Dystrophy Type 2I Research Articles

P.171 Detection of alpha-dystroglycan glycation in muscle biopsies using a multiplexed western blot method

Limb-girdle muscular dystrophy (LGMD) type 2I is an autosomal recessive muscle disease caused by partial loss of function mutations of the fukutin-related protein (FKRP) leading to hypo glycosylation of alpha dystroglycan (αDG). Diminished glycosylation of αDG leads to contraction induced injury in myocytes leading to impairment of muscle performance. Identify and develop a method for evaluating the extent of glycosylation of αDG in striated muscle biopsies from patients with LGMD2I. A multiplex western blot (WB) method was employed to assess the extent of glycosylation in αDG by detecting both total αDG and a specific glyco-epitope. In LGMD2I, the biochemical impairment of FKRP leads to reduced glycosylation, which can be measured by diminished levels of the glyco-epitope. The ability to detect both forms of αDG generates a ratio of αDG-glycan to total αDG as an estimate of the extent of glycosylation. Several commercially available antibodies were evaluated using several healthy control muscles to assess their utility in a multiplexed WB to detect total αDG and glycosylated αDG. Two suitable antibodies were identified that were compatible with the LiCor platform. The specificity of these antibodies was assessed using parental HEK293T and DAG1 HEK293T knockout cell lysates. The signal linearity in both detection channels was evaluated using a dilution series of the control tibialis anterior (TA) muscle and was found to have good linearity (r2 > 0.85) and dilutional linearity. A commercially available recombinantly produced human αDG (rh-αDG) with little to no detectable glyco-epitope was identified and evaluated. Healthy TA control muscle is present on each blot and used to normalize the calculated ratios from test articles. This WB method will be used to assess the extent of glycosylated αDG in a subset of participants in our ongoing LGMD2I natural history study and results will be presented. A multiplexed method was developed for determining the extent of glycosylation of αDG. This approach has the potential to inform on the extent of αDG glycosylation in LGMD2I patients and to assess cellular response to a therapeutic intervention.

O.18 Preliminary results from MLB-01-003: an open label phase 2 study of BBP-418 in patients with limb-girdle muscular dystrophy type 2I

O.18 Preliminary results from MLB-01-003: an open label phase 2 study of BBP-418 in patients with limb-girdle muscular dystrophy type 2I

BiAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

E-POSTERS – INFLAMMATORY MYOPATHIES AND MUSCULAR DYSTROPHIES – CASE REPORTS: EP.14Acute muscle weakness in paediatric age masking an underlying limb girdle muscular dystrophy type 2I

E-POSTERS – INFLAMMATORY MYOPATHIES AND MUSCULAR DYSTROPHIES – CASE REPORTS: EP.14Acute muscle weakness in paediatric age masking an underlying limb girdle muscular dystrophy type 2I

Limb-Girdle Muscular Dystrophy Type 2I in Norway

Limb-Girdle Muscular Dystrophy Type 2I in Norway

Obstetric management of a woman with limb-girdle muscular dystrophy type 2i and dilated cardiomyopathy.

Limb-girdle muscular dystrophy describes a clinical phenotype with progressive weakness and atrophy of the muscles of the shoulders and hips. One of the more common types, limb-girdle muscular dystrophy type 2i, is associated with impaired cardiac function and restrictive lung disease, typically disproportionate to muscular disease. This condition presents a number of complex challenges in pregnancy and there are few case reports of its successful management. Here we discuss the course of the first pregnancy of a 20-year-old woman with limb-girdle muscular dystrophy type 2i.

Magnetic Resonance Imaging Findings in the Muscle Tissue of Patients with Limb Girdle Muscular Dystrophy Type 2I Harboring the Founder Mutation c.545A>G in the FKRP Gene.

Limb girdle muscular dystrophy type 2I (LGMD2I) is an autosomal recessive muscular dystrophy that is rare in Asia and is caused by mutations in the fukutin-related protein gene (FKRP). The aim of this study was to determine if there are any characteristic features of muscle on magnetic resonance imaging (MRI) in patients with LGMD2I harboring the founder mutation c.545A>G in FKRP. Using MRI, we delineated changes in the thigh muscles of ten patients with genetically confirmed LGMD2I. The majority of muscle biopsy specimens showed reduced glycosylation of α-dystroglycan, decreased expression of laminin α2, and a dystrophic pattern. In our cohort, the muscles with the most severe fatty infiltration were adductor magnus and vastus intermedius, whereas the rectus femoris, sartorius, and gracilis muscles were relatively spared. In seven patients, we identified a concentric fatty infiltration pattern that was most pronounced in the vastus intermedius and vastus medialis muscles around the distal femoral diaphysis. In this disease, the initial fatty infiltration of the posterior thigh muscles gradually progresses anteriorly regardless of the founder mutation in FKRP. Muscle tissue in patients with LGMD2I who have the founder mutation c.545A>G in FKRP shows a distinctive concentric pattern of fatty infiltration and edema on MRI.

Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I.

α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP –ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a “glycan remodelling” process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.

Adeno-Associated Virus–Mediated Mini-Agrin Delivery Is Unable to Rescue Disease Phenotype in a Mouse Model of Limb Girdle Muscular Dystrophy Type 2I

Agrin is a basement membrane-specific proteoglycan that can regulate orientation of cytoskeleton proteins and improve function of dystrophic skeletal muscle. In skeletal muscle, agrin binds with high affinity to laminin(s) and α-dystroglycan (α-DG), an integral part of the dystrophin-glycoprotein complex. Miniaturized forms of agrin (mAgrin) have been shown to ameliorate disease pathology inalaminin-α2 knockout mouse model of muscular dystrophy, acting as a link between α-DG andlaminin(s). Here, we test whether mAgrin might also improve pathologies associated with FKRP-related dystroglycanopathies, another form of muscular dystrophy characterized by weak interactions between muscle and basement membranes. We demonstrate invitro that mAgrin enhances laminin binding to primary myoblasts and fibroblasts from an FKRP mutant mouse model and that this enhancement is abrogated when mAgrin is in molar excess relative to laminin. However, invivo delivery of mAgrin via adeno-associated virus (AAV) into FKRP mutant mice was unable to improve dystrophic phenotypes, both histologically and functionally. These results likely reflect insufficient binding of mAgrin to hypoglycosylated α-DG on muscle fibers and possibly abrogation of binding from molar excess of overexpressed AAV-delivered mAgrin. Further exploration of mAgrin modification is necessary to strengthen its binding to other membrane components, including hypoglycosylated α-DG, for potential therapeutic applications.

Improved Walking Capabilities after Eight Weeks of Hal® Exoskeleton-Supported Treadmill Therapy in a Patient with Limb-Girdle Muscular Dystrophy Type 2I

A 57-year-old woman suffering from limb-girdle muscular dystrophy type 2I (LGMD, fukutin-related protein gene mutation) took part in body-weight supported treadmill therapy with the voluntary driven exoskeleton HAL®. First clinical signs were evident 11 years ago. The diagnosis has been determined genetically in 2009. Clinical examination showed floppy quadriparesis with typical proximal pronouncement (MRC 3-4/5), positive Trendelenburg sign bilateral and lumbar hyperlordosis. Due to the muscular dystrophy, the patient is retired. She needs a wheeled walker to deal with longer distances, e. g shopping. Her muscle weakness causes falls regularly. The patient gave written informed consent to participate in a clinical application observation.

Trendelenburg-Like Gait, Instability and Altered Step Patterns in a Mouse Model for Limb Girdle Muscular Dystrophy 2i.

Limb-girdle muscular dystrophy type 2i (LGMD2i) affects thousands of lives with shortened life expectancy mainly due to cardiac and respiratory problems and difficulty with ambulation significantly compromising quality of life. Limited studies have noted impaired gait in patients and animal models of different muscular dystrophies, but not in animal models of LGMD2i. Our goal, therefore, was to quantify gait metrics in the fukutin-related protein P448L mutant (P448L) mouse, a recently developed model for LGMD2i. The Noldus CatWalk XT motion capture system was used to identify multiple gait impairments. An average galloping body speed of 35 cm/s for both P448L and C57BL/6 wild-type mice was maintained to ensure differences in gait were due only to strain physiology. Compared to wild-type mice, P448L mice reach maximum contact 10% faster and have 40% more paw surface area during stance. Additionally, force intensity at the time of maximum paw contact is roughly 2-fold higher in P448L mice. Paw swing time is reduced in P448L mice without changes in stride length as a faster swing speed compensates. Gait instability in P448L mice is indicated by 50% higher instances of 3 and 4 paw stance support and conversely, 2-fold fewer instances of single paw stance support and no instance of zero paw support. This leads to lower variation of normal step patterns used and a higher use of uncommon step patterns. Similar anomalies have also been noted in muscular dystrophy patients due to weakness in the hip abductor muscles, producing a Trendelenburg gait characterized by “waddling” and more pronounced shifts to the stance leg. Thus, gait of P448L mice replicates anomalies commonly seen in LGMD2i patients, which is not only potentially valuable for assessing drug efficacy in restoring movement biomechanics, but also for better understanding them.

