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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.

Most Mendelian disorders, including neuromuscular disorders, display extensive clinical heterogeneity that cannot be solely explained by primary genetic mutations. This phenotypic variability is largely attributed to the presence of disease modifiers, which can exacerbate or lessen the severity and progression of the disease. LAMA2-deficient congenital muscular dystrophy (LAMA2-CMD) is a fatal degenerative muscle disease resulting from mutations in the LAMA2 gene encoding Laminin-α2. Progressive muscle weakness is predominantly observed in the lower limbs in LAMA2-CMD patients, whereas upper limbs muscles are significantly less affected. However, very little is known about the molecular mechanism underlying differential pathophysiology between specific muscle groups. Here, we demonstrate that the triceps muscles of the dy2j/dy2j mouse model of LAMA2-CMD demonstrate very mild myopathic findings compared with the tibialis anterior (TA) muscles that undergo severe atrophy and fibrosis, suggesting a protective mechanism in the upper limbs of these mice. Comparative gene expression analysis reveals that S-Adenosylmethionine decarboxylase (Amd1) and Spermine oxidase (Smox), two components of polyamine pathway metabolism, are downregulated in the TA but not in the triceps of dy2j/dy2j mice. As a consequence, the level of polyamine metabolites is significantly lower in the TA than triceps. Normalization of either Amd1 or Smox expression in dy2j/dy2j TA ameliorates muscle fibrosis, reduces overactive profibrotic TGF-β pathway and leads to improved locomotion. In summary, we demonstrate that a deregulated polyamine metabolism is a characteristic feature of severely affected lower limb muscles in LAMA2-CMD. Targeted modulation of this pathway represents a novel therapeutic avenue for this devastating disease.

The purpose of this study was to determine whether thrombospondin (TSP)-1 promotes macrophage activity and disease progression in dysferlinopathy. First, we found that levels of TSP-1 are elevated in blood of non-ambulant dysferlinopathy patients compared with ambulant patients and healthy controls, supporting the idea that TSP-1 levels are correlated with disease progression. We then crossed dysferlinopathic BlaJ mice with TSP-1 knockout mice and assessed disease progression longitudinally with magnetic resonance imaging (MRI). In these mice, deletion of TSP-1 ameliorated loss in volume and mass of the moderately affected gluteal muscle but not of the severely affected psoas muscle. T2 MRI parameters revealed that loss of TSP-1 modestly inhibited inflammation only in gluteal muscle of male mice. Histological assessment indicated that deletion of TSP-1 reduced inflammatory cell infiltration of muscle fibers, but only early in disease progression. In addition, flow cytometry analysis revealed that, in males, TSP-1 knockout reduced macrophage infiltration and phagocytic activity, which is consistent with TSP-1-enhanced phagocytosis and pro-inflammatory cytokine induction in cultured macrophages. In summary, TSP-1 appears to play an accessory role in modulating Mp activity in BlaJ mice in a gender, age and muscle-dependent manner, but is unlikely a primary driver of disease progression of dysferlinopathy.

α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP –ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a “glycan remodelling” process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.

Repair of skeletal muscle after sarcolemmal damage involves dysferlin and dysferlin-interacting proteins such as annexins. Mice and patient lacking dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in dysferlinopathy.

Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

Adult-onset inherited myopathies with similar pathological features, including hereditary inclusion body myopathy (hIBM) and limb-girdle muscular dystrophy (LGMD), are a genetically heterogeneous group of muscle diseases. It is unclear whether these inherited myopathies initiated by mutations in distinct classes of genes are etiologically related. Here, we exploit a genetic model system to establish a mechanistic link between diseases caused by mutations in two distinct genes, hnRNPA2B1 and DNAJB6. Hrb98DE and mrj are the Drosophila melanogaster homologs of human hnRNPA2B1 and DNAJB6, respectively. We introduced disease-homologous mutations to Hrb98DE, thus capturing mutation-dependent phenotypes in a genetically tractable model system. Ectopic expression of the disease-associated mutant form of hnRNPA2B1 or Hrb98DE in fly muscle resulted in progressive, age-dependent cytoplasmic inclusion pathology, as observed in humans with hnRNPA2B1-related myopathy. Cytoplasmic inclusions consisted of hnRNPA2B1 or Hrb98DE protein in association with the stress granule marker ROX8 and additional endogenous RNA-binding proteins (RBPs), suggesting that these pathological inclusions are related to stress granules. Notably, TDP-43 was also recruited to these cytoplasmic inclusions. Remarkably, overexpression of MRJ rescued this phenotype and suppressed the formation of cytoplasmic inclusions, whereas reduction of endogenous MRJ by a classical loss of function allele enhanced it. Moreover, wild-type, but not disease-associated, mutant forms of MRJ interacted with RBPs after heat shock and prevented their accumulation in aggregates. These results indicate both genetic and physical interactions between disease-linked RBPs and DNAJB6/mrj, suggesting etiologic overlap between the pathogenesis of hIBM and LGMD initiated by mutations in hnRNPA2B1 and DNAJB6.

Consanguinity is known to be associated with an increase in the prevalence of autosomal recessive disorders such as autosomal recessive congenital ichthyosis (ARCI). ARCI often responds well to retinoid treatment. We describe a patient with ARCI who improved under isotretinoin treatment. The patient subsequently developed elevated levels of serum creatinine phosphokinase (CPK), which led to the diagnosis of a second autosomal recessive disorder, dysferlinopathy, a rare myopathy characterized by muscle weakness, decreased tendon reflexes and marked elevation of CPK levels. This report demonstrates the need for physicians to remain alert to the possible coexistence of rare and mutually relevant disorders in populations with a high rate of consanguinity.

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.

In 2001, we reported linkage of an autosomal dominant form of limb-girdle muscular dystrophy, limb-girdle muscular dystrophy 1F, to chromosome 7q32.1-32.2, but the identity of the mutant gene was elusive. Here, using a whole genome sequencing strategy, we identified the causative mutation of limb-girdle muscular dystrophy 1F, a heterozygous single nucleotide deletion (c.2771del) in the termination codon of transportin 3 (TNPO3). This gene is situated within the chromosomal region linked to the disease and encodes a nuclear membrane protein belonging to the importin beta family. TNPO3 transports serine/arginine-rich proteins into the nucleus, and has been identified as a key factor in the HIV-import process into the nucleus. The mutation is predicted to generate a 15-amino acid extension of the C-terminus of the protein, segregates with the clinical phenotype, and is absent in genomic sequence databases and a set of >200 control alleles. In skeletal muscle of affected individuals, expression of the mutant messenger RNA and histological abnormalities of nuclei and TNPO3 indicate altered TNPO3 function. Our results demonstrate that the TNPO3 mutation is the cause of limb-girdle muscular dystrophy 1F, expand our knowledge of the molecular basis of muscular dystrophies and bolster the importance of defects of nuclear envelope proteins as causes of inherited myopathies.

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.

Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.

Mutations in dysferlin cause an inherited muscular dystrophy because of defective membrane repair. Three interacting partners of dysferlin are also implicated in membrane resealing: caveolin-3 (in limb girdle muscular dystrophy type 1C), annexin A1, and the newly identified protein mitsugumin 53 (MG53). Mitsugumin 53 accumulates at sites of membrane damage, and MG53-knockout mice display a progressive muscular dystrophy. This study explored the expression and localization of MG53 in human skeletal muscle, how membrane repair proteins are modulated in various forms of muscular dystrophy, and whether MG53 is a primary cause of human muscle disease. Mitsugumin 53 showed variable sarcolemmal and/or cytoplasmic immunolabeling in control human muscle and elevated levels in dystrophic patients. No pathogenic MG53 mutations were identified in 50 muscular dystrophy patients, suggesting that MG53 is unlikely to be a common cause of muscular dystrophy in Australia. Western blot analysis confirmed upregulation of MG53, as well as of dysferlin, annexin A1, and caveolin-3 to different degrees, in different muscular dystrophies. Importantly, MG53, annexin A1, and dysferlin localize to the t-tubule network and show enriched labeling at longitudinal tubules of the t-system in overstretch. Our results suggest that longitudinal tubules of the t-system may represent sites of physiological membrane damage targeted by this membrane repair complex.

