Absence Of Dysferlin Research Articles

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.135 The 7-year progression of proximodistal dysferlinopathy in another Acadian patient from the Maritimes

Dysferlinopathies represent several pathologies caused by mutations in the DYSF gene. The two main phenotypes are Limb-girdle muscular dystrophy (LGMD) type 2B and distal Miyoshi's myopathy, but a proximodistal phenotype accounts for 6-35% of dysferlinopathies. The Acadians were French colonists who settled in the Canadian Maritimes and incurred the Great Deportation. Those who eluded capture remained mostly along the Maritime coastlines, preluding a higher regional prevalence of some recessive diseases. Dysferlinopathies are estimated to represent 35-45% of all LGMD's in the Acadian population. We report the case of a 19-year-old Acadian, who presented with substantially elevated serum creatine kinase levels at age 13, before the occurrence of rapidly declining distal posterior leg and limb-girdle myopathy during puberty. The patient has been followed for seven years providing clinical, imaging and laboratory documentation of disease progression. The absence of dysferlin, pathogenic homozygous DYSF mutations, and two homozygous TTN mutations were found. The parents were 3rd degree cousins and no extended family members were symptomatic. Finally, we compared the clinical phenotypes of nine unpublished cases of dysferlinopathy in Acadian patients from the Maritimes. Similar to previously reported cases of proximodistal dysferlinopathy, our patient showed macrophage infiltration on histology, and a fast disease progression supported by MRI series and clinical observations. The majority of Acadian cases, from three different DYSF mutations, also presented with a proximodistal phenotype. Modifier genes, environmental factors, or other unknown mutations have been proposed to explain phenotypic variability. Our patient had two homozygous mutations in A-band TTN, a gene region previously linked to distal myopathy. Proximodistal dysferlinopathy should be considered as a rare but plausible diagnosis in Acadian patients presenting with progressive distal and proximal myopathy.

Dysferlin-deficient myotubes show tethering of different membrane compartments characterized by TMEM16E and DHPRα

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.

Dysferlin links excitation-contraction coupling to structure and maintenance of the cardiac transverse-axial tubule system.

The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart. We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to β-adrenergic receptor stimulation. Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.

N-Acetylcysteine Reduces Skeletal Muscles Oxidative Stress and Improves Grip Strength in Dysferlin-Deficient Bla/J Mice.

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela’s inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.

"Boule du biceps" in dysferlinopathy.

Eymard et al.1 first described “boule du biceps” (bulging of biceps), a ball-like appearance caused by the selective atrophy of the distal half of the biceps brachii (BB) muscle, in patients with dysferliniopathy. This sign is also known as biceps lump. In our cohort of 24 patients with dysferlinopathy (22 symptomatic and 2 asymptomatic hyperCKemia), all confirmed both by the absence of dysferlin on muscle biopsy and by the presence of DYSF mutation on genetic analysis, except for 4 patients who were diagnosed only at the protein level. Among the 20 patients who were genetically confirmed, 18 were homozygotes for 3 different recurrent (c.342+1G>A, c.755C>T, and c.4431G>T) and 3 different nonrecurrent (c.1958delG, c.3944\_3948delinsG, and c.5983\_5984del) mutations, and 2 were compound heterozygotes of mutations different from those in other patients (c.2190dupA/c.3597G>A and c.4101G>A/c.6124C>T, respectively). Of note, we did not try to subclassify the clinical phenotype into Miyoshi myopathy and LGMD2B/LGMD-R2 dysferlin related as recent articles suggest that there is no essential difference in the pattern of muscle involvement, and thus, they are the same disease.2–5 The BB was carefully observed during contraction and relaxation to check for the sign. The function of the upper limb (UL) was assessed using the Medical Research Council (MRC) scale. Furthermore, the BB was observed in patients with other muscular dystrophy in our cohort (DMD/BMD 118 cases, sarcoglycanopathy 12 cases, FSHD 5 cases, LGMD2A 18 cases, and other LGMDs 5 cases). Boule du biceps was observed in 71% (17/24) of our patients with dysferlinopathy, including the 2 asymptomatic ones. The UL MRC score was 4 or higher in the 17 patients who showed boule du biceps, but was 3+ or lower in the 7 patients who lacked the sign. None of the controls showed boule du biceps. MRI of the BB of the asymptomatic patient during arm flexion showed that the distal part of the short head was partially infiltrated by fat, whereas the long head and the proximal part of the short head were spared. MRI of the symptomatic patient showed moderate fat infiltration in the long head and in the distal part of the short head, whereas the proximal part of the short head was spared (figure). The sign was absent in patients with more marked muscle weakness, suggesting that even the proximal portion of the BB may be affected in the advanced stages and thus can no longer show contraction. Five of 7 patients who did not show boule du biceps were nonambulant, indicating that this sign disappears in advanced stage of the disease. Our findings suggest that the distal portion of the short head of the BB is affected even in early stages, whereas the proximal part is spared, which gives rise to boule du biceps. This can be a valuable diagnostic clinical feature for dysferlinopathy from early stages of the disease.

