Limb-girdle Muscular Dystrophy 2I Research Articles

Phenotype Genotype Characterization of FKRP-related Muscular Dystrophy among Indian Patients.

The phenotypic spectrum of Fukutin-related protein (FKRP) mutations is highly variable and comprises of limb girdle muscular dystrophy (LGMD) R9 (previously LGMD 2I) and FKRP related congenital muscular dystrophies. To identify the distinct genotype phenotype pattern in Indian patients with FKRP gene mutations. We retrospectively reviewed the case files of patients with muscular dystrophy having a genetically confirmed FKRP mutation. All patients had undergone genetic testing using next-generation sequencing. Our patients included five males and four females presenting between 1.5 years and seven years of age (median age - 3 years). The initial symptom was a delayed acquisition of gross motor developmental milestones in seven patients and recurrent falls and poor sucking in one patient each. Two patients had a language delay, with both having abnormalities on the brain MRI. Macroglossia, scapular winging, and facial weakness were noted in one, three and four patients respectively. Calf muscle hypertrophy was seen in eight patients and ankle contractures in six. At the last follow-up, three patients had lost ambulation (median age - 7 years; range 6.5-9 years) and three patients had not attained independent ambulation. Creatine kinase levels ranged between 2793 and 32,396 U/L (mean 12,120 U/L). A common mutation - c.1343C>T was noted in 5 patients in our cohort. Additionally, four novel mutations were identified. Overall, six patients had an LGMD R9 phenotype, and three had a congenital muscular dystrophy phenotype. Patients with FKRP mutations can have varied presentations. A Duchenne-like phenotype was the most commonly encountered pattern in our cohort, with c.1343C>T being the most common mutation.

Ribitol dose-dependently enhances matriglycan expression and improves muscle function with prolonged life span in limb girdle muscular dystrophy 2I mouse model.

Limb Girdle Muscular Dystrophy 2I (LGMDR9) is one of the most common LGMD characterized by defects in glycosylation of α-dystroglycan (matriglycan) resulting from mutations of Fukutin-related protein (FKRP). There is no effective therapy currently available. We recently demonstrated that ribitol supplement increases levels of matriglycan in cells in vitro and in FKRP-P448L (P448L) mutant mouse model through drinking water administration. To be clinically relevant, we have now conducted a dose-escalating efficacy study by gavage in P448L mutant mice. Six months of ribitol treatment daily significantly rescued functions of skeletal, respiratory, and cardiac muscles dose-dependently. This was associated with a dose dependent increase in matriglycan and improvement in muscle pathology with reductions in muscle degeneration, inflammatory infiltration and fibrosis. Importantly, ribitol significantly increased life span and muscle functions of the female animals receiving treatment from 10 months of age. The only observed side effect was gastrointestinal tract bloating with loose stool and this effect is also dose dependent. The results validate the mechanism that ribitol as a pre-substrate of glycosyltransferase is able to compensate for the decreased function of mutant FKRP with restoration of matriglycan expression and provide a guidance for future clinical trial design.

FKRP mutations cause congenital muscular dystrophy 1C and limb-girdle muscular dystrophy 2I in Asian patients

Mutation in the fukutin-related protein (FKRP) gene causes alpha-dystroglycanopathies, a group of autosomal recessive disorders associated with defective glycosylated alpha-dystroglycan (α-DG). The disease phenotype shows a broad spectrum, from the most severe congenital form involving brain and eye anomalies to milder limb-girdle form. FKRP-related alpha-dystroglycanopathies are common in European countries. However, a limited number of patients have been reported in Asian countries. Here, we presented the clinical, pathological, and genetic findings of nine patients with FKRP mutations identified at a single muscle repository center in Japan. Three and six patients were diagnosed with congenital muscular dystrophy type 1C and limb-girdle muscular dystrophy 2I, respectively. None of our Asian patients showed the most severe form of alpha-dystroglycanopathy. While all patients showed a reduction in glycosylated α-DG levels, to variable degrees, these levels did not correlate to clinical severity. Fifteen distinct pathogenic mutations were identified in our cohort, including five novel mutations. Unlike in the populations belonging to European countries, no common mutation was found in our cohort.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

Efficient engraftment of pluripotent stem cell-derived myogenic progenitors in a novel immunodeficient mouse model of limb girdle muscular dystrophy 2I

