Dysferlin Gene Research Articles

609. Systemic Delivery of Dysferlin Overlap Vectors Mediates Functional Recovery of Dysferlin Deficiency

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene leading to absent or mutant protein. Dysferlin protein has been implicated in multiple functional roles specifically in membrane stabilization/repair, t-tubule formation and vesicle trafficking. The loss of dysferlin causes a progressive dystrophy characterized by chronic muscle fiber loss, fat replacements and fibrosis resulting in deteriorating muscle weakness. Efforts made in the gene therapy arena with dysferlin or surrogate gene replacement have shown some efficacy in restoring membrane repair, however, only delivery of full-length DYSF has been able to correct the underlying histopathology. Therefore, there is a strong rationale to develop therapies that deliver the entire DYSF cDNA. A potential issue with this is the size of the DYSF gene (6.5 kb) which is too large for canonical AAV packaging. To circumvent this, we have developed and previously shown efficacy with a unique dual vector system using AAV to deliver and express DYSF specifically in muscle cells. This two vector system (AAV.DYSF.DV) packaged in the rh. 74 serotype is defined by a 1 kb region of homology between the two vectors. Following delivery to muscle, this overlap serves as a substrate for recombination/repair to generate the full-length gene. Our previous work studied the efficacy of this treatment strategy through intramuscular and regional vascular delivery routes. However, as generalized muscle weakness is common in dysferlinopathies, therapies targeting all muscle groups are warranted to maximize clinical efficacy. In this update, we have treated dysferlin-deficient mice systemically by intravenous injection to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at 3 and 6 months post-treatment for dysferlin expression, restoration of membrane repair capability, diaphragm specific force measurements and muscle histology. Additional animals are awaiting MRI analysis following 1 year of treatment. A single systemic dose of 6×1012 vector genomes (3×1012 vg of each vector) resulted in widespread gene expression exceeding 30% of muscle fibers. Treated muscles showed a significant decrease in central nucleation and collagen deposition. Membrane repair ability was improved toward wild-type and force deficits in the diaphragm were restored to wildtype levels. The mice showed no evidence of local or systemic toxicity, further confirming previous safety data. This study, in conjunction with our previous work, lays the foundation for clinical trial.

404. Development of Human Artificial Chromosomes for Gene Cell Therapy of Muscular Dystrophies

Muscular dystrophies are clinically heterogeneous group of muscle diseases related to different genes defects. The results of defects impair myogenic differentiation and histophysiology of skeletal muscles. One of major forms of disease – limb-girdle muscular dystrophy 2B (LGMD2B), caused by dysferlin gene mutations. Both big size of gene and wide variety of mutations complicate development of genetically engineered drugs. Human artificial chromosomes (HAC) could be used as alternative approach. This vector can contain unlimitedly large DNA region for transfer, is retained during cell division, and does not have carcinogenic and immunogenic properties.HAC matrix was developed earlier by Larionov V. et al. (http://www.ncbi.nlm.nih.gov/pubmed/22123967). Optimized DYSF gene sequence (optDYSF) from pAd/CMV/V5-DEST vector (kindly provided by A.A. Rizvanov) was transferred into backbone vector also containing loxP sites for further transfer into HAC. Transfer correctness confirmed by sequencing. After that Chinese hamster ovary cells carrying HAC were transfected by backbone vector containing optDYSF. Selection performed in HAT medium. 7 clones were selected and analyzed at day 10. Expression of optDYSF observed in all obtained clones. Episomal HAC localization determined using fluorescent hybridization in sity by colocalization of label at optDYSF and a-satellite DNA. Episomal localization of HAC determined only in 2 clones from 7. Dysferlin production observed in all clones.On other hand described vector requires somatic myogenic vector cell. Human myoblasts could be successfully used for this purpose. The major source of myoblasts – skeletal muscle tissue is not optimal due to limited proliferative potential, especially in patients with muscular dystrophies. Our team revealed presence of myoblasts in gingival mucosa specimens and developed new method of myoblasts obtainment from it. Primary cultures of multipotent mesenchymal stromal cells (MSC) were derived from gingiva biopsy samples. The cells expressed typical mesenchymal stem cells markers and had ability for 3-way differentiation. Myogenic induction of gingiva derived MSC in culture was demonstrated. Gingiva derived MSC are capable to express markers of myogenic differentiation (skeletal actin, sceletal myosin, MyoD1) and form multicellular elongated fibers – myotubes. Ability of gingiva derived MSC cultures to differentiate in myogenic direction was preserved during up to 10th passage. Such features make this cell source attractive for using in treatment of inherited muscular degenerative diseases.In our future research we are planning to transfer HAC containing optDYSF gene into clonogenic gingiva derived MSC using microcell-mediated chromosome transfer. This method could be used for further development of gene cell therapy of LGMD2B.

