G.P.2.09 Glycosylation of α-dystroglycan in cultured cells and its restoration by glycosyltransferase

Abstracts / Neuromuscular Disorders 18 (2008) 724–833 abnormalities. Other abnormalities were also described including cleft lip and palate (CLP). Mutations in POMT1, POMT2, fukutin, FKRP and LARGE genes are found in 20–30% of children with WWS. Two brothers with WWS and CLP were born to healthy parents. They presented with hypotonia, hydrocephalus, cataracts, hydronephrosis and CLP. Muscle biopsy revealed variation in muscle fiber size and deficient a-dystroglycan staining with VIA4 antibodies indicating a glycosylation defect. Brain MRI showed lissencephaly, hydrocephalus and brainstem and cerebellar dysplasia. Genomic DNA from peripheral blood was obtained from the proband and the parents. Mutation analysis was performed by direct sequencing of the exons and intron/exon boundaries using an ABI Prism 3730 genetic analyzer. Sequence analysis of the proband identified two disease-causing mutations in exon 5 and 20 of the POMT1 gene. One mutation, c.291delC, not previously reported, is predicted to cause a frameshift resulting in premature stop codon 124 nucleotides downstream of the deletion. The second mutation, c.2167insG, which has been reported previously, causes a frameshift that is predicted to remove the 25 amino acids following Gly722. The mutations in exons 5 and 20 were inherited from the mother and the father, respectively. This is the first report of two sibs with WWS and CLP who were found to have a novel mutation in the POMT1 gene. This provides further evidence that WWS with CLP is associated with POMT1 mutations. We recommend POMT1 analysis in WWS cases associated with CLP when considering which gene to sequence first. doi:10.1016/j.nmd.2008.06.046

G.P.2.07 Alpha- dystroglycanopathy in an Italian patient due to large intragenic and single nucleotide deletions in the POMGnT1 gene S. Saredi 1; A. Ruggieri 1; C. Pantaleoni 2; S. D’Arrigo 2; E. Mottarelli 1; F. Blasevich 1; L. Morandi 1; I. Moroni 3; M. Mora 1 1 Fondazione IRCCS Istituto Neurologico C. Besta, Neuromuscular Diseases and Neuroimmunology, Milano, Italy; 2 Fondazione IRCCS Istituto Neurologico C. Besta, Developmental Neurology, Milano, Italy; 3 Fondazione IRCCS Istituto Neurologico C. Besta, Pediatric Neurology, Milano, Italy Background: Abnormal alpha-dystroglycan glycosylation is due to mutation in several genes coding putative or known glycosyltransferases. Clinical phenotypes are characterized mainly by congenital muscular dystrophy (CMD) and severe brain and eye abnormalities; however the spectrum of clinical findings is becoming much broader then previously thought. Objectives: To characterize the molecular defect leading to alpha-dystroglycan deficiency and severe muscle–eye–brain disease (MEB) phenotype in an Italian boy. Patient and methods: The patient, first-child of non consanguineous parents, had severe psychomotor delay, spastic tetraparesis and epilepsy. Neurological examination showed severe mental retardation and myopathy. Ophthalmic examination revealed severe myopia, retinopathy, and cataract. A muscle biopsy revealed severe dystrophic changes and alpha-dystroglycan deficiency. The patient died at the age of 17. Genomic DNA from the patient, his parents and a healthy sister was analysed by PCR and direct sequencing. Detection of the large POMGnT1 deletion was obtained by real time PCR amplification of all 22 exons and subsequent analysis of the breakpoint-spanning region by long range PCR amplification. Results: FKRP, POMT1 and POMT2 gene analysis was negative. The patient resulted to be a compound heterozygous for two novel mutations in the POMGnT1 gene: one was a single nucleotide deletion, inherited from the father, leading to frameshift and a stop codon after 26 residues; the second was an intragenic macrodeletion encompassing exons 2 and 8 and was inherited from the mother. Conclusions: Our study confirms the association between POMGnT1 mutations and MEB phenotype and widens the spectrum of molecular alterations in the POMGnT1 gene. doi:10.1016/j.nmd.2008.06.047