BackgroundDefects in α-dystroglycan (DG) glycosylation characterize a group of muscular dystrophies known as dystroglycanopathies. One of the key effectors in the α-DG glycosylation pathway is the glycosyltransferase fukutin-related protein (FKRP). Mutations in FKRP lead to a large spectrum of muscular dystrophies, including limb girdle muscular dystrophy 2I (LGMD2I). It remains unknown whether stem cell transplantation can promote muscle regeneration and ameliorate the muscle wasting phenotype associated with FKRP mutations.ResultsHere we transplanted murine and human pluripotent stem cell-derived myogenic progenitors into a novel immunodeficient FKRP-mutant mouse model by intra-muscular injection. Upon both mouse and human cell transplantation, we observe the presence of donor-derived myofibers even in absence of pre-injury, and the rescue of α-DG functional glycosylation, as shown by IIH6 immunoreactivity. The presence of donor-derived cells expressing Pax7 under the basal lamina is indicative of satellite cell engraftment, and therefore, long-term repopulation potential. Functional assays performed in the mouse-to-mouse cohort revealed enhanced specific force in transplanted muscles compared to PBS-injected controls.ConclusionsAltogether, our data demonstrate for the first time the suitability of a cell-based therapeutic approach to improve the muscle phenotype of dystrophic FKRP-mutant mice.

A novel noncoding FKRP mutation in early onset limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy 2I (LGMD2I, LGMD R9) (OMIM: muscular dystrophy—dystroglycanopathy [limb-girdle], type C, 5) is an autosomal recessive disorder caused by mutations in the fukutin-related protein ( FKRP ) gene.1 The phenotypic spectrum associated with FKRP is heterogeneous; the most severe phenotype, Walker-Warburg-like syndrome, is characterized by congenital hypotonia, progressive muscle weakness and atrophy, ocular and brain malformations, severe motor developmental delay, and profound intellectual disability. Milder phenotypes include the onset of disease from infancy to adulthood and variable clinical course including asymptomatic elevated serum creatinine kinase (CK), early onset rapidly progressive course with loss of ambulation in teenage years, late onset with slow progression, and primary cardiomyopathy with minimal skeletal muscle weakness.1,2 LGMD2I is typically characterized by proximal muscle weakness, calf hypertrophy, hypotonia, elevated CK level, normal cognition, and no structural brain abnormalities. Dilated cardiomyopathy and respiratory muscle weakness can be seen.1 Mutations in the FKRP gene reduce specific O-mannose-linked glycosylation of alpha-dystroglycan, leading to the instability of the linkage between the dystrophin–glycoprotein complex and laminin alpha 2 in the basement membrane of skeletal muscle.3 The most common FKRP mutation seen in LGMD2I is c.826C>A (p.Leu276Ile). Patients who are homozygous for this mutation typically have a milder phenotype, whereas compound heterozygotes have a relatively severe clinical course. In patients heterozygous for c.826C>A, clinical severity may correspond best to the mutation in the second allele.1,4 We describe a 9-year-old boy with LGMD2I who has the c.826C>A mutation in FKRP on 1 allele and a novel variant on the second allele. The authors acknowledge Ms. Terese Nelson who performed the histology and immunostaining and Mr. Joel Carl for assembling the figure

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.

Value of muscle magnetic resonance imaging in the differential diagnosis of muscular dystrophies related to the dystrophin-glycoprotein complex

BackgroundDystrophin-glycoprotein complex (DGC)-related muscular dystrophies may present similar clinical and pathological features as well as undetectable mutations thus being sometimes difficult to distinguish. We investigated the value of muscle magnetic resonance imaging (MRI) in the differential diagnosis of DGC-related muscular dystrophies and reported the largest series of Chinese patients with sarcoglycanopathies studied by muscle MRI.ResultsFifty-five patients with DGC-related muscular dystrophies, including 22 with confirmed sarcoglycanopathies, 11 with limb-girdle muscular dystrophy 2I (LGMD2I, FKRP-associated dystroglycanopathy), and 22 with dystrophinopathies underwent extensive clinical evaluation, muscle biopsies, genetic analysis, and muscle MRI examinations. Hierarchical clustering of patients according to the clinical characteristics showed that patients did not cluster according to the genotypes. No statistically significant differences were observed between sarcoglycanopathies and LGMD2I in terms of thigh muscle involvement. The concentric fatty infiltration pattern was observed not only in different sarcoglycanopathies (14/22) but also in LGMD2I (9/11). The trefoil with single fruit sign was observed in most patients with dystrophinopathies (21/22), and a few patients with sarcoglycanopathies (4/22) or LGMD2I (2/11). Hierarchical clustering showed that most patients with sarcoglycanopathies or LGMD2I can be distinguished from dystrophinopathies based on the concentric fatty infiltration pattern and trefoil with single fruit sign at the thigh level on muscle MRI.ConclusionsMuscle MRI at the thigh level potentially allows distinction of sarcoglycanopathies or FKRP-associated dystroglycanopathy from dystrophinopathies.