Myofiber Damage Precedes Macrophage Infiltration after in Vivo Injury in Dysferlin-Deficient A/J Mouse Skeletal Muscle

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies.

AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models.

ObjectiveDysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.MethodsTraditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.ResultsTreated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.InterpretationTreated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.

Identification of splicing defects caused by mutations in the dysferlin gene.

Missense, iso-semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5' or 3' splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5' or 3' splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.

G.P.288: Dysferlinopathy in Egypt: Clinical, Pathological and Genetic characteristics

Dysferlinopathy is caused by mutations of the dysferlin gene (DYSF) on chromosome 2p13. This gave rise to a spectrum of neuromuscular disorders including: limb-girdle (LGMD2B) and distal muscular dystrophies (Miyoshi myopathy: MM). Aim: to study the clinical, pathological and genetic characteristics of dysferlinopathy patients in Egypt. Subjects & Methods: Patients were selected from those with progressive muscular dystrophy referred to Muscle and Nerve Research Laboratory, Cairo, Egypt. We studied 77 patients with dystrophic muscle biopsy. Patients had neurological assessment, family pedigree study, Serum Creatine Kinase, ECHO cardiography, electromyography and Dystrophin gene testing. A battery of histochemical tests, immunohistochemistry using both fluorescent and automated methods against a panel of antibodies were done. Mini-multiplex Western Blotting was also done. Genetic study of Dysferlin gene was done for some patients. We found 40 patients with limb-girdle muscular dystrophy, 12 with dysferlinopathy, 6 with calpainopathy, 6 with sarcoglycanopathy and 16 with non-specified limb-girdle muscular dystrophy. Dysferlinopathy patients showed 3 patients with proximal type, 3 patients with Miyoshi myopathy and 6 patients with anterior compartment myopathy. Weakness was asymmetrical in 5 patients and symmetrical in 7 patients. Some muscles were uniformly affected in most patients: gluteus maximus, hamstrings, gastrocnemius, tibialis anterior and deltoids. Pathological study showed dystrophic changes of all biopsies, mitochondrial changes in 2 biopsies and inflammatory changes in 3 biopsies. Dysferlin gene analysis was done for only 6 patients of 4 families, showed homozygous mutations in two families, one heterozygous mutation in one family, and no mutation (only polymorphisms) in one family. Dysferlinopathy is a common condition in Egypt. Many clinical, pathological and genetic characteristics are found and help in its diagnosis.

G.P.284: Dysferlinopathy caused by protein misfolding: The novel murine animal model Dysf-MMex38

Limb-girdle muscular dystrophy 2B (LGMD2B) is caused by mutations in the dysferlin gene (DYSF). There are no mutational hotspots. Approximately one third of all mutations are missense mutations causing misfolding and protein aggregation, premature degradation and amyloid formation. There is no therapy. We could show that by treating patients’ primary myotubes with short dysferlin-specific peptides the endogenous produced missense mutant dysferlin can be relocated to the sarcolemma. There, it regains its proper function as a membrane repair protein. The human missense mutation DYSF p.L1341P causes all characteristics of a protein misfolding disease in dysferlinopathy. We therefore generated the first knock-in mouse model carrying the analogous murine missense mutation in exon 38 (Dysf p.L1360P, NP_001071162.1) named MMex38 (B6;129P2-Dysftm1.1Mdcb). Natural disease course was investigated at 8, 13 and 21 weeks of age (n = 36). We performed body composition, analyzed treadmill performance and voluntary running as well as protein analysis and histology of proximal and distal muscles. Evaluation of ER stress and membrane resealing assays complete the assessment. The targeted mutation leads to a dystrophic phenotype in 13 week old mice with a continuous disease progression in adulthood. Total body weight of 21 week old MMex38 was increased compared to WT controls without changes in the percentage of fat, free body fluid and lean tissue. Three weeks of treadmill exercise exposed significant endurance impairment in 19 to 21 week old MMex38 compared to controls. Histology revealed a distinct dystrophic pattern in 13 week old MMex38 and number of necrotic and regenerating fibres increased highly significant with age. In the immunofluorescence no dysferlin was detected at the sarcolemma. The successful generation of an animal model for protein misfolding in dysferlinopathy allows the investigation of pharmacokinetics and -dynamics of dysferlin-derived peptides in vivo.