The limb girdle muscular dystrophies (LGMDs) are a group of disorders with wide genetic and clinical heterogeneity. Recently, mutations in the ANO5 gene, which encodes a putative calcium-activated chloride channel belonging to the Anoctamin family of proteins, were identified in five families with one of two previously identified disorders, LGMD2L and non-dysferlin Miyoshi muscular dystrophy (MMD3). We screened a candidate group of 64 patients from 59 British and German kindreds and found the truncating mutation, c.191dupA in exon 5 of ANO5 in 20 patients, homozygously in 15 and in compound heterozygosity with other ANO5 variants in the rest. An intragenic SNP and an extragenic microsatellite marker are in linkage disequilibrium with the mutation, suggesting a founder effect in the Northern European population. We have further defined the clinical phenotype of ANO5 -associated muscular dystrophy. Patients show adult onset proximal lower limb weakness with highly raised creatinine kinase (CK) values (average 4500 IU/l) and frequent muscle atrophy and asymmetry of muscle involvement. Onset varies from the early 20s to 50s and the weakness is generally slowly progressive, with most patients remaining ambulant for several decades. Distal presentation is much less common but a milder degree of distal lower limb weakness is often observed. Upper limb strength is only mildly affected and cardiac and respiratory function is normal. Females appear less frequently affected. In the North of England population we have identified eight patients with ANO5 mutations, suggesting a minimum prevalence of 0.27/100 000, twice as common as dysferlinopathy. We suggest that mutations in ANO5 represent a relatively common cause of adult onset muscular dystrophy with high CK and that mutation screening, particularly of the common mutation c.191dupA, should be an early step in the diagnostic algorithm of adult LGMD patients. * Abbreviations : CK : creatinine kinase ECG : electrocardiogram EMG : electromyography IHC : immunohistochemistry LGMDs : limb-girdle muscular dystrophy MMD3 : Miyoshi muscular dystrophy MM : Miyoshi myopathy SNP : single-nucleotide polymorphism WB : western blot

Tcap/telethonin encodes a Z-disc protein that plays important roles in sarcomere assembly, sarcomere-membrane interaction and stretch sensing. It remains unclear why mutations in Tcap lead to limb-girdle muscular dystrophy 2G (LGMD2G) in human patients. Here, we cloned tcap in zebrafish and conducted genetic studies. We show that tcap is functionally conserved, as the Tcap protein appears in the sarcomeric Z-disc and reduction of Tcap resulted in muscular dystrophy-like phenotypes including deformed muscle structure and impaired swimming ability. However, the observations that Tcap integrates into the sarcomere at a stage after the Z-disc becomes periodic, and that the sarcomere remains intact in tcap morphants, suggest that defective sarcomere assembly does not contribute to this particular type of muscular dystrophy. Instead, a defective interaction between the sarcomere and plasma membrane was detected, which was further underscored by the disrupted development of the T-tubule system. Pertinent to a potential function in stretch sensor signaling, zebrafish tcap exhibits a variable expression pattern during somitogenesis. The variable expression is inducible by stretch force, and the expression level of Tcap is negatively regulated by integrin-link kinase (ILK), a protein kinase that is involved in stretch sensing signaling. Together, our genetic studies of tcap in zebrafish suggested that pathogenesis in LGMD2G is due to a disruption of sarcomere–T-tubular interaction, but not of sarcomere assembly per se. In addition, our data prompted a novel hypothesis that predicts that the transcription level of Tcap can be regulated by the stretch force to ensure proper sarcomere–membrane interaction in striated muscles.

Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence of or defective muscular proteins. The murine model for limb-girdle muscular dystrophy 2B (LGMD2B), the SJL mice, carries a deletion in the dysferlin gene that causes a reduction in the protein levels to 15% of normal. The mice show muscle weakness that begins at 4–6 weeks and is nearly complete by 8 months of age. The possibility of restoring the defective muscle protein and improving muscular performance by cell therapy is a promising approach for the treatment of LGMDs or other forms of progressive muscular dystrophies. Here we have injected human adipose stromal cells (hASCs) into the SJL mice, without immunosuppression, aiming to assess their ability to engraft into recipient dystrophic muscle after systemic delivery; form chimeric human/mouse muscle fibers; express human muscle proteins in the dystrophic host and improve muscular performance. We show for the first time that hASCs are not rejected after systemic injection even without immunosuppression, are able to fuse with the host muscle, express a significant amount of human muscle proteins, and improve motor ability of injected animals. These results may have important applications for future therapy in patients with different forms of muscular dystrophies. Disclosure of potential conflicts of interest is found at the end of this article.

Muscular dystrophies with reduced glycosylation of alpha-dystroglycan (alpha-DG), commonly referred to as dystroglycanopathies, are a heterogeneous group of autosomal recessive conditions which include a wide spectrum of clinical severity. Reported phenotypes range from severe congenital onset Walker-Warburg syndrome (WWS) with severe structural brain and eye involvement, to relatively mild adult onset limb girdle muscular dystrophy (LGMD). Specific clinical syndromes were originally described in association with mutations in any one of six demonstrated or putative glycosyltransferases. Work performed on patients with mutations in the FKRP gene has identified that the spectrum of phenotypes due to mutations in this gene is much wider than originally assumed. To further define the mutation frequency and phenotypes associated with mutations in the other five genes, we studied a large cohort of patients with evidence of a dystroglycanopathy. Exclusion of mutations in FKRP was a prerequisite for participation in this study. Ninety-two probands were screened for mutations in POMT1, POMT2, POMGnT1, fukutin and LARGE. Homozygous and compound heterozygous mutations were detected in a total of 31 probands (34 individuals from 31 families); 37 different mutations were identified, of which 32 were novel. Mutations in POMT2 were the most prevalent in our cohort with nine cases, followed by POMT1 with eight cases, POMGnT1 with seven cases, fukutin with six cases and LARGE with only a single case. All patients with POMT1 and POMT2 mutations had evidence of either structural or functional central nervous system involvement including four patients with mental retardation and a LGMD phenotype. In contrast mutations in fukutin and POMGnT1 were detected in four patients with LGMD and no evidence of brain involvement. The majority of patients (six out of nine) with mutations in POMT2 had a Muscle-Eye-Brain (MEB)-like condition. In addition we identified a mutation in the gene LARGE in a patient with WWS. Our data expands the clinical phenotypes associated with POMT1, POMT2, POMGnT1, fukutin and LARGE mutations. Mutations in these five glycosyltransferase genes were detected in 34% of patients indicating that, after the exclusion of FKRP, the majority of patients with a dystroglycanopathy harbour mutations in novel genes.

Diagnosis of limb girdle muscular dystrophy type 2A can be complex due to phenotypic variability, lack of precision of protein analysis in muscle biopsies, and absence of mutational hot spots in the CAPN3 gene. The aim of this study was to review clinical and biopsy data from a group of patients with known CAPN3 genetic status to validate and refine our current diagnostic strategy, which combines clinical information and protein analysis to direct gene testing. We analysed 85 patients in whom CAPN3 gene sequencing had been performed. Forty-two patients had two mutations, 15 a single mutation and in 28 no mutation was found. We identified clinical features that clearly discriminated the LGMD2A patients. These were: presence of scapular winging, contractures and normal respiratory function. In addition, a typical pattern of muscle weakness on manual muscle testing could be confirmed. Interpretation of protein expression obtained by Western blot was complex and involved the analysis of a number of bands detected by two antibodies for calpain 3. Loss of all calpain 3 bands was 100% specific for LGMD2A, but this pattern was found in only 23%. Absence or reduction of the approximately 60 kDa bands was also highly specific for LGMD2A, while increased abundance was highly predictive of no mutations being found even where other bands were reduced, suggesting that this is the most sensitive marker of artefactual protein degradation. Twenty-three percent of the patients with two mutations had normal full-sized calpain 3 protein, consistent with the finding of mutations localized in parts of the gene likely or proven to be involved in autolytic activity. Clinical and biochemical findings in patients with only one mutation were similar to patients with two mutations, indicating that other gene analysis techniques should be used before excluding the diagnosis. Our analysis confirms that our strategy is still valid to prioritize genetic testing in this complex group of patients, provided patients with normal protein but a suggestive clinical phenotype are not excluded from genetic testing.

