Dysferlin Protein Research Articles

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.

Annexin A1 is a biomarker of T‐tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy

Skeletal muscle complaints are a common consequence of cholesterol-lowering therapy. Transverse tubular (T-tubular) vacuolations occur in patients having statin-associated myopathy and, to a lesser extent, in statin-treated patients without myopathy. We have investigated quantitative changes in T-tubular morphology and looked for early indicators of T-tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol-lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin-naive subjects. In addition, morphometry of the T-tubular system was performed. Only the gene expression for annexin A1 was up-regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin-treated patients, the volume fraction of the T-tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca(2+)-dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T-tubular sites. The up-regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T-tubular repair.

Treatment of dysferlinopathy with deflazacort: a double-blind, placebo-controlled clinical trial

BackgroundDysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B).MethodsWe assessed the one-year-natural course of dysferlinopathy, and the safety and efficacy of deflazacort treatment in a double-blind, placebo-controlled cross-over trial. After one year of natural course without intervention, 25 patients with genetically defined dysferlinopathy were randomized to receive deflazacort and placebo for six months each (1 mg/kg/day in month one, 1 mg/kg every 2nd day during months two to six) in one of two treatment sequences.ResultsDuring one year of natural course, muscle strength declined about 2% as measured by CIDD (Clinical Investigation of Duchenne Dystrophy) score, and 76 Newton as measured by hand-held dynamometry. Deflazacort did not improve muscle strength. In contrast, there is a trend of worsening muscle strength under deflazacort treatment, which recovers after discontinuation of the study drug. During deflazacort treatment, patients showed a broad spectrum of steroid side effects.ConclusionDeflazacort is not an effective therapy for dysferlinopathies, and off-label use is not warranted. This is an important finding, since steroid treatment should not be administered in patients with dysferlinopathy, who may be often misdiagnosed as polymyositis.Trial registrationThis clinical trial was registered at http://www.ClincalTrials.gov, identifier: NCT00527228, and was always freely accessible to the public.

Evaluating Cellular Repair Potential - Lessons from Skeletal Muscle

The ability to repair disruptions in the plasma membrane is critical for maintaining cellular integrity and health; this is especially true for cells that routinely experience membrane damage as a result of activity such as skeletal muscle. Dysferlinopathies are a class of late onset muscle wasting diseases associated with the absence or truncation of the protein dysferlin. Dysferlin is postulated to have a role in membrane repair and the absence of dysferlin in both humans and animal models is associated with muscle damage. We are developing techniques to evaluate the repair potential of dysferlin null skeletal muscle fibers and myotubes using fluorescence assays of dye-leakage and calcium activity. In isolated muscle fibers or myotubes containing fluorescent dye and subjected to IR laser wounding, both the rate and duration of dye-leakage are measured by confocal microscopy. Upon wounding repair-competent fibers a “puff” of dye is released at the wound site and dye leakage ceases within 60 seconds. Under conditions where resealing is inhibited (i.e. low extracellular [Ca2+]) a large amount of dye is lost; leakage continues for >10 minutes. Changes in intracellular [Ca2+]free are measured using a combination of the calcium indicator dyes fluo-4 and Fura Red. Upon wounding, a rise in [Ca2+]free is observed at the wound site and either returns to the pre-wound level following successful resealing, or in the absence of resealing remains high and spreads through the entire fiber or mytube. Dysferlin null muscle, is repair competent but may differ in the developed calcium load, contractile response, and wounding threshold compared to dysferlin positive muscle. Since our overall goal is to develop interventions that boost cellular repair potential, quantifiable metrics of this complicated cellular process are required when testing the efficacy of repair-targeted treatments.

T.P.21 Exon skipping for dysferlinopathies

Abstract Mutations in the dysferlin encoding DYSF gene have been reported for limb-girdle muscular dystrophy 2B, miyoshi myopathy and distal myopathy with anterior tibial onset patients. Most patients have small mutations within exons, inducing premature stops or amino acid substitutions, which lead to protein instability or mislocalization. The dysferlin protein has a suggested function in membrane repair and vesicle trafficking. It contains six or seven calcium-dependent C2 lipid binding (C2) domains that probably mediate vesicle fusion. Based on reports of mild patients lacking one or more C2 domains, it has been suggested some of the C2 domains are redundant. The exon skipping approach employs antisense oligonucleotides (AONs) to hide exons from the splicing machinery during pre-mRNA splicing. This allows reframing of transcripts (as is currently tested in phase 3 clinical trials for Duchenne muscular dystrophy) but might also be a viable therapeutic approach for dysferlinopathies. Here the mutated exon is targeted to bypass the mutation. For in-frame exons the open reading frame will be maintained, allowing the production of an internally deleted but hopefully partially functional dysferlin. For out-of-frame exons, skipping of additional flanking exons would be needed. We here further explored this approach for dysferlinopathies by testing antisense oligonucleotides (AONs) to induce the skipping of exons 8, 9, 20, 21, 33, 38 and 43 in control myogenic cells. In addition, we have tested AONs targeting exon 30 in myoblast cultures from a patient with a mutation in exon 30, to confirm dysferlin restoration, and proper localization. Finally, we have tested AONs targeting exon 30 in mice to obtain in vivo proof of concept.