G.P.285: Membrane and phospholipid binding properties of dysferlin

Dysferlin is a protein of the ferlin family predominantly expressed in heart and skeletal muscle. The 230kDa type II transmembrane protein contains seven C2 domains and localizes to the T-tubule system and the plasma membrane in skeletal muscle. Mutations in the dysferlin gene lead to LGMD2B and Miyoshi Myopathy. Dysferlin is involved in skeletal muscle membrane repair, regeneration and biogenesis of the T-tubule system but the precise molecular function of dysferlin is so far unknown. Here we show that full-length dysferlin is able to bind phospholipids, especially PI (4,5) P₂ (PIP2), an important phospholipid within the T-tubule membrane. In C2C12 myotubes dysferlin colocalized with PIP2 at the T-tubule system. This T-tubule localization of dysferlin was altered after depletion of PIP2 indicating importance of PIP2 for T-tubule integrity. In non-muscle cells co-expression of dysferlin with a sensor for PIP2 lead to recruitment of PIP2 from the plasma membrane to an intracellular localization and colocalization with dysferlin. Furthermore, co-expression of a constitutively active mutant of Arf6 or the PIP2 kinase with dysferlin induced dysferlin recruitment to PIP2-containing vacuoles. These results indicate PIP2-binding of dysferlin which was corroborated by PIP strip analysis and liposome flotation experiments <i>in vitro</i>. Truncated dysferlins and pathogenic mutations of dysferlin failed to be recruited to PIP2-containing structures and did not bind membranes in liposome flotation experiments indicating that the full-length protein is necessary for PIP2 binding. Taken together, these results suggest that full-length dysferlin is able to bind lipid membranes with preference of the negatively charged phospholipid PIP2. Binding to PIP2 was abolished by truncations and pathogenic mutations of the dysferlin gene which indicates that PIP2-binding may be important in dysferlin-deficient muscle pathology.

G.P.287: Genetic and epigenetic determinants of low dysferlin expression in monocytes

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle and peripheral blood monocytes. Dysferlin expression in skeletal muscle and monocytes strongly correlate in the healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While eighteen of these individuals with protein levels in the range of 40–64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of these 18 volunteers detected missense variants in only four. Analysis of DNA methylation patterns at DYSF locus showed no changes in DNA methylation levels at CpG island and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, results of the monocyte assay can not be used when determining carrier status and molecular analysis of DYSF must be performed.

Genetic and epigenetic determinants of low dysferlin expression in monocytes.

Dysferlinopathies are autosomal recessive inherited muscular dystrophies caused by mutations in the gene DYSF. Dysferlin is primarily expressed in skeletal muscle, cardiac muscle, and peripheral blood monocytes. Expression in skeletal muscle and monocytes strongly correlates in healthy and disease states. We evaluated the efficiency of the monocyte assay to detect carriers and to determine the carrier frequency of dysferlinopathies in the general population. We enrolled 149 healthy volunteers and collected peripheral blood samples for protein analysis. While 18 of these individuals with protein levels in the range of 40%-64% were predicted to be carriers by the monocyte assay, subsequent DYSF sequencing analysis in 14 of 18 detected missense variants in only four. Analysis of DNA methylation patterns at the DYSF locus showed no changes in methylation levels at CpG islands and shores between samples. Our results suggest that: (1) dysferlin expression can also be regulated by factors outside of the dysferlin gene, but not related to DNA methylation; (2) carrier frequency and therefore the number of affected individuals could be higher than previously estimated; and (3) although reliable for evaluating dysferlinopathies, the monocyte assay cannot be used to determine the carrier status; for this, a molecular analysis of DYSF must be performed.

Cis-splicing and Translation of the Pre-Trans-splicing Molecule Combine With Efficiency in Spliceosome-mediated RNA Trans-splicing

Muscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity.

Clinical heterogeneity and a high proportion of novel mutations in a Chinese cohort of patients with dysferlinopathy.

Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene. This study presents clinical features and the mutational spectrum in the largest cohort of Chinese patients analyzed to date. A total of 36 unrelated Chinese patients with diagnostic suspicion of dysferlinopathy were clinically and genetically characterized. Patients were divided into five phenotypes: 19 patients with limb girdle muscular dystrophy (LGMD) type 2B, 10 with Miyoshi myopathy (MM), 1 with distal anterior compartment myopathy (DACM), 3 with exercise intolerance, and 3 with asymptomatic hypercreatine phosphokinasemia (hyperCPKemia). Thirty-one patients showed an absence or drastic reduction of dysferlin expression by Westernblot. Forty-three mutations were identified in DYSF, including 31 novel. Our study underlines clinical heterogeneity and a high proportion of novel mutations in Chinese patients affected with dysferlinopathy.