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of pathologies. We have identified a cohort of 14 French-Canadian patients from eight different families displaying a novel form of LGMD with an autosomal recessive inheritance. These patients share some features with previously described cases of 'quadriceps myopathy' that evolved into an LGMD. All demonstrate quadriceps femoris asymmetrical atrophy. Creatine kinase values were variable from normal to 6000 U/l. Clinical evaluations and MRI studies demonstrate a variable intrafamilial and interfamilial phenotype. Asymmetrical muscle involvement was clinically observed and confirmed by imaging. MRI studies suggest that the hamstrings and the adductor magnus are the first limb muscles to demonstrate fatty infiltration. Muscle pathology shows no sign of active inflammation but increased endomysial connective tissue associated with basal lamina duplication and collagen disorganization. A genome-wide scan using the two largest families uncovered linkage to marker D11S1360 on chromosome 11p12 [multipoint logarithm of the odds (LOD) score of 2.78]. Further genotyping for the eight families confirmed linkage to this new LGMD locus (multipoint LOD score of 4.56). Fine mapping subsequently defined a less than 3.3 cM candidate interval on 11p13-p12. Haplotype analysis of carrier chromosomes suggests that the most frequent mutation may account for up to 81.3% of French-Canadian mutations. In this study, we describe the chromosomal locus of a new form of recessive LGMD with prominent quadriceps femoris atrophy.

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with limb-girdle muscular dystrophy type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), three similar adult-onset, progressive and autosomal dominant muscular dystrophies. To further investigate myotilin's role in the pathogenesis of these muscle diseases, we have characterized three independent lines of transgenic mice expressing mutant (T57I) myotilin under the control of the human skeletal actin promoter. Similar to LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. We show that the mutant myotilin protein properly localizes to the Z-disc and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared with littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. In sum, we have established a promising patho-physiological mouse model that unifies the phenotypes of LGMD1A, MFM and SBM.

Although there is still a long way to go, our understanding of the genetic basis of cardiomyopathies--dilated or hypertrophic--has significantly improved over the past decade. This new and intriguing era of cardiogenetics has already answered some important questions concerning the pathophysiology of these disorders, but it has also raised some new questions: how do we define "presymptomatic" mutation carriers? Should we treat them? Do we have any diagnostic tools to identify the presymptomatic subjects in those families where the underlying mutation has not been identified yet? To address at least part of these questions, there is a clear need for screening techniques in the early stage of the disease which have to be sensitive and non-invasive. In recent years Tissue Doppler Imaging (TDI) has emerged as a well suited technique for these purposes and several interesting papers on this issue have been published. This paper reviews the findings from TDI in several forms of inherited cardiomyopathy. Although the implementation of this technique in everyday clinical practice still requires some refinement, the results from these studies are encouraging and TDI is likely to be complementary to other established screening tools such as ECG and conventional echocardiography.

