A homozygous DPM3 mutation in a patient with alpha-dystroglycan-related limb girdle muscular dystrophy

Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 P.256 Severe Walker–Warburg syndrome associated with new mutation in ISPD gene identified with whole exome sequencing A. Kaçar Bayram 1, H. Per 1, H. Gumus 1, S. Kumandas 1, A. Çag˘layan 2 1 School of Medicine, Erciyes University, Kayseri, Turkey; 2 School of Medicine, Istanbul Bilim University, Istanbul, Turkey Walker–Warburg syndrome (WWS) is an autosomal recessive congenital muscular dystrophy characterized by hydrocephalus, cobblestone lissencephaly, brainstem and cerebellar hypoplasia, and eye abnormalities. Dystroglycan plays an important role in anchorage of muscle fibers to the extracellular matrix and also in the development for neuroblast migration. The molecular level of WWS is attributed to abnormal glycosylation of alpha-dystroglycan. Recently, the ISPD gene has been implicated in the etiology of WWS, which plays an important role in alpha-dystroglycan glycosylation. We present a girl with WWS associated with a novel nonsense mutation in the ISPD gene. She had been diagnosed with hydrocephalus and agenesis of the corpus callosum by fetal MRI. The newborn required neonatal care unit due to poor sucking, hypotonia, and respiratory distress. A ventriculoperitoneal shunt was performed on the first week. Three weeks later, she underwent cataract surgery. There was absent deep tendon reflexes. Serum creatine kinase level was 1152 U/l. She developed generalized tonic-clonic seizures in the second month. Her EEG recording was compatible with hypsarrhythmia. Postnatal MRI revealed hydrocephalus, agenesis of corpus callosum, retrocerebellar cyst associated with the fourth ventricle, cerebellar dysplasia and hypoplasia, type II lissencephaly, and Z-shaped hypoplastic brainstem. Despite all treatments, she passed away at the age of 5 months. In this study, we have identified a novel mutation in ISPD gene with whole exome sequencing associated with severe phenotype of WSS. Our findings contribute to a possible relationship between ISPD mutation and severe phenotype of WWS. Further studies are required in order to identify the impact of ISPD gene mutations in severe phenotypes of WSS. http://dx.doi.org/10.1016/j.nmd.2016.06.286

P.257 Congenital mirror movements in alpha-dystroglycanopathy (ADG) due to SGK196 mutation D. Ardicli 1, R. Gocmen 2, S. Cirak 3, B. Talim 1, G. Haliloglu 1, H. Topaloglu 1 1 Hacettepe University Children’s Hospital, Ankara, Turkey; 2 Hacettepe University, Ankara, Turkey; 3 Institute of Human Genetics, University Hospital Cologne, Cologne, Germany Congenital mirror movements either isolated or in association with several genetic syndromes can be defined as inability to perform unimanual movements. Alpha-dystroglycan plays a key role in development and maintenance of basement membranes in muscle and non-muscle tissues. Defect in axonal guidance results in disturbances of neuronal migration/positioning. A 17-year-old right-handed boy was referred to our clinic with childhood onset muscle weakness, easy fatigue, clumsiness, and difficulty in running and climbing. Muscle weakness was stable in childhood and phenotype was consistent with limb-girdle muscular dystrophy. Past medical history revealed mirror movements in the upper limbs, starting from early infancy. Parents were first cousins and his brother had also similar mirror movements. On examination, he had mild cognitive dysfunction, short stature, truncal obesity, calf pseudohypertrophy, hyporeflexia, proximal muscle weakness (4 + /5), Gowers sign, pes cavus deformity, and mirror movements in the upper limbs. Laboratory, muscle biopsy, and structural MRI findings were consistent with ADG, and further molecular work-up revealed a homozygous missense mutation in SGK196 (POMK) gene. In addition to structural MRI showing cerebellar cortical disorganisation with microcysts and brainstem hypoplasia, functional MRI study demonstrated bilateral symmetrical activation in the precentral and postsentral gyrus during both ipsilateral and contralateral handclenching tasks. To date, congenital mirror movements were previously