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G.P.2.08 LARGE overexpression in transgenic mice: Implications for therapeutic interventions in muscular dystrophy M. Brockington 1; S. Torelli 1; S. Cirak 1; S.C. Brown 2; D.J. Wells 2; F. Muntoni 1 1 UCL Institute of Child Health, Dubowitz Neuromuscular Centre, London, United Kingdom; 2 Imperial College London, Charing Cross Campus, Department of Cellular and Molecular Neuroscience, London, United Kingdom Much attention has recently been focused on the biochemical properties of LARGE and its potential as a therapeutic agent in some forms of muscular dystrophy. LARGE is one of a number of known or putative glycosyltransferase enzymes that underlie a group of muscular dystrophies commonly referred to as the ‘‘dystroglycanopathies”. They share the common pathological feature of a hypoglycosylated form of a-dystroglycan. The forced overexpression of LARGE results in hyperglycosylation of adystroglycan in cell lines from both normal and dystroglycanopathy patients where ligand binding is restored. To address the potential of LARGE as a therapeutic agent we have generated several mouse lines expressing a human LARGE transgene tagged with the V5 epitope under the control of a synthetic CMV/chicken b-actin promoter (pCAGGS). We have now characterised these mice in detail in order to assess the consequence of a-dystroglycan hyperglycosylation in vivo. Phenotypically, mice harbouring the LARGE transgene were indistinguishable from their wild type litter mates at the gross morphological level. Western blot analysis showed a surprisingly variable pattern of transgene expression: highest in skeletal muscle and cardiac muscle with little or no expression in brain, kidney and liver. High levels of transgene expression were correlated with a-dystroglycan hyperglycosyaltion as determined either by labelling with antibody IIH6 or increased laminin binding on an overlay assay. Transgene protein expression was substantiated using quantitative real time PCR analysis. Immunohistochemical analysis of sections from skeletal muscle, cardiac muscle, brain, kidney and liver showed a similar pattern of transgene expression. In skeletal muscle, expression was highly variable between fibres. Staining for other components of the DGC and extracellular matrix ligands was normal. Neuromuscular junction formation was not grossly perturbed. Having established that a-dystroglycan hyperglycosylation in LARGE transgenic mice has no obvious detrimental effects we are now in a position to cross these mice with models of dystroglycanopathy and determine if skeletal muscle function is restored. doi:10.1016/j.nmd.2008.06.048

G.P.2.09 Glycosylation of a-dystroglycan in cultured cells and its restoration by glycosyltransferase F. Saito; Y. Arai; H. Hagiwara; T. Shimizu; K. Matsumura Teikyo University, Department of Neurology, Tokyo, Japan Dystroglycan (DG) is a central component of dystrophin–glycoprotein complex that links extracellular matrix and cytoskeleton. a-Dystroglycanopathy is a disorder characterized by muscular dystrophy often associated with brain anomaly, mental retardation and eye abnormalities. Mutations of known or putative glycosyltransferases, POMGnT1, POMT1, POMT2, fukutin, FKRP and LARGE, were identified and defective glycosylation of a-DG has been implicated in the pathogenesis of this disorder. Recently, it was reported that overexpression of LARGE recover a function of a-dystroglycan and bypasses the defective glycosylation of a-DG in cultured cells deficient in fukutin, POMGnT1 or POM1. In this study, we characterized glycosylation status of a-DG in several cultured cell lines and examined the restorative effect of glycosyltransferases on the defective glycosylation of a-DG. We demonstrated that benign tumor or embryonic cell lines such as RT4, HEK293 or C2C12 express a-DG detectable by an antibody against sugar chain of a-DG, whereas malignant carcinoma cell

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Abstracts / Neuromuscular Disorders 18 (2008) 724–833

lines such as HeLa or MCF7 did not. Using an antibody against core protein, a-DG with molecular mass of 75 kDa was detected in these carcinoma cells, suggesting that functional a-dystroglycan is lost in carcinoma cells. Next, we examined if the function of a-DG is restored in these cells by gene transfer of glycosyltransferases, mutation of which leads to a-dystroglycanopathy. Transfection of the cells with LARGE greatly restored the laminin binding activity of a-DG, whereas other glycosyltransferases including POMGnT1, POMT1, POMT2, fukutin or FKRP did not. These results demonstrate that the restoration of functional a-DG by LARGE is a possible molecular target when developing therapeutic strategies for a-dystroglycanopathy. doi:10.1016/j.nmd.2008.06.049

G.P.2.10 Characterisation of the brain and eye phenotype of a FKRP knock-down mouse model of Muscle–Eye–Brain disease M.R. Ackroyd 1; L. Skordis 1; M. Kaluarachchi 1; S. Prior 1; F. Muntoni 2; S.C. Brown 1 1 Imperial College, Department of Neuroscience, London, United Kingdom; 2 Institute of Child Health, University College London, London, United Kingdom Mutations in the gene encoding for fukutin related protein (FKRP) are responsible for a spectrum of disorders that are classified within the group now known as the dystroglycanopathies. These range from limb-girdle muscular dystrophies through to Muscle–Eye–Brain disease (MEB) and Walker–Warburg syndrome (WWS). We have created a novel mouse model with reduced FKRP expression that resembles MEB and has severe structural brain defects indicative of disordered neuronal migration. Nuclei counts across the cerebral cortex illustrate only a slight reduction in overall number in the mutant compared to wild type but a significant disruption in the later formed cortical layers where there were significantly fewer nuclei in affected mice. Immunolabelling with the neuronal marker NeuN which identifies a population of post mitotic neurons confirmed that many of these mis-placed nuclei were of neuronal origin. These data are consistent with a defective pial basement membrane resulting from the hypoglycosylation of alpha-dystroglycan which subsequently disrupts the radial glial scaffold. We have now shown that there is a similar disruption in the cerebellar basement membrane and will present BrdU birth dating together with immunocytochemical analyses to compare modes of neuronal migration in the cortex and cerebellum. We have also identified a disruption of the inner limiting basement membrane of the eye with an associated alteration in positioning of the ganglion cell layer. Detailed immunocytochemical studies suggest that this is due to a novel interaction between alpha-dystroglycan and components of the extracellular matrix. These studies identify some of the key pathological mechanisms of disease associated with a reduction in the expression of FKRP. doi:10.1016/j.nmd.2008.06.050

