao Xiao, PhD, University of North Carolina, Chapel Hill, USA
6. Website For a full description of the workshop and program, please visit http://www.carolinasmedicalcenter.org/LGMD.
7. McColl–Lockwood Laboratory for Muscular Dystrophy Research The McColl–Lockwood Laboratory was established in 2005 with a generous research endowment grant jointly created by the
[1] Blake DJ, Esapa CT, Martin-Rendon E, McIlhinney RA. Glycosylation defects and muscular dystrophy. Adv Exp Med Biol 2005;564:97–8. [2] Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164(2):727–37. [3] Toda T, Chiyonobu T, Xiong H, et al. Fukutin and alpha-dystroglycanopathies. Acta Myol 2005;24(2):60–3. [4] Matsumoto H, Hayashi YK, Kim DS, et al. Congenital muscular dystrophy with glycosylation defects of alpha-dystroglycan in Japan. Neuromuscul Disord 2005;15(5):342–8. [5] Kobayashi K, Nakahori Y, Miyake M, et al. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 1998;394(6691):388–92. [6] Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10(25):2851–9. [7] Yoshida A, Kobayashi K, Manya H, et al. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 2001;1(5):717–24. [8] Beltran-Valero de Bernabe D, Currier S, Steinbrecher A, et al. Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker–Warburg syndrome. Am J Hum Genet 2002; 71(5):1033–43. [9] Longman C, Brockington M, Torelli S, et al. 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 2003;12(21):2853–61. [10] van Reeuwijk J, Janssen M, van den Elzen C, et al. POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker–Warburg syndrome. J Med Genet 2005;42(12):907–12. [11] Sasaki T, Yamada H, Matsumura K, Shimizu T, Kobata A, Endo T. Detection of Omannosyl glycans in rabbit skeletal muscle alpha-dystroglycan. Biochim Biophys Acta 1998;1425(3):599–606. [12] Michele DE, Barresi R, Kanagawa M, et al. Post-translational disruption of dystroglycan–ligand interactions in congenital muscular dystrophies. Nature 2002;418(6896):417–22. [13] Martin PT. Dystroglycan glycosylation and its role in matrix binding in skeletal muscle. Glycobiology 2003;13(8):55R–66R. [14] Grewal PK, Hewitt JE. Glycosylation defects: a new mechanism for muscular dystrophy? Hum Mol Genet 2003;12:R259–64.
Contents lists available at ScienceDirect
Neuromuscular Disorders journal homepage: www.elsevier.com/locate/nmd
Workshop report
International workshop: Glycosylation defects in muscular dystrophies – Enhancing glycosylation to fight muscle diseases, 15–16 May, 2008, Charlotte, USA Yiumo Michael Chan a,*, Susan Brown b, Qi Lu a a b
McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte, NC 28231, USA Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Campus, UK
1. Introduction
3. Report summary
The first international workshop on glycosylation defects in muscular dystrophies (IWGDMD) took place on May 15 and 16 in Charlotte, North Carolina, USA. The meeting was organized by the McColl–Lockwood Laboratory for Muscular Dystrophy Research at the Carolinas Medical Center (CMC) and assembled 15 clinicians and scientists from US, Europe and Japan. In addition, the workshop was joined by participants from Carolinas Medical Center, University of North Carolina at Charlotte, University of North Carolina at Chapel Hill, Wake Forest University and Virginia Tech. The goals of the workshop are (1) to examine the current status of research in a new class of muscular dystrophies that are primarily associated with glycosylation defects, (2) to facilitate international collaborations in developing treatment plans.
3.1. Clinical and laboratory diagnosis
2. Background Glycosylation is a common post-translational modification event for proteins to maintain essential cellular functions. Recently, a new class of genes has been implicated in different types of muscular dystrophies [1]. Despite the variations in the clinical phenotypes, they are often associated with abnormal glycosylation of a-dystroglycan in muscle, therefore commonly referred as ‘‘dystroglycanopathies” [2–4]. As of today, mutations in six genes have been identified in patients with different dystroglycanopathies; these are fukutin-related protein (FKRP), fukutin, POMT1, POMT2, POMGnT1 and LARGE [5–10]. All these genes encode known or putative glycosyltransferases that are thought to be involved in the O-mannosylation (a special type of O-linked glycosylation) of a-dystroglycan [11]. It has been suggested that aberrant glycosylation of a-dystroglycan disrupts its binding to several proteins in the extracellular matrix including laminin, agrin and perlecan, resulting in muscle weakness and cell death [12–14]. However, the exact disease mechanism is not fully understood and currently, there is no effective treatment available.