MRI – MUSCLE IMAGING: P.297Which muscle segmentation approach to use for obtaining the highest discriminate power in natural history neuromuscular diseases using quantitative NMRI?

The degree of fatty infiltration, as measured using water-fat NMR imaging separation techniques (such as Dixon) is now a widely accepted muscle imaging biomarker and an often proposed outcome measure in many neuromuscular disease longitudinal studies. The question remains which is the specific muscle (group) to evaluate during the study period. We aimed to compare different segmentation approaches (global vs individual muscles) in a variety of neuromuscular disorders: immune-mediated necrotizing myopathy (IMNM), inclusion body myositis (IBM), GNE myopathy (GNEM), Duchenne muscular dystrophy (DMD), limb-girdle muscular dystrophy types 2B and 2I, Pompe disease (GSD2) and spinal muscular atrophy (SMA). We used the standardized response mean (SRM) of the fat fraction (FF) change after 1 year to assess the sensitivity of the different approaches. In 109 patients (21 IMNM, 21 IBM, 10 GNEM, 20 DMD, 12 LGMD2B, 7 LGMD2I, 13 GSD2, 5 SMA) two NMR scans were obtained with one year between them; on a clinical 3T Siemens system. A fat-water separation 3-point Dixon NMR sequence (TE1/TE2/TE3= 2.75/3.95/5.15 ms; TR = 10 ms) was performed in 5 slices at the level of the thigh as well as at the level of the leg. Regions of interest were drawn in individual muscles, muscle groups and in the whole segment. Global segmentation gave a satisfactory SRM (≥0.8) in thigh for IMNM, IBM, GNEM, LGMD2B and DMD. In leg, this was the case for IMNM, GNEM, LGMD2B and DMD. In some diseases, higher SRMs were found in individual muscles or muscle groups (including for LGMD2I and SMA). We can conclude that global segmentation is in some cases at least as sensitive as individual muscle or muscle group segmentation, implying that individual muscle segmentation is not always mandatory. A significant increase in FF does not automatically imply a high SRM, which means that the best muscle candidates to detect a treatment effect are not systematically the ones experiencing the most and severe and fastest disease progression. Generalization of the concept is premature. Additionally, the extent of intermuscular fat needs to be further investigated as changes in the nutritional status will likely bias the evaluation of muscular FF. Also other NMR outcome measures might be interesting to look into in such as water T2 and muscle cross-sectional area.

Demembranated skeletal and cardiac fibers produce less force with altered cross-bridge kinetics in a mouse model for limb-girdle muscular dystrophy 2i.

Limb-girdle muscular dystrophy 2i (LGMD2i) is a dystroglycanopathy that compromises myofiber integrity and primarily reduces power output in limb muscles but can influence cardiac muscle as well. Previous studies of LGMD2i made use of a transgenic mouse model in which a proline-to-leucine (P448L) mutation in fukutin-related protein severely reduces glycosylation of α-dystroglycan. Muscle function is compromised in P448L mice in a manner similar to human patients with LGMD2i. In situ studies reported lower maximal twitch force and depressed force-velocity curves in medial gastrocnemius (MG) muscles from male P448L mice. Here, we measured Ca2+-activated force generation and cross-bridge kinetics in both demembranated MG fibers and papillary muscle strips from P448L mice. Maximal activated tension was 37% lower in MG fibers and 18% lower in papillary strips from P448L mice than controls. We also found slightly faster rates of cross-bridge recruitment and detachment in MG fibers from P448L than control mice. These increases in skeletal cross-bridge cycling could reduce the unitary force output from individual cross bridges by lowering the ratio of time spent in a force-bearing state to total cycle time. This suggests that the decreased force production in LGMD2i may be due (at least in part) to altered cross-bridge kinetics. This finding is notable, as the majority of studies germane to muscular dystrophies have focused on sarcolemma or whole muscle properties, whereas our findings suggest that the disease pathology is also influenced by potential downstream effects on cross-bridge behavior.

