Limb-girdle Muscular Dystrophy Type 2I Research Articles

Oral ribose supplementation in dystroglycanopathy: A single case study.

Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein (ISPD), fukutin-related protein (FKRP), and fukutin (FKTN) genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous FKRP mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation (p < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects.

Analysis of clinical features and genetic variants in three Chinese pedigrees affected with Limb girdle muscular dystrophy type 2I

To analyze the clinical features and genetic variants of three Chinese pedigrees affected with Limb girdle muscular dystrophy type 2I (LGMD2I). Clinical data and peripheral blood samples of the three probands and their family members were collected. Whole exome sequencing was carried out for the probands. Candidate variants were verified by Sanger sequencing of their family members. Probands 1 and 2 both featured weakness in the lower limbs. Proband 1 had lost walking ability and had pulmonary ventilation dysfunction. Proband 3 had lower limb pain, palpitations and asthma after exercise. Genetic sequencing revealed that proband 1 harbored compound heterozygous c.545A>G (p.Y182C) and c.1391A>T (p.N464I) variants of the FKRP gene, proband 2 harbored compound heterozygous c.545A>G (p.Y182C) and c.941C>T (p.T314M) variants of the FKRP gene, and proband 3 harbored compound heterozygous c.545A>G (p.Y182C) and c.161G>A (p.R54Q) variants. Among these, the c.161G>A (p.R54Q) variant was unreported previously. Compound heterozygous variants of the FKRP gene probably underlay the LGMD2I in the three patients. Whole exome sequencing is crucial for the diagnosis of LGMD2I. The identification of the novel variant also broadened the mutational spectrum of the FKRP gene.

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

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.

Duchenne muscular dystrophy\u2013like phenotype in an LGMD2I patient with novel FKRP gene variants

A 32-year-old man initially received a diagnosis of Duchenne muscular dystrophy (DMD). Genetic analysis revealed two novel heterozygous FKRP variants: c.169G>A (p.Glu57Lys) and c.692G>A (p.Trp231*). These results indicated that the patient had limb-girdle muscular dystrophy type 2I (LGMD2I) caused by recessive FKRP variants. Patients with LGMD2I and DMD have many overlapping phenotypes. LGMD2I should be considered in patients who have a DMD phenotype but not a DMD pathogenic variant.

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.

P.180Paraspinal muscle affection in limb-girdle muscular dystrophy type 2I patients

The autosomal recessive limb-girdle muscular dystrophy type 2I (LGMD2I) is characterized by progressive weakness of shoulder- and hip-girdles. Affection of the proximal extremity muscles is well-described whereas description of paraspinal muscle involvement is lacking. In this study, we investigate the involvement of paraspinal muscles in patients with LGMD2I. We used quantitative MRI (Dixon technique) to investigate fat replacement in leg and paraspinal muscles: erector spinae at C6, Th12 and L4/L5 and multifidus at Th12 and L4/L5. Back strength was measured by Biodex stationary dynamometry. The study plans to include 20 LGMD2I patients and 24 matched healthy controls (HC). Currently, 10 LGMD2I patients (mean age 37.6 (range 19-60), M:F=6:4) and 24 HC (mean age 41 (25-62), M:F=12:8) have been studied. MRI has been performed in all LGMD2I patients and 20 HC. Stationary dynamometry was done in all LGMD2I patients and 11 HC. LGMD2I patients have a significantly increased mean fat fraction (mFF) in paraspinal muscles. At cervical level: mFF is 29.9% vs 14.3% in HC (p=0.00017), thoracic level: 32.8% vs 8.7% (p=4.5•10-6), and lumbar level: 58.8% vs 24.6% (p=3.02•10-6). The psoas major muscle was also severely affected with a mFF of 54.8% vs 9.3% in HC (p=5.2•10-8). In the legs, especially the hamstrings have increased mFF (53.2% vs 9.4% (p=2.6•10-8)). Data shows a tendency of reduced back strength with increased mFF. Mean trunk extension peak torque is 96.6 Nm vs 296.9 Nm in HC (p=2.1•10-6). In conclusion, the results suggest that patients with LGMD2I have affection of the paraspinal muscles in addition to the well-described involvement of the shoulder- and hip-girdle. Stabilization and mobility of the spine is dependent on the paraspinal muscles. Affection of these muscles might compromise balance, posture and the ability to walk, which should be taken into account in the clinical evaluation and management of LGMD2I patients.

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.