Mutations in the non-lysosomal cysteine protease calpain 3 cause limb-girdle muscular dystrophy type 2A (LGMD2A). Our previous studies of the calpain 3 knockout mouse (C3KO) suggested a role for calpain 3 in sarcomere formation and remodeling. Calpain 3 may mediate remodeling by cleavage and release of myofibrillar proteins, targeting them for ubiquitination and proteasomal degradation. Loss of proper protein turnover may be the basis for this muscle disease. To test this hypothesis in vivo, we used an experimental model of hindlimb unloading and reloading that has been shown to induce sarcomere remodeling. We showed that the rate of atrophy and especially the rate of growth are decreased in C3KO muscles under conditions promoting sarcomere remodeling. In wild-type mice, an elevated level of ubiquitinated proteins was observed during muscle reloading, which is presumably necessary to remove atrophy-specific and damaged proteins. This increase in ubiquitination correlated with an increase in calpain 3 expression. C3KO muscles did not show any increase in ubiquitination at the reloading stage, suggesting that calpain 3 is necessary for ubiquitination and that it acts upstream of the ubiquitination machinery. We found upregulation of heat shock proteins in C3KO muscles following challenge with a physiological condition that requires highly increased protein degradation. Furthermore, old C3KO mice show evidence of insoluble protein aggregate formation in skeletal muscles. These studies suggest that accumulation of aged and damaged proteins can lead to cellular toxicity and a cell stress response in C3KO muscles, and that these characteristics are pathological features of LGMD2A.

Mutations in myotilin gene (MYOT) have been associated with variable syndromes including limb girdle muscular dystrophy type 1A (LGMD1A) and a subgroup of myofibrillar myopathy (MFM/MYOT). We studied six Spanish patients from three unrelated kindreds and seven patients without family history. Three previously reported and two novel disease-associated MYOT mutations were identified in this group of patients. The disease is characterized by the onset at the age of 42-77 years with muscle weakness initially in distal or proximal leg muscles, eventually spreading to other muscle groups of the lower and upper extremities. Associated signs of cardiomyopathy, respiratory failure and peripheral neuropathy are present in a fraction of patients. Myopathological features of focal myofibrillar destruction resulting in intracytoplasmic deposits, strongly immunoreactive to myotilin, multiple rimmed and centrally or subsarcolemmally located non-rimmed vacuoles and streaming Z-lines, were observed in each patient studied. The Spanish cohort, the largest group of patients studied so far, shares phenotypic features with both LGMD1A and MFM/MYOT variants thus establishing a continuum of phenotypic manifestations characteristic of myotilinopathy, an emerging neuromuscular disorder.

Sir, We read with great interest the work recently published in Brain (Vissing et al. , 2016), in which the findings on limb girdle muscular dystrophy families from the UK, Denmark and Sweden are described. The cases showed a segregation pattern compatible with a dominant transmission. All the affected cases except for one share a heterozygous mutation in the CAPN3 gene previously described in recessive forms of limb girdle muscular dystrophy 2A (LGMD2A) (Richard et al. , 1997; Groen et al. , 2007). The mutation located within a common haplotype, suggests a common ancestral origin of these families, probably spread over a territory that shared a past Viking settlement as described for other mutations (Pliner et al. , 2014). The possibility of an alternative inheritance pattern of the same phenotype, associated with mutations in the same gene, is a well-known scenario in several myopathies. Besides the myotonia congenita, the collagen-related myopathies and the desminopathies referred to by the authors (Vissing et al. , 2016), there are also other well characterized examples such as RYR1 mutations associated to a central core myopathy (Klein et al. , 2012; Snoeck et al. , 2015) and mutations in the TTN gene responsible for dominant distal myopathy or a recessive variant of limb girdle muscular dystrophy (LGMD2J) (Hackman et al. , 2002, 2003; Udd et al. , 2005). Furthermore, in other recessive limb girdle muscular dystrophies, heterozygous carriers may present subclinical symptoms [creatine kinase (CK) elevations and/or alterations in MRI] associated with a reduction of the protein (Fischer et al. , 2003; Brummer et al. , 2005; Illa et al. , 2007). In this context, the understanding of this phenomenon in LGMD2A would be of great interest to shed some light on the knowledge of its pathophysiology, …