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reported in only one patient with ADG caused by LARGE mutation. To our knowledge, this is the first patient in the literature with mirror movements and SGK196 (POMK) mutation. There may be more patients like this within the disease spectrum. http://dx.doi.org/10.1016/j.nmd.2016.06.287

P.258 Clinical, radiological, and genetic survey of patients with muscle–eye–brain disease caused by mutations in POMGNT1 U. Yis¸ 1, G. Uyanık 2, D. Rosendahl 3, K. Çarman 4, E. Bayram 1, M. Heise 2, G. Cömertpay 5, S. Kurul 1 1 Dokuz Eylül Üniversity School of Medicine, I˙zmir, Turkey; 2 Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 3 Institute of Human Genetics, Albert-Ludwigs University Medical Centre Freiburg, Freiburg, Germany; 4 Division of Child Neurology, Gaziantep Children’s Hospital, Gaziantep, Turkey; 5 Department of Medical Biology, Faculty of Medicine, Balcali Hospital, Adana, Turkey To evaluate clinical, genetic, and radiologic features of our patients with muscle-eye-brain disease. The data of patients who were diagnosed with muscle–eye–brain disease from a cohort of patients with congenital muscular dystrophy in the division of pediatric neurology of Dokuz Eylül University School of Medicine and Gaziantep children’s hospital between 2005 and 2013 were analyzed retrospectively. From a cohort of 34 patients with congenital muscular dystrophy, 12 patients from 10 families were diagnosed with muscle– eye–brain disease. The mean age of the patients was 9 ± 5.5 years (2–19 years). Mean serum creatine kinase value was 2485.80 ± 1308.54 IU/L (700–4267 IU/ L). All patients presented with muscular hypotonia at birth followed by varying degrees of spasticity and exaggerated deep tendon reflexes in later stages of life. Three patients were able to walk. The most common ophthalmologic and radiologic abnormalities were cataracts, retinal detachment, periventricular white matter abnormalities, ventriculomegaly, pontocerebellar hypoplasia, and multiple cerebellar cysts. All of the patients had mutations in the POMGNT1 gene. The most common mutation detected in 66% of patients was c.1814 G > A (p.R605H). Two novel mutations were identified. We suggest that muscle–eye–brain disease is a relatively common muscular dystrophy in Turkey. It should be suspected in patients with muscular hypotonia, increased creatine kinase, and structural eye and brain abnormalities. The c.1814 G > A mutation in exon 21 of the POMGNT1 gene is apparently a common mutation in the Turkish population. http://dx.doi.org/10.1016/j.nmd.2016.06.288

P.259 A homozygous DPM3 mutation in a patient with alpha-dystroglycan-related limb girdle muscular dystrophy P. Van den Bergh 1, Y. Sznajer 1, V. Van Parijs 1, W. van Tol 2, R. Wevers 2, D. Lefeber 2, L. Xu 3, M. Lek 3, D. MacArthur 3, L. Xu 4, M. Lek 4, D. MacArthur 4, K. Johnson 5, L. Phillips 5, A. Topf 5, V. Straub 5 1 University Hospitals St-Luc, Brussels, Belgium; 2 RadboudUMC, Nijmegen, Netherlands; 3 Massachusetts General Hospital, Boston, USA; 4 Broad Institute of Harvard and MIT, Cambridge, USA; 5 Institute of Genetic Medicine, Newcastle upon Tyne, UK A female patient presented at age 30 with right brachial plexopathy. The origin was inflammatory and full recovery occurred. As serum CK activity was elevated at 4310 IU/L (N < 200), a deltoid muscle biopsy was performed. It only showed mild nonspecific myopathic changes. At age 42, the patient had difficulty rising from a chair and an unsteady gait with tendency to fall. Because of CK at 2732 IU/L, a quadriceps muscle biopsy was performed that showed a dystrophic pattern and alpha-dystroglycan (aDG) deficiency on immunoblotting. At age 57, proximal leg weakness had only mild progressed.

