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At least 19 recordsLinked to original sources

PubMed2001

Dysferlin protein analysis in limb-girdle muscular dystrophies.

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD21 (19q13.3). We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency. In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B. Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients. Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle. Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin. Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (approximately 14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Adult↗

PubMed2001

Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy.

BACKGROUND: Mutations in the genes encoding for calpain-3 and dysferlin are responsible for limb-girdle muscular dystrophy (LGMD) type 2A and 2B, the most common forms of autosomal recessive LGMD. OBJECTIVE: To identify calpain-3 or dysferlin deficiency in a large cohort of patients with as yet unclassified LGMD and myopathy through candidate protein analysis. METHODS: The authors' muscle biopsy database search identified 407 candidate muscle biopsies with normal dystrophin and sarcoglycan. Calpain-3 and dysferlin were studied by Western blotting and immunohistochemistry. RESULTS: Combined calpain-3 and dysferlin Western blot analysis identified calpain-3 deficiency in 66 (16%) muscle biopsies. In 31 cases (47%), the protein was absent, and in 35 (53%), it was severely reduced in amount (3 to 50% of control). Dysferlin deficiency was found in 26 (6.5%) muscle biopsies. In 9, the protein was absent (35%), and in 17 (65%), it was severely reduced in amount (traces to 20% of control). Twenty-eight percent (53/191) of patients with LGMD phenotype had calpain-3 deficiency. Sixty percent (21/35) of patients with distal myopathy had dysferlin deficiency. Dysferlin immunohistochemistry showed, in the completely dysferlin-deficient patients, absent reaction at the sarcolemma but positive nuclear membrane labeling and, in the partially dysferlin-deficient patients, scattered granular positive cytoplasmic areas and diffuse reaction in regenerating fibers. CONCLUSION: About 25% of previously unclassified dystrophy/myopathy cases are due to calpain-3 or dysferlin protein deficiency. These results suggest that immunoblot analysis may be used to define patients for calpain-3 and dysferlin gene mutation studies.

Biopsy↗

PubMed2002

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice.

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.

Amino Acid Sequence↗

PubMed2002

Miyoshi myopathy patients with novel 5' splicing donor site mutations showed different dysferlin immunostaining at the sarcolemma.

We analyzed five clinically defined cases of Miyoshi myopathy both genetically and immunologically. Western blot of muscle specimens confirmed that all of these patients had dysferlin deficiency. Immunohistochemistry revealed that two of the five patients showed positive dysferlin immunostaining. Subsequent mutation analysis of the dysferlin gene in these two patients revealed that both had novel 5' splicing donor site mutations. One patient with a homozygous G to C substitution at nucleotide 1036+1 exon 6 splicing donor site showed patchy sarcolemmal dysferlin immunostaining. The second patient with both a heterozygous G to A substitution at nucleotide 1310+1 exon 10 splicing donor site and a heterozygous C to G substitution at nucleotide 1939 (which induces Tyr 522 Stop of exon 18) showed both patchy sarcolemmal and diffuse cytoplasmic dysferlin immunostaining. In contrast to Becker muscular dystrophy, the clinical course and severity of dysferlin staining positive patients was not clearly different from negative patients. These results suggest that a splicing mutation of the dysferlin gene may have the potential to cause decreased dysferlin expression but may not be related to the milder clinical phenotype.

Adolescent↗

PubMed2003

Defective membrane repair in dysferlin-deficient muscular dystrophy.

Muscular dystrophy includes a diverse group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle. Mutations in dysferlin are linked to two clinically distinct muscle diseases, limb-girdle muscular dystrophy type 2B and Miyoshi myopathy, but the mechanism that leads to muscle degeneration is unknown. Dysferlin is a homologue of the Caenorhabditis elegans fer-1 gene, which mediates vesicle fusion to the plasma membrane in spermatids. Here we show that dysferlin-null mice maintain a functional dystrophin-glycoprotein complex but nevertheless develop a progressive muscular dystrophy. In normal muscle, membrane patches enriched in dysferlin can be detected in response to sarcolemma injuries. In contrast, there are sub-sarcolemmal accumulations of vesicles in dysferlin-null muscle. Membrane repair assays with a two-photon laser-scanning microscope demonstrated that wild-type muscle fibres efficiently reseal their sarcolemma in the presence of Ca2+. Interestingly, dysferlin-deficient muscle fibres are defective in Ca2+-dependent sarcolemma resealing. Membrane repair is therefore an active process in skeletal muscle fibres, and dysferlin has an essential role in this process. Our findings show that disruption of the muscle membrane repair machinery is responsible for dysferlin-deficient muscle degeneration, and highlight the importance of this basic cellular mechanism of membrane resealing in human disease.