Contractile properties are disrupted in Becker muscular dystrophy, but not in limb girdle type 2I.

We investigated whether a linear relationship between muscle strength and cross-sectional area (CSA) is preserved in calf muscles of patients with Becker muscular dystrophy (BMD, n = 14) and limb-girdle type 2I muscular dystrophy (LGMD2I, n = 11), before and after correcting for muscle fat infiltration. The Dixon magnetic resonance imaging technique was used to quantify fat and calculate a fat-free contractile CSA. Strength was assessed by dynamometry. Muscle strength/CSA relationships were significantly lower in patients versus controls. The strength/contractile-CSA relationship was still severely lowered in BMD, but was almost normalized in LGMD2I. Our findings suggest close to intact contractile properties in LGMD2I, which are severely disrupted in BMD. Ann Neurol 2016;80:466-471.

377. AAV-Mediated Transfer of FKRP Shows Therapeutic Efficacy in a Murine Model of Limb-Girdle Muscular Dystrophy Type 2i, but Requires Tight Control of Gene Expression

Limb Girdle Muscular Dystrophies (LGMD) type 2I, a recessive autosomal muscular dystrophy, is caused by mutations in the Fukutin Related Protein (FKRP) gene. It has been proposed that FKRP, whose function remains unclear, is a participant in α-dystroglycan (αDG) glycosylation, which is important to ensure the cell/matrix anchor of muscle fibers. A knock-in mouse model of LGMD2I was generated to express the most frequent mutation (L276I) encountered in patients. The introduction of the mutation did not alter the expression of FKRP, neither at transcriptional nor at translational levels, but did alter its function since abnormal glycosylation of αDG was observed. In this model, skeletal muscles were functionally impaired from 2 months of age and a moderate dystrophic pattern was evident by histology starting from 6 months of age. Gene transfer with a rAAV2/9 vector expressing Fkrp restored the biochemical defects, corrected the histological abnormalities and improved the resistance to eccentric stress in the mouse model was obtained. However, injection of high doses of the vector induced a decrease of αDG glycosylation and laminin binding. Finally, we showed that intravenous injection of the rAAV-Fkrp vector into a dystrophic mouse model suffering of dystroglycanopathy due to skeletal muscle-specific Fukutin (Fktn) knock-out caused toxicity. The dose-dependent worsening of the dystrophic phenotype suggests requirement for a precise control of its expression.

A New Mouse Model of Limb-Girdle Muscular Dystrophy Type 2I Homozygous for the Common L276I Mutation Mimicking the Mild Phenotype in Humans

Limb-girdle muscular dystrophy type 2I (LGMD2I) is caused by mutations in the Fukutin-related protein (FKRP) gene, leading to inadequate glycosylation of α-dystroglycan, an important protein linking the extracellular matrix to the cytoskeleton. We created a mouse model of the common FKRP L276I mutation and a hemizygous FKRP L276I knockout model. We studied histopathology and protein expression in the models at different ages and found that homozygous FKRP L276I mice developed a mild progressive myopathy with increased muscle regeneration and fibrosis starting from 1 year of age. This was likely caused by progressive loss of α-dystroglycan–specific glycosylation, which was decreased by 78% at 20 months. The homozygous FKRP knockout was embryonic lethal, but the hemizygous L276I model resembled the homozygous FKRP L276I model at comparable ages. These models emphasize the importance of FKRP in maintaining proper glycosylation of α-dystroglycan. The mild progression in the homozygous FKRP L276I model resembles that in patients with LGMD2I who are homozygous for the L276I mutation. This animal model could, therefore, be relevant for understanding the pathophysiology of and developing a treatment strategy for the human disorder.

A New Mouse Model of Limb-Girdle Muscular Dystrophy Type 2I Homozygous for the Common L276I Mutation Mimicking the Mild Phenotype in Humans.

Limb-girdle muscular dystrophy type 2I (LGMD2I) is caused by mutations in the Fukutin-related protein (FKRP) gene, leading to inadequate glycosylation of α-dystroglycan, an important protein linking the extracellular matrix to the cytoskeleton. We created a mouse model of the common FKRP L276I mutation and a hemizygous FKRP L276I knockout model. We studied histopathology and protein expression in the models at different ages and found that homozygous FKRP L276I mice developed a mild progressive myopathy with increased muscle regeneration and fibrosis starting from 1 year of age. This was likely caused by progressive loss of α-dystroglycan-specific glycosylation, which was decreased by 78% at 20 months. The homozygous FKRP knockout was embryonic lethal, but the hemizygous L276I model resembled the homozygous FKRP L276I model at comparable ages. These models emphasize the importance of FKRP in maintaining proper glycosylation of α-dystroglycan. The mild progression in the homozygous FKRP L276I model resembles that in patients with LGMD2I who are homozygous for the L276I mutation. This animal model could, therefore, be relevant for understanding the pathophysiology of and developing a treatment strategy for the human disorder.