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Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212

Brain MRI, cardiac and ophthalmologic work-up were unremarkable. There was no family history of neuromuscular disease. Exome sequencing (MYOSEQ) revealed a homozygous c.131T > G (p.Leu44Pro) variant in DPM3, encoding dolichol-P-mannose synthase (DPMS) subunit 3. Leu44 is an evolutionary highly conserved amino acid and the change is predicted to be pathogenic (SIFT database). DMPS is composed of 3 subunits. DPM3 tethers the catalytic subunit (DPM1) to the ER membrane. As DPMS is required in different glycosylation pathways (N-glycosylation, C- and O-mannosylation, GPI-anchor formation), it is not surprising that also O-mannosylation of aDG is compromised when one of the subunit encoding genes is mutated. DPM1 and DPM2 mutations have been reported in patients with aDG-type congenital muscular dystrophy without (Yang et al., 2013) and with central nervous system involvement (Barone et al., 2012). To the best of our knowledge, only 1 patient with mild LGMD and DPM3 mutations has been reported in an 11 year-old female, who at age 20 years developed dilated cardiomyopathy. Because evidence of abnormal N-glycosylation was also found, the authors proposed to use label CDG-Io nomenclature (Lefeber et al., 2009). Our findings provide further evidence that abnormal aDG glycosylation related to DPM3 may lead to an LGMD phenotype. Results of segregation and functional studies will be presented. http://dx.doi.org/10.1016/j.nmd.2016.06.289

P.260 Mobility shift of beta-dystroglycan combined with reduced laminin alpha2 expression is a marker of genetic defects in the GMPPB gene S. Torelli 1, A. Sarkozy 1, R. Mein 2, M. Yau 2, S. Hammans 3, A. Radunovic 4, M. Henderson 5, M. Walker 6, S. Marino 7, S. Winder 8, L. Feng 1, R. Phadke 1, C. Sewry 1, K. Bushby 9, F. Muntoni 1, R. Barresi 9 1 University College London, London, UK; 2 Guy’s Hospital, London, UK; 3 Southampton General Hospital, Southampton, UK; 4 Royal London Hospital, London, UK; 5 Muscle Immunoanalysis Unit, Dental Hospital, Newcastle upon Tyne, UK; 6 Southampton University Hospitals, Southampton, UK; 7 Queen Mary University of London, London, UK; 8 The University of Sheffield, Sheffield, UK; 9 University of Newcastle, Newcastle upon Tyne, UK The dystroglycanopathies are a genetically heterogeneous group of muscle disorders, with a phenotypic spectrum ranging from severe congenital muscular dystrophies with brain and eye involvement, to milder adult onset limb girdle muscular dystrophies. They share the common pathological feature of a hypoglycosylated form of alpha-dystroglycan (α-DG) on skeletal muscle biopsy. Dystroglycan (DG) is a protein encoded by a single gene (DAG1) and cleaved into two (α-DG and β-DG) subunits following post-translational processing. Up until today mutations in 18 genes (mainly affecting α-DG glycosylation) have been reported to be involved in the pathogenesis of the dystroglycanopathies. Despite the genetic heterogeneity of this group of diseases, muscle biopsies from all affected individuals show variable reduction of immunolabeling to monoclonal antibodies specific for glyco-epitopes of alpha-DG (clone IIH6 and clone VIA4-1). Currently the only way to achieve a precise molecular diagnosis is through the screening of all the genes responsible for this group of conditions. In all these diseases β-DG was described to be normal. However, we now report that western blot analysis performed on the muscle samples from 12 cases with mutations in GDP-mannose Pyrophosphorylase B (GMPPB) consistently demonstrated a shift in the mobility of β-DG. This was not observed in any other dystroglycanopathy sample analyzed including cases with mutations in dystroglycanopathy genes (such as FKRP, POMT1, POMGnT1, ISPD) and it was always accompanied by reduction of laminin α2. The significance of this alteration of beta-DG is currently under investigation. Our data demonstrate that a change in beta-DG electrophoretic mobility in patients with dystroglycanopathy is a clear and specific indicator of a molecular defect in GMPPB. http://dx.doi.org/10.1016/j.nmd.2016.06.290