G.P.40 LGMD2G with clinical presentation of congenital muscular dystrophy: A rare phenotype

LGMD2G is a relatively rare and mild autosomal recessive form of progressive neuromuscular disorder with a wide spectrum of inter and intrafamilial clinical variability. The age at onset ranges from 9 to 15 years old and loss of ambulation is uncommon, or in the third or fourth decade of life. LGMD2G is caused by mutations in the TCAP gene, causing the deficiency of the sarcomeric protein telethonin in muscle of affected patients. Up to now, only a few families have been described, mainly in Brazil. In a recent screening for mutation in the TCAP gene, including 200 additional LGMD adult patients, we identified six new cases, confirming the rarity of the disease also in Brasil. All these patients carried the same c.157C>T (Q53X) homozygous missense mutation, identified in the first families, suggesting a common origin of this mutation. In addition,we studied a five years-old patient with clinical diagnosis of congenital muscular dystrophy. Immunohistochemical analysis for muscle protein excluded the deficiency of dystrophin, the four sarcoglycans, α2-laminin, dysferlin and calpain3 proteins. Surprisingly, telethonin analysis showed a total deficiency, both through immune fluorescence and western blot analyses. Sequencing of the TCAP gene identified the common c.157C>T mutation in a homozygous state in the patient and in heterozygosity in both non-consanguineous parents. This called our attention to the fact that the rarity of the disease could be due to the screening of the improper patients. Telethonin deficiency could be, in fact, the cause of more severe forms of muscular dystrophies. To test this hypothesis, we analyzed telethonin deficiency in the muscle of 118 patients with clinical diagnosis of congenital MD, from which we selected sixteen patients for gene sequencing. However, no alteration was found in any of them. Therefore, mutation in the TCAP gene is not a common cause of severe forms of MD. FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.

G.P.36 An autosomal dominant distal myopathy

The distal myopathies are a clinically and pathologically heterogeneous group of genetic disorders in which the distal muscles of limbs are selectively or disproportionately affected. Until date, at least 20 genes have been shown to be involved in distal myopathy. A 26-year-old Chinese male presented with progressive weakness of distal limbs for 2years. His mother has the same symptoms. Neurological examination revealed wading gait, with no ataxia, predominantly distal limb weakness of four limbs was noticed. Sensory examination was unremarkable, no evidence of pyramidal tract signs. Serum creatine kinase was 656IU/l. Electrocardiogram test was normal. Nerve conduction study showed normal sensory and motor nerve conduction. Needle electromyography showed decreased amplitude and short-duration motor unit action potentials and some myotonic discharges in some tested muscles. MRI scans of his legs revealed significantly fatty infiltration of anterior tibial muscles. Muscle biopsy of anterior tibial muscle revealed degenerating and necrotic fibers and many rimmed vacuoles were observed; type-fibers are predominant; glycogen and lipid contents appear normal; anti-dysferlin immunohistochemical stain was normal. MYH7 gene and Mex5–6 region of the TTN gene were sequenced. No pathogenic variant was detected. The patients we reported with distal muscular dystrophy was of early adult onset and presumably inherited in an autosomal dominant inheritance. It was characterized by a selective involvement of anterior tibial muscle, finger extensors muscle, and neck extension muscles. Serum CK level was mildly elevated. Histopathological findings revealed autophagic vacuoles and normal expression of dysferlin protein. Laing distal myopathy or tibial muscular dystrophy (TMD) was suspected according clinic, electrophysiological, and pathological findings. But no pathogenic variant was detected in MYH7 gene and Mex5–6 region of th.

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5Å resolution X-ray structure shows that a single region from the AHNAK Cterminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane.