G.P.2.11

burg syndrome (WWS), which are associated with brain and eye abnormalities. The defective glycosylation of a-dystroglycan in these disorders leads to a failure of a-dystroglycan to bind to extracellular matrix components and previous attempts to model these disorders have shown that the generation of fukutin and Pomt1 deficient knockout mice results in early embryonic lethality due to basement membrane defects. We have used the zebrafish as an animal model to investigate the pathological consequences of down-regulating the expression of the putative glycosyltransferase gene fukutin-related protein (FKRP) on embryonic development. We have found that down-regulating FKRP in the zebrafish results in embryos which develop a range of abnormalities reminiscent of the developmental defects observed in human muscular dystrophies associated with mutations in the FKRP gene. FKRP morphant embryos showed a spectrum of phenotypic severity involving alterations in somitic structure and muscle fibre organisation as well as defects in developing neuronal structures and eye morphology. The pathological phenotype was found to correlate with a reduction in a-dystroglycan glycosylation and reduced laminin binding. Further characterisation of the developmental processes affected in FKRP morphant embryos may lead to a better understanding of the pathological spectrum observed in muscular dystrophies associated with mutations in the human FKRP gene. doi:10.1016/j.nmd.2008.06.051

G.P.2.12 Generation of a model mouse for Fukuyama congenital muscular dystrophy carrying a retrotransposal insertion in the 30 UTR in the fukutin gene: Therapeutic benefit of enhanced dystroglycan glycosylation to dystroglycanopathy T. Toda 1; M. Kanagawa 1; A. Nishimoto 1; T. Chiyonobu 1; S. Takeda 2 1 Osaka University Graduate School of Medicine, Division of Clinical Genetics, Suita, Osaka, Japan; 2 Otsuka GEN Research Institute, Tokushima, Japan Fukuyama congenital muscular dystrophy (FCMD) is characterized by severe congenital muscular dystrophy with brain anomaly. FCMD is caused by mutations in fukutin and the major mutation is a retrotransposal insertion in the 30 untranslated region. In FCMD, dystroglycan (DG), a cell surface receptor for matrix proteins, is hypoglycosylated and loses the laminin-binding activity. To understand the pathogenesis and develop a therapeutic strategy, we generated a model mouse carrying the retrotransposal insertion. The targeting vector was generated using mutant lox system. Exon 10 of mouse fukutin was substituted by exon 10 of human patient’s fukutin with the retrotransposal insertion. Mice homozygous for the mutation show no typical sign of muscular dystrophy. Western blotting analysis shows that the majority of DG species are hypoglycosylated but functionally glycosylated forms are also present. Solid-phase binding assays indicated that more than 50% of laminin-binding activity remained in the mutant skeletal muscle. Finally, we show that abnormally glycosylated DG species were restored after adenoviral gene transfer of the fukutin gene. Taken together, these data demonstrate that the presence of only a small population of functionally glycosylated DG is sufficient to prevent disease progression, suggesting a possibility of glycotherapy to a group of congenital muscular dystrophy.

Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of FKRP P. Thornhill; D. Bassett; H. Lochmu¨ller; K. Bushby; V. Straub Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom

doi:10.1016/j.nmd.2008.06.052

A number of muscular dystrophies (MD) are associated with the defective glycosylation of a-dystroglycan and many of which are now known to result from mutations in a number of genes encoding putative or known glycosyltransferases. These diseases include severe forms of congenital muscular dystrophy (CMD) such as Fukuyama type congenital muscular dystrophy (FCMD), Muscle–Eye–Brain disease (MEB) and Walker–War-

T.P.1.01

CLINICAL ASSESSMENT TOOLS; POSTER PRESENTATIONS

Use of the 6 min walk test as an endpoint in clinical trials for neuromuscular diseases J.M. Florence 1; A. van der Ploeg 2; P.R. Clemens 3; D.M. Escolar 4; P. Laforet 5; B. Rosenbloom 6; M. Wasserstein 7; A. Skrinar 8; A. Pestronk 1; J.E. Mayhew 4