* Corresponding author. Tel.: +1 704 355 6427; fax: +1 704 355 1679. E-mail address: [email protected] (Y.M. Chan).
Herbert Bonkovsky, Vice President of Research in CMC, delivered the opening remarks for the conference and welcomed the delegates to Charlotte and Carolinas Medical Center. The first session, chaired by Benjamin Brooks, Director of the Carolinas neuromuscular/ALS-MDA Center at CMC, provided an overview of current clinical diagnosis and management of the diseases. Susan Sparks discussed the challenge in determining genotype– phenotype correlation in limb girdle muscular dystrophy type 2I (LGMD2I) due to the highly variable clinical spectrums associated with FKRP mutations. Although there seems to be a general correlation between the amount of residual glycosylation of a-dystroglycan and severity, exceptions were observed in some patients. To better define and characterize LGMD2I, microarray technology was used to identify molecular networks that are specifically altered in LGMD2I but not in other types of muscular dystrophies. This approach has the potential to allow identification of critical downstream elements that can be targeted for therapies in the future. Pascale Guicheney also reiterated that the spectrum of clinical phenotypes associated with the dystroglycanopathies is broader than expected. Noticeably, some patients have mild phenotypes despite the severe reduction of a-dystroglycan glycosylation. It was proposed that a combined approach using genetics, immunohistochemistry and measurement of glycosyltransferase activities (namely POMT and POMGnT1) in immortalized lymphoblastoid cell lines from patients is helpful in the identification and characterization of dystroglycanopathies. Ichizo Nishino concluded the morning session with his work on Fukuyama congenital muscular dystrophy (FCMD), which is caused by mutations in the fukutin gene. He pointed out that the frequency of each subtype of a-dystroglycanopathies varies among different ethnic groups, with FCMD being the most predominant form of congenital muscular dystrophy in Japan. Interestingly, despite extensive genetic analysis, there are still patients without mutations in the six known genes responsible for dystroglycanopathies, indicating the existence of yet-to-be-identified genes.
0960-8966/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.09.007
Y.M. Chan et al. / Neuromuscular Disorders 18 (2008) 1002–1004
3.2. Pathogenesis and animal models The afternoon session devoted to the critical role of glycosylation in muscular dystrophies. Participants reviewed the use of different animal models and other essential tools to investigate the diseases. Jane Hewitt used the myodystrophy (myd) mice to study the LARGE gene family. Several studies have suggested that up-regulation of LARGE can be used as a potential therapy to compensate for the loss of other glycosyltransferases in dystroglycanopathies. The role and function of LARGE2, a separate but similar gene to LARGE, was discussed. In addition, zebrafish was explored as a useful alternative model for the diseases since it appeared that the glycosylation pathway for a-dystroglycan is highly conserved in this organism. Genri Kawahara discussed the development of a zebrafish model for LGMD2I. When FKRP expression was knocked down with morpholinos, zebrafish displayed abnormal formation of muscle, heart and head, similar to human patients. Given that several of the mouse models for dystroglycanopathies are embryonic lethal, the use of zebrafish as an alternative model clearly provides a major benefit and advantage. Susan Brown then presented a new exciting mouse model for FKRP-related muscular dystrophies. A FKRP mutant line of mice was generated and developed a phenotype reminiscent of muscle-eye-brain disease. These mice display a severe reduction of a-dystroglycan offering new and valuable insights into the function of FKRP. One of the key issues will be to validate and develop these mice into a useful model that can be used to test potential therapeutic compounds in the future. Tatsushi Toda discussed his work on fukutin and FCMD. By generating different lines of fukutin mutant mice, he suggested that a small amount of functional glycosylated a-dystroglycan might be sufficient to prevent disease progress. To develop effective therapies, a major consideration is the level of the functional dystroglycan and glycosyltransferase in muscle required for normal function. Another key question is how to document the putative enzymatic activities for fukutin and FKRP. Surprisingly, Dr. Toda demonstrated an unexpected role of fukutin in interacting with POMGnT1 and regulating its glycosyltransferase activity. Kevin Campbell further emphasized the importance of dystroglycan in the disease process. He reported the identity of specific conserved residues that are important for functional dystroglycan glycosylation by LARGE. The basic mechanism underlying this subset of muscular dystrophies seems to be hypoglycosylation of a-dystroglycan, resulting in disrupting interaction with its ligand, namely, laminin in