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.

6th Dysferlin Conference, 3–6 April 2013, Arlington, Virginia, USA

The 2013 Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from April 3–6, 2013 in Arlington, VA. Participants included 34 researcher speakers, 5 dysferlinopathy patients and all 8 members of the Jain Foundation team. Dysferlinopathy is a rare disease that typically robs patients of mobility during their second or third decade of life. The goals of these Dysferlin Conferences are to bring experts in the field together so that they will collaborate with one another, to quicken the pace of understanding the biology of the disease and to build effective platforms to ameliorate disease. This is important because the function of dysferlin and how to compensate for its absence is still not well understood, in spite of the fact that the dysferlin gene was identified more than a decade ago. The objective of this conference, therefore, was to share and discuss the newest unpublished research defining the role of dysferlin in skeletal muscle, why its absence causes muscular dystrophy and possible therapies for dysferlin-deficient muscular dystrophy patients.

Membrane damage-induced vesicle–vesicle fusion of dysferlin-containing vesicles in muscle cells requires microtubules and kinesin

Mutations in the dysferlin gene resulting in dysferlin-deficiency lead to limb-girdle muscular dystrophy 2B and Myoshi myopathy in humans. Dysferlin has been proposed as a critical regulator of vesicle-mediated membrane resealing in muscle fibers, and localizes to muscle fiber wounds following sarcolemma damage. Studies in fibroblasts and urchin eggs suggest that trafficking and fusion of intracellular vesicles with the plasma membrane during resealing requires the intracellular cytoskeleton. However, the contribution of dysferlin-containing vesicles to resealing in muscle and the role of the cytoskeleton in regulating dysferlin-containing vesicle biology is unclear. Here, we use live-cell imaging to examine the behavior of dysferlin-containing vesicles following cellular wounding in muscle cells and examine the role of microtubules and kinesin in dysferlin-containing vesicle behavior following wounding. Our data indicate that dysferlin-containing vesicles move along microtubules via the kinesin motor KIF5B in muscle cells. Membrane wounding induces dysferlin-containing vesicle-vesicle fusion and the formation of extremely large cytoplasmic vesicles, and this response depends on both microtubules and functional KIF5B. In non-muscle cell types, lysosomes are critical mediators of membrane resealing, and our data indicate that dysferlin-containing vesicles are capable of fusing with lysosomes following wounding which may contribute to formation of large wound sealing vesicles in muscle cells. Overall, our data provide mechanistic evidence that microtubule-based transport of dysferlin-containing vesicles may be critical for resealing, and highlight a critical role for dysferlin-containing vesicle-vesicle and vesicle-organelle fusion in response to wounding in muscle cells.

FER-1/Dysferlin promotes cholinergic signaling at the neuromuscular junction in C. elegans and mice

SummaryDysferlin is a member of the evolutionarily conserved ferlin gene family. Mutations in Dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), an inherited, progressive and incurable muscle disorder. However, the molecular mechanisms underlying disease pathogenesis are not fully understood. We found that both loss-of-function mutations and muscle-specific overexpression of C. elegans fer-1, the founding member of the Dysferlin gene family, caused defects in muscle cholinergic signaling. To determine if Dysferlin-dependent regulation of cholinergic signaling is evolutionarily conserved, we examined the in vivo physiological properties of skeletal muscle synaptic signaling in a mouse model of Dysferlin-deficiency. In addition to a loss in muscle strength, Dysferlin −/− mice also exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials following repetitive nerve stimulation, which was observed in another Dysferlin mouse model but not in a Dysferlin-independent mouse model of muscular dystrophy. Oral administration of Pyridostigmine bromide, a clinically used acetylcholinesterase inhibitor (AchE.I) known to increase synaptic efficacy, reversed the action potential defect and restored in vivo muscle strength to Dysferlin −/− mice without altering muscle pathophysiology. Our data demonstrate a previously unappreciated role for Dysferlin in the regulation of cholinergic signaling and suggest that such regulation may play a significant pathophysiological role in LGMD2B disease.