P.261 Identification of sequence variants in eight genes associated with dystroglycanopathies using whole exome sequencing L. Phillips 1, A. Töpf 1, K. Johnson 1, M. Bertoli 1, L. Xu 2, M. Lek 2, K. Claeys 3, P. Van den Bergh 4, J. Vissing 5, J. Colomer 6, C. Wallgren-Patterson 7, A. Lopez de Munain 8, J. Vilchez 9, A. Kostera-Pruszczyk 10, D. MacArthur 2, V. Straub 1 1 Newcastle University, Newcastle upon Tyne, UK; 2 Broad Institute of Harvard and MIT, Cambridge, USA; 3 RWTH Aachen University Hospital, Aachen, Germany; 4 Cliniques universitaires St-Luc, Brussels, Belgium; 5 University of Copenhagen, Copenhagen, Denmark; 6 Hospital Universitari Sant Joan de Déu, Esplugues de Llobregat, Spain; 7 The Folkhaelson Department of Medical Genetics, Helsinki, Finland; 8 The Basque Foundation for Health Innovation and Research (BIOEF), San Sebastian, Spain; 9 Fundacion para la Investigacion del Hospital Universitario, Valencia, Spain; 10 Medical University of Warsaw, Warsaw, Poland The MYO-SEQ sequencing project aims to carry out whole exome sequencing (WES) on 1000 patients with limb girdle weakness and an elevated serum creatine kinase activity from around Europe, helping to facilitate the integration of next generation sequencing technologies into healthcare. To date, WES and data analysis were carried out on 591 – from this cohort, 28 potential causal variants in 8 genes associated with dystroglycanopathy have been identified in 15 patients. Dystroglycanopathies are a group of heterogeneous muscular dystrophies which result from an abnormal function of α-dystroglycan (α-DG). This peripheral membrane receptor, which links the subsarcolemmal cytoskeleton to components of the extracellular matrix, is highly glycosylated and abnormal glycosylation results in the disruption of this link. To date a total of at least 17 genes involved in the glycosylation pathway of α-dystroglycan have been identified as secondary dystroglycanopathy genes. The phenotypic spectrum of dystroglycanopathy patients varies in severity from Walker Warburg Syndrome to mild forms of limb girdle muscular dystrophy (LGMD). In MYO-SEQ we identified one LGMD2U (ISPD) patient with one missense and one novel frameshift variant; two LGMD2I (FKRP) patients, one with the common homozygous founder mutation and the other one being compound heterozygous; three LGMD2T (GMPPB) patients, two with two identical missense variants and one with a frameshift variant and one LGMD2K (POMT1) case with a missense and one reported frameshift variant. A total of six LGMD2N (POMT2) cases were identified carrying one novel essential splice site and 10 missense variants, two of them previously reported. One patient had a missense and one nonsense mutation in POMK; one a homozygous missense variant in B3GNT1, and one patient a homozygous missense variant in the DPM3 gene. The clinical presentations of the patients were generally on the mild end of the dystroglycanopathy spectrum. http://dx.doi.org/10.1016/j.nmd.2016.06.291

P.262 Prednisolone therapy reduces incidence of mononuclear T-cell infiltrates after AAV transduction of GALGT2 in the rhesus macaque M. Cramer 1, P. Martin 2 1 The Ohio State University, Columbus, OH, USA; 2 The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA Overexpression of the GALGT2 gene results in the protection of skeletal myofibers from injury and the inhibition of muscular dystrophy in the mdx mouse model for DMD, the Sgca−/− model for LGMD2D, and the dyW/dyW model for MDC1A. We have developed a gene therapy, rAAVrh74.MCK.GALGT2 that can induce GALGT2 overexpression in skeletal and cardiac muscle and is now beginning clinical trials in DMD patients. In our preclinical studies of systemic gene delivery in rhesus macaques, we noticed that AAV muscle transduction led to development of occasional CD8+ T-cell infiltrates. Here, we show that daily treatment of macaques with prednisolone (0.75 mg/kg/day) for 14 days prior to injection and thereafter significantly reduced the presence of T-cell infiltrates in transduced skeletal muscles. In the