Sarcolemmal Repair Is a Slow Process and Includes EHD2

Skeletal muscle is continually subjected to microinjuries that must be repaired to maintain structure and function. Fluorescent dye influx after laser injury of muscle fibers is a commonly used assay to study membrane repair. This approach reveals that initial resealing only takes a few seconds. However, by this method the process of membrane repair can only be studied in part and is therefore poorly understood. We investigated membrane repair by visualizing endogenous and GFP-tagged repair proteins after laser wounding. We demonstrate that membrane repair and remodeling after injury is not a quick event but requires more than 20 min. The endogenous repair protein dysferlin becomes visible at the injury site after 20 seconds but accumulates further for at least 30 min. Annexin A1 and F-actin are also enriched at the wounding area. We identified a new participant in the membrane repair process, the ATPase EHD2. We show, that EHD2, but not EHD1 or mutant EHD2, accumulates at the site of injury in human myotubes and at a peculiar structure that develops during membrane remodeling, the repair dome. In conclusion, we established an approach to visualize membrane repair that allows a new understanding of the spatial and temporal events involved.

Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

Lack of Correlation between Outcomes of Membrane Repair Assay and Correction of Dystrophic Changes in Experimental Therapeutic Strategy in Dysferlinopathy

Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.

Abstract 5180: RNAi-mediated diminution of myoferlin increases adhesion of breast cancer cells

Abstract Introduction: Ferlins are a family of proteins that play a variety of roles associated with plasma membrane dynamics in eukaryotes, including plasma membrane repair, regulation of membrane expression of receptors, and endocytosis. Dysferlin was recently reported to regulate platelet endothelial cellular adhesion molecule-1 (PECAM-1) in endothelial cells (1). We have shown that ablation of myoferlin (MYOF) in breast tumor cells attenuates invasion in vitro (in review, J Cell Sci). Moreover, loss of MYOF was found to be associated with reduced phosphorylation of a number of receptor tyrosine kinases (in press, PNAS), and others have reported on the role of MYOF in tyrosine kinase receptors expression at the plasma membrane (2). These results prompted us to hypothesize that MYOF may be involved in cell-matrix adhesion. Methods & Results: We used lentiviral-mediated RNA interference (RNAi) gene silencing to generate stable MYOF-deficient MDA-MB-231 cells and confirmed MYOF reduction with immunoblotting and qRT-PCR. RNAi control cells were generated in tandem with a non-human gene targeting construct. Relative adhesion and spreading of the wild-type, control, and MYOF-deficient epithelial cells were evaluated using a real-time electrical impedance measurement system (xCELLigence, Roche Applied Science). Results show a significant increase in the adhesion and spreading of the MYOF-deficient cells in comparision to wild-type and control cells. A trypsin assay developed using the same electrical impedance system demonstrated greater adhesion strength in the MYOF-deficient cells. This was measured by the duration of time necessary to reduce the pre-trypsin-treatment impedance by one-half. These results were further validated using a conventional parallel plate device, in which MYOF-deficient cells were found to be more adherent than controls when subjected to a range of shear stresses (1-10 dynes/cm2). PCR array screening of extracellular matrix related genes (SABiosciences) in MYOF-deficient and RNAi control MDA-MB-231 cells indicated that intercellular adhesion molecule 1 (ICAM-1/CD54) and vitronectin mRNA levels are increased following MYOF-diminution, and may potentially contribute to the mechanism behind the altered adhesion in MYOF-deficient cells. Conclusion: Myoferlin is important for the regulation of breast cancer epithelial cell adhesion. Since dysregulation of adhesion is often associated with cancer invasion and metastasis, myoferlin may play a role in these processes in breast carcinoma. (1) Sharma A, Yu C, Leung C, Trane A, Lau M, Utokaparch S, et al. A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis. Arterioscler Thromb Vasc Biol 2010 Nov;30 (11):2196-204. (2) Yu C, Sharma A, Trane A, Utokaparch S, Leung C, Bernatchez P. Myoferlin gene silencing decreases Tie-2 expression in vitro and angiogenesis in vivo. Vascul Pharmacol 2011 May 6. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5180. doi:1538-7445.AM2012-5180

Proteasomal Inhibition Restores Biological Function of Mis-sense Mutated Dysferlin in Patient-derived Muscle Cells

Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations.

Dysferlin-deficient immortalized human myoblasts and myotubes as a useful tool to study dysferlinopathy.

Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well as to test therapeutic strategies at the cellular level.

UMD-DYSF, a novel locus specific database for the compilation and interactive analysis of mutations in the dysferlin gene

Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD-DYSF), a Locus-Specific Database developed with the UMD® software. The main objective of UMD-DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense- or isosemantic-exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD-DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease-causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD-DYSF.