A promotive effect for halofuginone on membrane repair and synaptotagmin-7 levels in muscle cells of dysferlin-null mice.

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy.

Purpose: It is challenging to deliver the full-length dysferlin gene or protein to restore cellular functions of dysferlin-deficient (DYSF-/-) myofibres in dysferlinopathy, a disease caused by the absence of dysferlin, which is currently without effective treatment. Exosomes, efficient membranous nanoscale carriers of biological cargoes, could be useful.Experimental design: Myotube- and human serum-derived exosomes were investigated for their capabilities of restoring dysferlin protein and cellular functions in murine and human DYSF-/- cells. Moreover, dysferlinopathic patient serum- and urine-derived exosomes were assessed for their abilities as diagnostic tools for dysferlinopathy.Results: Here we show that exosomes from dysferlin-expressing myotubes carry abundant dysferlin and enable transfer of full-length dysferlin protein to DYSF-/- myotubes. Exogenous dysferlin correctly localizes on DYSF-/- myotube membranes, enabling membrane resealing in response to injury. Human serum exosomes also carry dysferlin protein and improve membrane repair capabilities of human DYSF-/- myotubes irrespective of mutations. Lack of dysferlin in dysferlinopathic patient serum and urine exosomes enables differentiation between healthy controls and dysferlinopathic patients.Conclusions: Our findings provide evidence that exosomes are efficient carriers of dysferlin and can be employed for the treatment and non-invasive diagnosis of dysferlinopathy.

Involvement of oxidative stress, Nuclear Factor kappa B and the Ubiquitin proteasomal pathway in dysferlinopathy

AimsDysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. Main methodsFourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. Key findingsDysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. SignificanceAnti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.

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).

The Phenotype of Dysferlin-Deficient Mice Is Not Rescued by Adeno-Associated Virus–Mediated Transfer of Anoctamin 5

Mutations in dysferlin and anoctamin 5 are the cause of muscular disorders, with the main presentations as limb-girdle muscular dystrophy or Miyoshi type of distal myopathy. Both these proteins have been implicated in sarcolemmal resealing. On the basis of similarities in associated phenotypes and protein functions, we tested the hypothesis that ANO5 protein could compensate for dysferlin absence. We first defined that the main transcript of ANO5 expressed in skeletal muscle is the 22-exon full-length isoform, and we demonstrated that dysferlin-deficient (Dysf (prmd)) mice have lower Ano5 expression levels, an observation that further enhanced the rational of the tested hypothesis. We then showed that AAV-mediated transfer of human ANO5 (hANO5) did not lead to apparent toxicity in wild-type mice. Finally, we demonstrated that AAV-hANO5 injection was not able to compensate for dysferlin deficiency in the Dysf (prmd) mouse model or improve the membrane repair defect seen in the absence of dysferlin. Consequently, overexpressing hANO5 does not seem to provide a valuable therapeutic strategy for dysferlin deficiency.

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.

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.

Myogenesis in dysferlin-deficient myoblasts is inhibited by an intrinsic inflammatory response

Limb-girdle muscular dystrophy type 2B results from mutations in dysferlin, a membrane-associated protein involved in cellular membrane repair. Primary myoblast cultures derived from dysferlinopathy patients show reduced myogenic potential, suggesting that dysferlin may regulate myotube fusion and be required for muscle regeneration. These observations contrast with the findings that muscle develops normally in pre-symptomatic dysferlinopathy patients. To better understand the role of dysferlin in myogenesis, we investigated this process in vitro using cells derived from two mouse models of dysferlinopathy: SJL/J and A/J mice. We observed that myotubes derived from dysferlin-deficient muscle were of significantly smaller diameters, contained fewer myonuclei, and displayed reduced myogenic gene expression compared to dysferlin-sufficient cells. Together, these findings suggest that the absence of dysferlin from myoblasts is detrimental to myogenesis. Pro-inflammatory NFκB signaling was upregulated in dysferlin-deficient myotubes; the anti-inflammatory agent celastrol reduced the NFκB activation and improved myogenesis in dysferlin-deficient cultures. The results suggest that decreased myotube fusion in dysferlin deficiency is attributable to intrinsic inflammatory activation and can be improved using anti-inflammatory mediators.