347. Tagging FKRP and LARGE by CRISPR/Cas9 for Monitoring Expression and Localization

Limb-girdle muscular dystrophy type 2I is caused by mutations in the gene for fukutin-related protein (FKRP). This protein is a putative glycosyltransferase shown to play an important role in the addition of glycans to the muscle membrane protein alpha-dystroglycan (α-DG). The glycosylation of α-DG is accomplished through the function of more than 17 putative and known glycosylatransferases including LARGE, which, when overexpressed, has been shown to compensate for the loss of function in FKRP and Fukutin. However, the exact localization of the endogenous proteins is unknown and no reliable antibody is available for monitoring the changes of their expression. The use of CRISPR/Cas9 for gene editing has been growing in populatiry as a method for introducing mutations into endogenous proteins or as a way of introducing specific sequences, such as a reporter gene. Here we present the use of the CRISPR/Cas9 gene editing system for the addition of a GFP tag to endogenous FKRP and LARGE for the identification of localization as well as to act as a marker for examining changes in expression as a results of treatment with specific compounds. We have applied several techniques to determine the effectiveness of the CRISPR/Cas9 system and encountered many different barriers. The results of these experiments provide valuable information in understanding the role of FKRP in α-DG glycosylation as well as new ways of examining the effects of experimental therapies targeting this glycosylation. With the new knowledge and methods obtained it will be easier to eventually move into in vivo studies to examine their effects in an animal model.

A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients

We generated a novel monoclonal antibody, DAG-6F4, against alpha-dystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On Western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated alpha-dystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect alpha-dystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.

P.8.1 Muscle biopsy findings in Limb Girdle muscle Dystrophy 2I (LGMD2I)

To assess potential correlation of severity of Limb Girdle Muscular Dystrophy type 2I (LGMD2I) with morphological, immunohistochemical and immunoblot alterations, in muscle biopsies from 27 patients with (LGMD2I). Mutations in the FKRP (Fukutin Related Protein) gene produce a range of clinical phenotypes including Limb Girdle Muscular Dystrophy Type 2I (LGMD2I), which belong to the mild end of the clinical spectrum. Seven different FKRP mutations have been detected among Norwegian LGMD2I patients of whom the majority were homozygous for the common c.826C>A mutation, and presented with a milder phenotype. Muscle biopsies were obtained from 27 patients. Quantitative evaluation of morphological alterations in muscle cross- sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy. The following myopathic changes were graded in the scoring system: fibrosis, regeneration, atrophy, centralized nuclei, necrosis, and inflammation. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG epitope. Muscle biopsies from 27 patients with LGMD2I presented large variation in morphological features (Table 1).

Protein Turnover and Cellular Stress in Mildly and Severely Affected Muscles from Patients with Limb Girdle Muscular Dystrophy Type 2I

Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4) and healthy subjects (n = 4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

Limb-girdle muscular dystrophy type 2I is not rare in Taiwan

Alpha-dystroglycanopathy is caused by the glycosylation defects of α-dystroglycan (α-DG). The clinical spectrum ranges from severe congenital muscular dystrophy (CMD) to later-onset limb girdle muscular dystrophy (LGMD). Among all α-dystroglycanopathies, LGMD type 2I caused by FKRP mutations is most commonly seen in Europe but appears to be rare in Asia. We screened uncategorized 40 LGMD and 10 CMD patients by immunohistochemistry for α-DG and found 7 with reduced α-DG immunostaining. Immunoblotting with laminin overlay assay confirmed the impaired glycosylation of α-DG. Among them, five LGMD patients harbored FKRP mutations leading to the diagnosis of LGMD2I. One common mutation, c.948delC, was identified and cardiomyopathy was found to be very common in our cohort. Muscle images showed severe involvement of gluteal muscles and posterior compartment at both thigh and calf levels, which is helpful for the differential diagnosis. Due to the higher frequency of LGMD2I with cardiomyopathy in our series, the early introduction of mutation analysis of FKRP in undiagnosed Taiwanese LGMD patients is highly recommended.