the extracellular matrix. The selection of appropriate promoters for generating mouse models was mentioned in the presentation since the use of different promoters might affect the expression of the genes they regulate, thereby changing the outcomes and interpretations. During the discussion, the issue regarding the glycoepitopes recognized by the two most commonly used antibodies against a-dystroglycan (IIH6C4 and VIA4-1) was raised. The characterization of these epitopes will be essential for understanding dystroglycan glycosylation and the nature of the aberrantly glycosylated dystroglycan. Derek Blake has investigated the role of FKRP in LGMD2I from a different perspective. Using a heterologous cell expression system, his group found that many of the disease-causing FKRP mutations caused the proteins to misfold in specific manners, leading to their retention in the endoplasmic reticulum. Similar to the mislocalization of CFTR mutant protein documented in cystic fibrosis, the folding, processing and trafficking of FKRP might contribute to the pathogenesis of muscular dystrophies. The possibility of identifying proteins that can correct the folding and trafficking of mutant FKRP was discussed. One of the approaches discussed was to utilize mass spectrometry.
1003
Tamao Endo provided an extensive review of the complexity of the O-mannosylation pathway with respect to a-dystroglycan. To gain insight into the regulation of dystroglycan glycosylation, the specific amino acids on a-dystroglycan recognized by POMT1 and 2 were determined. It also appeared that POMT1 and POMT2 form an active enzyme complex essential for glycosyltransferase activity. The ability to assay this activity will be an important part of work focused on identifying additional O-mannosylated proteins and their functional role in muscular dystrophies. The evening was concluded with welcome addresses by James McDeavitt, Senior Vice President in CMC and Mr. Hugh McColl, Jr., the founding donor of the Carolinas Muscular Dystrophy Research Endowment. 3.3. Experimental therapies The second day’s session focused on potential treatments for dystroglycanopathies. Speakers reviewed and discussed different treatment strategies. Key areas of emphasis included how to effectively disseminate current knowledge/technology into the development of applicable therapies. John Vissing discussed the potential benefits of endurance training for LGMD2I patients. It was suggested that aerobic training, such as cycling, is safe and provides some benefit to patients. The potential benefit of other types of exercises, for example, strengthen training is also being investigated. Discussions highlighted the possibility of combination therapies using both drugs and physical exercise for muscular dystrophies in the future. In addition, Dr. Vissing reported that the L276I FKRP mutation has a very high prevalence in Denmark, suggesting a founder effect for this particular mutation. Paul Martin provided evidence that overexpression of cytotoxic T cell GalNAc transferase (Galgt2) has therapeutic values in several animal models of muscular dystrophies, including DMD, CMD and sarcoglycan-deficient LGMD. The possibility of GalgT2 being a suitable molecular target for therapy in dystroglycanopathies was discussed but it was pointed out that initial efforts must be addressed to increase efficiency and level of expression. Drug screening strategy to identify compounds that can up-regulate GalgT2 activity in human was also examined. Xiao Xiao discussed the various adeno-associated virus (AAV)based vectors and approaches to improve the efficiency of gene transfer and to reduce the immune response associated with gene therapy. Dr. Xiao’s recently studies have demonstrated successful systematic delivery of (1) a miniature dystrophin gene to DMD mouse and dog models, (2) a d-sarcoglycan gene to a LGMD2F hamster model, (3) an agrin gene to a congenital muscular dystrophy mouse model. These vectors are being further developed for systemic delivery of FKRP gene. In addition, they can be used to deliver small hairpin interference RNA post-natally in mice to knock down FKRP expression to generate LGMD2I phenotypes for functional studies. Ellen Welch took a different approach with regard to developing a potential treatment for muscular dystrophies. The read-through strategy allows translational machinery in cells to ignore premature stop codons due to nonsense mutations, leading to production of near-normal protein. Currently, clinical trials for a subset of DMD patients with nonsense mutations are being conducted using read-through compounds developed by PTC Therapeutics. This strategy clearly has the potential to be used to treat dystroglycanopathies caused by nonsense mutations. The key question to be addressed is whether the read-through compounds can result in sufficient amounts of glycosyltransferases to restore normal muscle function. In addition, considerable efforts have been made to identify small molecules that can up-regulate specific protein targets known to be involved in muscular dystrophies.