Full-length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

The protein dysferlin is abundantly expressed in skeletal and cardiac muscles, where its main function is membrane repair. Mutations in the dysferlin gene are involved in two autosomal recessive muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Development of effective therapies remains a great challenge. Strategies to repair the dysferlin gene by skipping mutated exons, using antisense oligonucleotides (AONs), may be suitable only for a subset of mutations, while cell and gene therapy can be extended to all mutations. AON-treated blood-derived CD133+ stem cells isolated from patients with Miyoshi myopathy led to partial dysferlin reconstitution in vitro but failed to express dysferlin after intramuscular transplantation into scid/blAJ dysferlin null mice. We thus extended these experiments producing the full-length dysferlin mediated by a lentiviral vector in blood-derived CD133+ stem cells isolated from the same patients. Transplantation of engineered blood-derived CD133+ stem cells into scid/blAJ mice resulted in sufficient dysferlin expression to correct functional deficits in skeletal muscle membrane repair. Our data suggest for the first time that lentivirus-mediated delivery of full-length dysferlin in stem cells isolated from Miyoshi myopathy patients could represent an alternative therapeutic approach for treatment of dysferlinopathies.

P.1.12 Whole exome sequencing as a genetic diagnostic tool for congenital muscular dystrophies

Congenital muscular dystrophies (CMDs) are a genetically heterogeneous group of neuromuscular disorders that include a wide spectrum of clinical and pathological features. Mutations in several genes that encode extracellular matrix, nuclear envelope, sarcolemmal proteins and glycosylation enzymes are known to be responsible for CMDs. The large overlap of clinical presentations resulting from different gene mutations poses a challenge for clinicians in determining disease etiology for each patient. The aim of this study was to investigate the use of whole exome sequencing (WES) to identify the causative gene(s) in three CMD families with similar clinical features, including early-onset generalized weakness, severely elevated serum CPK levels, and rapid progression of disease. The highly consanguineous first family had two affected children diagnosed with CMD at the age of 1.5 years. Serum CK levels in the older sibling were 728 and 4909 IU/L at 2 and 6 years. WES of DNA from one affected child identified a known homozygous frameshift mutation in the dysferlin gene ( DYSF ): c.2779delG, p.A927LfsX21 in the patient. Reexamination of patient muscle biopsy in light of the genetic findings revealed absence of dysferlin protein. The second family had one affected child with CK levels of 2675 and 4025 IU/L at 1.5 and 3 years. WES revealed compound heterozygous missense mutations in the fukutin ( FKTN ) gene: a novel mutation (c.G915C, p.W305C) and a known mutation (c.G920A, p.R307Q) in the patient. All known muscle disease genes were excluded by WES in one affected individual from the third family with two affected children. Studies are ongoing for identifying the causative gene in this family. Our results highlight the genetic heterogeneity in patients diagnosed with CMD and suggest that WES may represent a powerful diagnostic tool for neuromuscular diseases with shared clinical and pathological presentations.

P.5.5 Clinical and genetic characterization of a cohort of 30 Chilean patients with dysferlinopathy

Mutations in the dysferlin gene lead to LGMD2B and Miyoshi myopathy among other phenotypes. We describe a cohort of 30 patients, from 24 non-related Chilean families, harbouring point mutations in the DYSF gene. Diagnosis was based on clinical findings or absence of dysferlin in muscle biopsy. Assessment workup consisted of clinical evaluation, Motor Function Measure (MFM) scale, CK level, electrodiagnostic testing, whole body MRI, echocardiogram, spirometry and DYSF gene direct sequencing. Nine mutations were consistently found in the cohort, four of which (c.5979dupA; c.2858dupT; c.2779delG and c.4390G>T) accounted for 77% of the total. In four patients only one mutation was found after complete DYSF gene sequencing. The age at symptom onset ranged from 10 to 33years (mean 20.7), symptoms manifesting invariably as weakness in the legs, distally (21/30) or proximally (9/30), progressing later to the upper limbs. Mean serum CK level was increased 58(±36) times above normal values. Electrodiagnostic assessment showed normal NCV and repetitive stimulation testing, with distinct degrees and distribution of myopathic changes on needle EMG. Single fibre EMG was normal in four confirmed dysferlinopathy patients. Muscle MRI done in 27/30 patients showed impairment with a similar distribution in all patients despite clinical phenotype. MFM scale score in 27/30 patients at different disease stages showed major impairment for standing and transfers and axial and proximal motor function, with relative sparing of distal motor function. Spirometry showed a mild restrictive defect in 3/17 patients at late stages of disease. Echocardiogram performed in 22/30 patients was within normal range. The clinical spectrum of dysferlinopathy in the series is in agreement with similar cohorts reported. The relative high frequency of some mutations suggests a founder effect for such mutations in the Chilean population. FONDECYT#1110159 (Conicyt, Chile).