Histological assessment of SJL/J mice treated with the antioxidants coenzyme Q10 and resveratrol

The muscular dystrophies (MDs) are genetic disorders of muscle degeneration due to mutations in genes that encode a wide variety of proteins. Dysferlinopathy are characterized by the absence of dysferlin in skeletal muscle and an autosomal recessive mode of inheritance. Both histological and ultrastructural pathology have been well established in dysferlinopathy patients and dysferlin-deficient animal models. To our knowledge the effect of antioxidant supplementation on this level has not been described previously. This article therefore focuses on the histopathology to reveal the effect of antioxidant supplementation. The study aimed to determine, at cellular level, the histopathological changes in the SJL/J mouse model following a 90 day trial with antioxidant supplementation. Markedly reduced inflammatory insult in the more affected quadriceps muscles of animals treated with high doses of CoQ10 and a combination of resveratrol/CoQ10 were observed. The outcome provides evidence that high doses of antioxidant supplementation resulted in decreased dystrophic markers and enhanced tissue integrity at cellular level.

Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages.

Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy

Skeletal muscle requires an efficient and active membrane repair system to overcome the rigours of frequent contraction. Dysferlin is a component of that system and absence of dysferlin causes muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels and a prominent inflammatory infiltrate. We have observed that dysferlinopathy patient biopsies show an excess of immature fibres and therefore investigated the role of dysferlin in muscle regeneration. Using notexin-induced muscle damage, we have shown that regeneration is attenuated in a mouse model of dysferlinopathy, with delayed removal of necrotic fibres, an extended inflammatory phase and delayed functional recovery. Satellite cell activation and myoblast fusion appear normal, but there is a reduction in early neutrophil recruitment in regenerating and also needle wounded muscle in dysferlin-deficient mice. Primary mouse dysferlinopathy myoblast cultures show reduced cytokine release upon stimulation, indicating that the secretion of chemotactic molecules is impaired. We suggest an extension to the muscle membrane repair model, where in addition to fusing patch repair vesicles with the sarcolemma dysferlin is also involved in the release of chemotactic agents. Reduced neutrophil recruitment results in incomplete cycles of regeneration in dysferlinopathy which combines with the membrane repair deficit to ultimately trigger dystrophic pathology. This study reveals a novel pathomechanism affecting muscle regeneration and maintenance in dysferlinopathy and highlights enhancement of the neutrophil response as a potential therapeutic avenue in these disorders.

Dysferlinopathy in Chile: Evidence of Two Novel Mutations in the First Reported Cases

We describe two Chilean patients with dysferlinopathy, a 32-year-old man with Miyoshi's distal myopathy and a 29-year-old woman with a proximodistal phenotype. Absence of dysferlin in frozen muscle biopsy allowed diagnostic confirmation. In these two patients, two mutations not previously identified in other populations were found: a homozygous c.1948delC (p.Leu650TyrfsX6) was found in the male patient; the heterozygous mutation c.1276G>A (p.Gly426Arg) was found in the female patient in association with the previously reported c.2858dupT (p.Phe954ValfsX2). To our knowledge, this is the first time that mutations in DYSF are identified in native Chileans. Our findings suggest the possibility that mutations in the DYSF gene were present in the Native American population before colonization.

The susceptibility to experimental autoimmune encephalomyelitis is not related to dysferlin-deficiency

Recent observations suggested that dysferlin might play a role in the development of autoimmune central nervous system (CNS) inflammation. To address this issue, we studied the induction and effector phase of experimental autoimmune encephalomyelitis in C57BL/10 mice producing intact or functionally deficient dysferlin. We found that both types of mice showed identical T-cell and antibody responses against the immunogen, and developed CNS inflammation with identical clinical courses, frequencies, lesion distributions, sizes and compositions. These findings suggest that the presence or absence of dysferlin does not have any consequences for the triggering or effector phase of autoimmune CNS inflammation.

A new phenotype of dysferlinopathy with congenital onset

We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene ( DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.

Dysferlin Deficiency Shows Compensatory Induction of Rab27A/Slp2a That May Contribute to Inflammatory Onset

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.