Comparison of Dysferlin Expression in Human Skeletal Muscle with That in Monocytes for the Diagnosis of Dysferlin Myopathy

BackgroundDysferlinopathies are caused by mutations in the dysferlin gene (DYSF). Diagnosis is complex due to the high clinical variability of the disease and because dysferlin expression in the muscle biopsy may be secondarily reduced due to a primary defect in some other gene. Dysferlin is also expressed in peripheral blood monocytes (PBM). Studying dysferlin in monocytes is used for the diagnosis of dysferlin myopathies. The aim of the study was to determine whether dysferlin expression in PBM correlates with that in skeletal muscle.Methodology/Principal FindingsUsing western-blot (WB) we quantified dysferlin expression in PBM from 21 pathological controls with other myopathies in whom mutations in DYSF were excluded and from 17 patients who had dysferlinopathy and two mutations in DYSF. Results were compared with protein expression in muscle by WB and immunohistochemistry (IH). We found a good correlation between skeletal muscle and monocytes using WB. However, IH results were misleading because abnormal expression of dysferlin was also observed in 13/21 pathological controls.Conclusions/SignificanceThe analysis of dysferlin protein expression in PBM is helpful when: 1) the skeletal muscle IH pattern is abnormal or 2) when muscle WB can not be performed either because muscle sample is lacking or insufficient or because the muscle biopsy is taken from a muscle at an end-stage and it mainly consists of fat and fibrotic tissue.

5th Annual Dysferlin Conference 11–14 July 2011, Chicago, Illinois, USA

The fifth Annual Dysferlin Conference, sponsored and organized by the Jain Foundation, was held from July 11 to 14, 2011 in Chicago, Illinois. Participants included 34 speakers, 31 poster presenters, 14 dysferlinopathy patients and family members, 5 representatives from other muscular dystrophy foundations, and 12 members of the Jain Foundation team. The objective of the dysferlin conference is to discuss progress towards developing therapies for the dysferlinopathies, Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MMD1). These diseases manifest progressive muscle loss, usually beginning in the late teenage years, and are caused by mutations in the gene encoding dysferlin. The dysferlin protein is required for effective repair of muscle fiber membranes, but little is known about how this defect leads to the muscle loss experienced by patients, or if dysferlin is involved in other cellular processes that contribute to the pathology. The meeting addressed these and other issues specific to dysferlin deficiency, including the best tools for studying dysferlin and possible causes and interventions for the disease. Four dysferlinopathy patients from the Jain Foundation’s patient registry also shared their experiences with this disease, including the risk of misdiagnosis with polymyositis and the damaging outcome of prednisone treatment.

Unmasking Potential Intracellular Roles For Dysferlin through Improved Immunolabeling Methods

Mutations in the DYSF gene that severely reduce the levels of the protein dysferlin are implicated in muscle-wasting syndromes known as dysferlinopathies. Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers. The aim of this article is to study the localization of dysferlin in skeletal muscle through optimized immunolabeling methods. We studied the localization of dysferlin in control rat skeletal muscle using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimized immunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control and dysferlin-deficient mouse muscle. Our data suggest that dysferlin is present in a reticulum of the sarcoplasm, similar but not identical to those containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein dystrophin. Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin. They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.

Non-invasive protein analysis in the first dysferlinopathy Croatian families

Abstract Mutations in human dysferlin (DYSF) gene cause both limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), also named dysferlinopathy. They are autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal limb girdle muscles caused by partial or complete absence of a sarcolemmal protein dysferlin. The size and large mutational spectrum of DYSF impose a multistep diagnosis strategy before gene analysis. Here we report the first three patients from two unrelated Croatian families in which diagnosis of dysferlinopathy was suggested on the basis of clinical picture, family history and linkage analysis. In order to confirm the presumed diagnosis, we performed a blood-based assay in which dysferlin expression is screened in blood monocytes. All three tested patients showed complete absence of dysferlin expression, giving strong evidence of dysferlinopathy that was recently confirmed by mutation analysis. In conclusion, we would suggest the presented diagnostic strategy as a reliable and non-invasive method to be used as an alternative to muscle tissue protein analysis in routine diagnostics of dysferlinopathies, prior to the more complex and demanding search for causative DYSF mutations. This non-aggressive approach seems especially useful in situation in which multiplex Western blot (WB) analysis of different muscular dystrophy proteins on muscle sample is not available.

Translational Research and Therapeutic Perspectives in Dysferlinopathies

Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development.