Animals↗

PubMed2001

The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle.

Dysferlin is a surface membrane protein in skeletal muscle whose deficiency causes distal and proximal, recessively inherited, forms of muscular dystrophy designated Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B), respectively. The function of dysferlin is not defined. Caveolin-3 is another skeletal muscle membrane protein which is important in the formation of caveolae and whose mutations cause dominantly inherited limb girdle muscular dystrophy type 1C (LGMD1C). We report that dysferlin co-immunoprecipitates with caveolin-3 from biopsied normal human skeletal muscles. We also describe abnormal localization of dysferlin in muscles from patients with LGMD1C including novel missense mutation (T64P) in the human caveolin-3 gene (CAV3). The immunoprecipitation data are consistent with the parallel observation that dysferlin immunostaining is not normal in LGMD1C muscles. Amino acid sequence analysis of the dysferlin protein reveals seven sites that correspond to caveolin-3 scaffold-binding motifs, and one site that is a potential target to bind the WW domain of the caveolin-3 protein. This is the first description of a possible dysferlin interacting protein; it suggests the hypothesis that one function of dysferlin may be to interact with caveolin-3 to subserve signaling functions of caveolae.

Caveolin 3↗

PubMed2001

Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy.

OBJECTIVE: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. METHODS: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. RESULTS: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 +/- 3.9 years. CONCLUSION: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Adolescent↗

PubMed2000

Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb--girdle muscular dystrophies.

Dysferlin has recently been identified as a novel gene involved in limb-girdle muscular dystrophy type 2B (LGMD2B) and its allelic disease, Miyoshi myopathy. The predicted structure of dysferlin suggests that it is a transmembrane protein possibly involved in membrane fusion. Thus, unlike previously identified structural proteins in muscular dystrophy, dysferlin is likely involved in a novel pathogenic mechanism for this disease. In this study, we have analyzed the expression of dysferlin in skeletal muscle of patients with disruptions in the dystrophin-glycoprotein complex and patients with a clinical diagnosis of LGMD2B or Miyoshi myopathy. We show expression of dysferlin at the sarcolemma in normal muscle and reduced sarcolemmal expression along with accumulation of intracellular staining in dystrophic muscle. Electron microscopy in Miyoshi myopathy biopsies suggests that the cytoplasmic staining could be a result of the abundance of intracellular vesicles. Our results indicate that dysferlin expression is perturbed in LGMD and that both mutations in the dysferlin gene and disruption of the dystrophin-glycoprotein complex can lead to the accumulation of dysferlin within the cytoplasm.

Dysferlin↗

PubMed2003

Dysferlin expression in tubular aggregates: their possible relationship to endoplasmic reticulum stress.

Dysferlin is a newly identified sarcolemmal protein related to Miyoshi myopathy and limb-girdle muscular dystrophy. Although its function is still unknown, it is inferred from the presence of C2 domains and a transmembrane domain in its sequence that dysferlin may be expressed or located not only at the sarcolemma but also in other membranous organelles to interact with Ca(2+). Tubular aggregates (TAs) are derived from sarcoplasmic reticulum (SR) and found in various myopathies, especially in those related to disturbed intra-sarcoplasmic Ca(2+) homeostasis. To clarify the expression of dysferlin in TAs and the relationship among TA formation, dysferlin expression, and endoplasmic reticulum (ER) stress, we examined the expression of dysferlin and other sarcolemmal proteins by immunohistochemistry in 12 muscle biopsy specimens with TAs from 11 cases of periodic paralysis and 1 case of myalgia/cramps syndrome. Moreover, the expression of glucose-regulated protein 78 (GRP78) and GRP94, which are up-regulated under ER stress, was also examined by immunohistochemistry and immunoblotting. TAs showed strong expression of dysferlin. GRP78 and GRP94 were also intensely expressed in TAs. Total amounts of GRP78 and GRP94 were significantly increased in muscles with TAs compared with normal controls. These results indicate that muscles with TAs seem to be under ER stress, probably resulting from disturbed intra-sarcoplasmic Ca(2+) homeostasis. Strong expression of dysferlin in TAs suggests the possibility that it is located not only at the sarcolemma but also in the SR, at least in the pathological conditions.