1004
Y.M. Chan et al. / Neuromuscular Disorders 18 (2008) 1002–1004
Qi Lu provided a detailed overview of the diseases and other potential treatments currently pursued in the laboratory. It has become apparent that an important approach will be to artificially increase a-dystroglycan glycosylation. A drug screening system has been established to search for small molecules with the potential to enhance glycosylation of the targeted a-dystroglycan. The laboratory is also collaborating with PTC Therapeutics to determine the potential value of candidate read-through drugs for LGMD2I. To conclude the workshop, Dr. Lu stated that ‘‘It is clear that wider collaborations among the limited number of laboratories engaged in the study of dystroglycanopathies are important to further our understanding of the disease mechanisms and development of animal models and effective treatments’. 4. The workshop organizers Yiumo Michael Chan, PhD, Carolinas Medical Center, US, Jeannie Maggio, Carolinas Medical Center, US, Qi Lu, MD, PhD, Carolinas Medical Center, US, Herbert Bonkovsky, MD, Carolinas Medical Center, US, Derek Blake, PhD, Cardiff University, UK, Francesco Muntoni, MD, Imperial College, UK.
McColl and Lockwood families as well as Carolinas HealthCare Foundation. The laboratory focuses on the translational research for limb girdle muscular dystrophy type 2I. Acknowledgements This workshop was made possible by the financial support of the Carolinas Muscular Dystrophy Research Endowment at the Carolinas HealthCare Foundation and the Muscular Dystrophy Association, USA. Special thanks are due to Mr. and Mrs. Luther Lockwood for their substantial support of this workshop through their successful charity event, ‘‘Jeans, Genes and Geniuses”. In additional, the Organizing Committee thank Jeannie Maggio for working meticulously on every aspect of the meeting. Gracious support was also provided by Dr. Lu, Dr. Blake and Dr. Muntoni. The Organizing Committee extend appreciation to Libby Anderson, Ashley Ginn and Denise Moseley at the Marketing Department for website design and marketing materials and Frederick Jones and Trey Maggio for audio-visual assistance. References
5. List of participants 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Derek Blake, PhD, Cardiff University, UK Susan Brown, PhD, Imperial College, UK Kevin Campbell, PhD, University of Iowa, USA Tamao Endo, PhD, TMIG, Japan Pascale Guicheney, PhD, INSERM, France Jane Hewitt, PhD, University of Nottingham, UK Genri Kawahara, PhD, Children’s Hospital Boston, USA Qi Lu, MD, PhD, Carolinas Medical Center, USA Paul Martin, PhD, Ohio State University, USA Ichizo Nishino, MD, PhD, NCNP, Japan Susan Sparks, MD, PhD, Children’s National Medical Center, USA Tatsushi Toda, MD, PhD, Osaka University, Japan John Vissing, MD, DMSci, University of Copenhagen, Denmark Ellen Welch, PhD, PTC Therapeutics, USA Xiao Xiao, PhD, University of North Carolina, Chapel Hill, USA
6. Website For a full description of the workshop and program, please visit http://www.carolinasmedicalcenter.org/LGMD.
7. McColl–Lockwood Laboratory for Muscular Dystrophy Research The McColl–Lockwood Laboratory was established in 2005 with a generous research endowment grant jointly created by the
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