A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.

Skeletal muscle contractile properties in a novel murine model for limb girdle muscular dystrophy 2i.

Limb-girdle muscular dystrophy (LGMD) 2i results from mutations in fukutin-related protein and aberrant α-dystroglycan glycosylation. Although this significantly compromises muscle function and ambulation, the comprehensive characteristics of contractile dysfunction are unknown. Therefore, we quantified the in situ contractile properties of the medial gastrocnemius in young adult P448L mice, an affected muscle of a novel model of LGMD2i. Normalized maximal twitch force, tetanic force, and power were significantly smaller in P448L mice, compared with sex-matched, wild-type mice. These differences were consistent with the replacement of contractile fibers by passive tissue. The shape of the active force-length relationships were similar in both groups, regardless of sex, consistent with an intact sarcomeric structure in P448L mice. Passive force-length curves normalized to maximal isometric force were steeper in P448L mice, and passive elements contribute disproportionately more to total contractile force in P448L mice. Sex differences were mostly noted in the force-velocity curves, as normalized values for maximal and optimal velocities were significantly slower in P448L males, compared with wild-type, but not in P448L females. This suggests that the dystrophic phenotype, which may include possible changes in cross-bridge kinetics and fiber-type proportions, progresses more quickly in P448L males. These results together indicate that active force and power generation are compromised in both sexes of P448L mice, while passive forces increase. More importantly, the results identified several functional markers of disease pathophysiology that could aid in developing and assessment of novel therapeutics for LGMD2i and possibly other dystroglycanopathies as well. NEW & NOTEWORTHY Comprehensive assessments of muscle contractile function have, until now, never been performed in an animal model for any dystroglycanopathy. This study suggests that skeletal muscle contractile properties are significantly compromised in a recently developed model for limb-girdle muscular dystrophy 2i, the P448L mouse. It further identifies novel pathological markers of muscle function that are suitable for developing therapeutics and for better understanding of disease pathogenesis.

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.

Childhood activity on progression in limb girdle muscular dystrophy 2I (P1.116)

Childhood activity on progression in limb girdle muscular dystrophy 2I (P1.116)

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.

B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I

Overexpression of B4GALNT2 (previously GALGT2) inhibits the development of muscle pathology in mouse models of Duchenne muscular dystrophy, congenital muscular dystrophy 1A, and limb girdle muscular dystrophy 2D. In these models, muscle GALGT2 overexpression induces the glycosylation of α dystroglycan with the cytotoxic T cell glycan and increases the overexpression of dystrophin and laminin α2 surrogates known to inhibit disease. Here, we show that GALGT2 gene therapy significantly reduces muscle pathology in FKRP P448Lneo(-) mice, a model for limb girdle muscular dystrophy 2I. rAAVrh74.MCK.GALGT2-treated FKRP P448Lneo(-) muscles showed reduced levels of centrally nucleated myofibers, reduced variance, increased size of myofiber diameters, reduced myofiber immunoglobulin G uptake, and reduced muscle wasting at 3 and 6 months after treatment. GALGT2 overexpression in FKRP P448Lneo(-) muscles did not cause substantial glycosylation of α dystroglycan with the cytotoxic T cell glycan or increased expression of dystrophin and laminin α2 surrogates in mature skeletal myofibers, but it increased the number of embryonic myosin-positive regenerating myofibers. These data demonstrate that GALGT2 overexpression can reduce the extent of muscle pathology in FKRP mutant muscles, but that it may do so via a mechanism that differs from its ability to induce surrogate gene expression.

Glucocorticoid Steroid and Alendronate Treatment Alleviates Dystrophic Phenotype with Enhanced Functional Glycosylation of α-Dystroglycan in Mouse Model of Limb-Girdle Muscular Dystrophy with FKRPP448L Mutation