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.98AAV-mediated gene transfer of FKRP for therapy of LGMD2I

Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan α, a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins whose functions are not fully elucidated. In particular, mutations in the FKRP gene encoding Fukutin related protein lead to hypoglycosylation of α, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We and others have published the proof of concept of FKRP gene transfer using an AAV vector for treating FKRP deficiencies. As the knock-in mouse models used in these studies are not severe enough to evaluate properly the dose-effect of AAV-FKRP administration, we developed a muscle specific FKRP knock-down mouse model. This new mouse model, named HSA-FKRPdel, presents a much more severe phenotype than observed in knock-in mouse. Defects of glycosylation of αDG and of its binding to laminin were observed, as well as histological dystrophic signs as centronucleation and inflammation. Functional evaluation also showed a reduced force of HSA-FKRPdel mice. AAV-FKRP was systemically administrated to this new mouse model at different doses. Depending of the dose, positive effects were observed at the molecular, histological and functional levels. Full animal characterization and therapeutic effects will be presented. Development of AAV production at the GMP level is now on going. These data will be included in an IND for AAV-mediated transfer of FKRP in patients.

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.

Limb-girdle muscular dystrophy type 2I: two Chinese families and a review in Asian patients

ABSTRACTBackground: Limb-girdle muscular dystrophy type 2I (LGMD2I) is an autosomal recessive hereditary disorder caused by mutations in the fukutin-related protein (FKRP) gene. Although the features of the disorder in European patients have been summarized, Asian patients with LGMD2I have rarely been reported. Thus, the clinical differences in LGMD2I between Asian and European patients and the associated genetic changes remain unclear.Methods: We reported detailed clinical data as well as results from muscle biopsy, muscle MRI and genetic analysis of the FKRP gene in two unrelated Chinese families with LGMD2I. Additionally, a review of the literature focusing on the clinical and mutational features of LGMD2I in Asian patients was performed.Results: The muscle biopsy results showed dystrophic features. Immunohistochemical staining revealed decreased glycosylations on α-dystroglycan. The muscle MRI results showed that the gluteus maximus, adductor, biceps femoris, vastus intermedius and vastus lateralis were severely affected. The patients in the two families harbored the same compound heterozygous mutations (c.545A>G and c.948delC). One patient showed significant clinical improvement after corticosteroid treatment.Conclusion: Our study expanded the reported spectrum of Asian LGMD2I patients. Our literature review revealed that pathogenic mutations in the FKRP gene in Asian LGMD2I patients are compound heterozygous rather than homozygous. Compound heterozygous Asian patients have a mild phenotype but frequently show respiratory and cardiac impairments. Corticosteroids may be beneficial for the treatment of LGMD2I and should be further investigated.

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.

Efficacy of Gene Therapy Is Dependent on Disease Progression in Dystrophic Mice with Mutations in the FKRP Gene

Loss-of-function mutations in the Fukutin-related protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) and other forms of congenital muscular dystrophy-dystroglycanopathy that are associated with glycosylation defects in the α-dystroglycan (α-DG) protein. Systemic administration of a single dose of recombinant adeno-associated virus serotype 9 (AAV9) vector expressing human FKRP to a mouse model of LGMD2I at various stages of disease progression was evaluated. The results demonstrate rescue of functional glycosylation of α-DG and muscle function, along with improvements in muscle structure at all disease stages versus age-matched untreated cohorts. Nevertheless, mice treated in the latter stages of disease progression revealed a decrease in beneficial effects of the treatment. The results provide a proof of concept for future clinical trials in patients with FKRP-related muscular dystrophy and demonstrate that AAV-mediated gene therapy can potentially benefit patients at all stages of disease progression, but earlier intervention would be highly preferred.

Limb girdle muscular dystrophy type 2I: No correlation between clinical severity, histopathology and glycosylated α-dystroglycan levels in patients homozygous for common FKRP mutation

Limb girdle muscular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C>A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability.FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is associated with loss of interaction with laminin α2, which in turn results in laminin α2 depletion. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C>A is related to alterations in muscle fibre pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harbouring the c.826C>A/c.826C>A genotype by histological examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease.

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.

FKRP mutations, including a founder mutation, cause phenotype variability in Chinese patients with dystroglycanopathies.

Mutations in the fukutin-related protein (FKRP) gene have been associated with dystroglycanopathies, which are common in Europe but rare in Asia. Our study aimed to retrospectively analyze and characterize the clinical, myopathological and genetic features of 12 Chinese patients with FKRP mutations. Three patients were diagnosed with congenital muscular dystrophy type 1C (MDC1C) and nine patients were diagnosed with limb girdle muscular dystrophy type 2I (LGMD2I). Three muscle biopsy specimens had dystrophic changes and reduced glycosylated α-dystroglycan staining, and two showed reduced expression of laminin α2. Two known and 13 novel mutations were identified in our single center cohort. Interestingly, the c.545A>G mutation was found in eight of the nine LGMD2I patients as a founder mutation and this founder mutation in Chinese patients differs from the one seen in European patients. Moreover, patients homozygous for the c.545A>G mutation were clinically asymptomatic, a less severe phenotype than in compound heterozygous patients with the c.545A>G mutation. The 13 novel mutations of FKRP significantly expanded the mutation spectrum of MDC1C and LGMD2I, and the different founder mutations indicate the ethnic difference in FKRP mutations.

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.