Fukutin-related protein-muscular dystrophy is characterized by defects in glycosylation of α-dystroglycan with variable clinical phenotypes, most commonly as limb-girdle muscular dystrophy 2I. There is no effective therapy available. Glucocorticoid steroids have become the standard treatment for Duchenne and other muscular dystrophies with serious adverse effects, including excessive weight gain, immune suppression, and bone loss. Bisphosphonates have been used to treat Duchenne muscular dystrophy for prevention of osteoporosis. Herein, we evaluated prednisolone and alendronate for their therapeutic potential in the FKRPP448L-mutant mouse representing moderate limb-girdle muscular dystrophy 2I. Mice were treated with prednisolone, alendronate, and both in combination for up to 6 months. Prednisolone improved muscle pathology with significant reduction in muscle degeneration, but had no effect on serum creatine kinase levels and muscle strength. Alendronate treatment did not ameliorate muscle degeneration, but demonstrated a limited enhancement on muscle function test. Combined treatment of prednisolone and alendronate provided best improvement in muscle pathology with normalized fiber size distribution and significantly reduced serum creatine kinase levels, but had limited effect on muscle force generation. The use of alendronate significantly mitigated the bone loss. Prednisolone alone and in combination with alendronate enhance functionally glycosylated α-dystroglycan. These results, for thefirst time, demonstrate the efficacy and feasibility of this alliance treatment of the two drugs for fukutin-related protein-muscular dystrophy.

Analysis of the Specific Immune Response in Skeletal Muscle in LGMD2I Patients

Background/Purpose: Limb-girdle muscular dystrophy 2I (LGMD2I) is one of the most frequently encountered muscular dystrophies worldwide. Caused by mutations in the fukutin-related protein (FKRP) gene, it leads to a typical clinical limb girdle weakness and reduced or absent sarcolemmal staining of α- dystroglycan in skeletal muscle fibers. Inflammatory infiltrates have been detected on biopsies before, but a detailed characterization is lacking so far. Hence, we analyzed biopsy specimens from three university hospital institutions of genetically confirmed LGMD2I patients, focusing on markers of adaptive immunity and fibrosis.

Muscle and Heart Function Restoration in a Limb Girdle Muscular Dystrophy 2I (LGMD2I) Mouse Model by Systemic FKRP Gene Delivery

Mutations in fukutin-related protein (FKRP) gene cause a wide spectrum of disease phenotypes including the mild limb-girdle muscular dystrophy 2I (LGMD2I), the severe Walker-Warburg syndrome, and muscle-eye-brain disease. FKRP deficiency results in α-dystroglycan (α-DG) hypoglycosylation in the muscle and heart, which is a biochemical hallmark of dystroglycanopathies. To study gene replacement therapy, we generated and characterized a new mouse model of LGMD2I harboring the human mutation leucine 276 to isoleucine (L276I) in the mouse alleles. The homozygous knock-in mice (L276I(KI)) mimic the classic late onset phenotype of LGMD2I in both skeletal and cardiac muscles. Systemic delivery of human FKRP gene by AAV9 vector in the L276I(KI) mice, at either neonatal age or at the age of 9 months, rendered body wide FKRP expression and restored glycosylation of α-DG in both skeletal and cardiac muscles. FKRP gene therapy ameliorated dystrophic pathology and cardiomyopathy such as muscle degeneration, fibrosis, and myofiber membrane leakage, resulting in restoration of muscle and heart contractile functions. Thus, these results demonstrated that the treatment based on FKRP gene replacement was effective.

G.P.279: Efficient AAV-mediated transfer of FKRP in a new mouse model of Limb Girdle Muscular Dystrophy 2I

Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan (aDG), 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 aDG, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We generated a knock-in mouse model of LGMD2I, carrying the most frequent mutation (L276I) encountered in LGMD2I patients. Molecular characterization of this mouse model showed that the introduction of the mutation did not alter the expression of FKRP. However, the protein appears to have altered function since abnormal glycosylation of aDG and reduction of laminin binding was observed. Histologically, the muscles of this model show a dystrophic pattern starting from 6 months of age, consisting both in the presence of central nuclei and in fiber size variability. Interestingly, functional muscle impairment can be observed as early as 2 months of age by a decrease of the muscle resistance to eccentric mechanical stress. To evaluate gene transfer as a therapeutic approach, we cloned the FKRP cDNA in an AAV vector under the transcriptional control of the desmin promoter. The recombinant AAV2/9 vector was injected intramuscularly or intravenously in the mouse model. Expression of the FKRP transgene was obtained, both at RNA and protein levels. The glycosylation of aDG was restored as well as laminin binding. A histological rescue was observed by the decrease of fibers with centrally located nuclei. The AAV vector also improved the muscle function, since it conferred a better resistance to eccentric stress to the injected muscles.