Adolescent↗

PubMed1999

Dysferlin is a plasma membrane protein and is expressed early in human development.

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

Amino Acid Sequence↗

PubMed2000

Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation.

Mutations in dysferlin were recently described in patients with Miyoshi myopathy, a disorder that preferentially affects the distal musculature, and in patients with Limb-Girdle Muscular Dystrophy 2B, a disorder that affects the proximal musculature. Despite the phenotypic differences, the types of mutations associated with Miyoshi myopathy and Limb-Girdle Muscular Dystrophy 2B do not differ significantly. Thus, the etiology of the phenotypic variability associated with dysferlin mutations remains unknown. Using genetic linkage and mutation analysis, we identified a large inbred pedigree of Yemenite Jewish descent with limb-girdle muscular dystrophy. The phenotype in these patients included slowly progressive, proximal, and distal muscular weakness in the lower limbs with markedly elevated serum creatine kinase (CK) levels. These patients had normal development and muscle strength and function in early life. Muscle biopsies from 4 affected patients showed a typical dystrophic pattern but interestingly, in 2, an inflammatory process was seen. The inflammatory infiltrates included primarily CD3 positive lymphocytes. Associated with this phenotype, we identified a previously undescribed frameshift mutation at nucleotide 5711 of dysferlin. This mutation produced an absence of normal dysferlin mRNA synthesis by affecting an acceptor site and cryptic splicing. Thus, splice site mutations that disrupt dysferlin may produce a phenotype associated with inflammation.

Alternative Splicing↗

PubMed1998

Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.

Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).

Adult↗

PubMed2001

Cloning of the mouse dysferlin gene and genomic characterization of the SJL-Dysf mutation.

The SJL mouse strain has been widely used as an animal model for experimental autoimmune encephalitis (EAE), inflammatory muscle disease and lymphomas and has also been used as a background strain for the generation of animal models for a variety of diseases including motor neurone disease, multiple sclerosis and atherosclerosis. Recently the SJL mouse was shown to have myopathy due to dysferlin deficiency, so that it can now be considered a natural animal model for limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). We have cloned the mouse dysferlin cDNA and analysis of the sequence shows that the mouse dysferlin gene is characterized by six C2 domain sequences and a C-terminal anchoring domain, with the human and the mouse dysferlin genes sharing > 90% sequence homology overall. Genomic analysis of the SJL mutation confirms that the 171 bp RNA deletion has arisen by exon skipping resulting from a splice site mutation. The identification of this mutation has implications for the various groups using this widely available mouse stock.

Amino Acid Sequence↗

PubMed1999

Dysferlin is a surface membrane-associated protein that is absent in Miyoshi myopathy.

Recently we reported that mutations in a muscle protein "dysferlin" are present in limb girdle muscular dystrophy-2B and a related, adult-onset, distal dystrophy known as Miyoshi myopathy (MM). We report that antibodies to dysferlin identify a protein of approximately 230 kDa and show that dysferlin is located in the muscle membrane. This protein is absent in MM and LGMD-2B muscle. By contrast, dystrophin and other dystrophin-associated proteins are normal in these patients. Thus, dysferlin is a membrane-associated protein that is not likely to be an integral component of the dystrophin complex. Although it is not essential for initial myogenesis, it appears to be critical for sustained normal function in mature muscle.

Dysferlin↗

PubMed2000

The third human FER-1-like protein is highly similar to dysferlin.

Dysferlin, the protein product of the gene mutated in patients with an autosomal recessive limb-girdle muscular dystrophy type 2B (LGMD2B) and a distal muscular dystrophy, Miyoshi myopathy, is homologous to a Caenorhabditis elegans spermatogenesis factor, FER-1. Analysis of fer-1 mutants and of sequence predictions of the FER-1 and dysferlin ORFs has predicted a role in membrane fusion. Otoferlin, another human FER-1-like protein (ferlin), has recently been shown to be responsible for autosomal recessive nonsyndromic deafness (DFNB9). In this report we describe the third human ferlin gene, FER1L3, which maps to chromosome 10q23.3. Expression analysis of the orthologous mouse gene shows ubiquitous expression but predominant expression in the eye, esophagus, and salivary gland. All the ferlins are characterized by sequences corresponding to multiple C2 domains that share the highest level of homology with the C2A domain of rat synaptotagmin III. They are predicted to be Type II transmembrane proteins, with the majority of the protein facing the cytoplasm anchored by the C-terminal transmembrane domain. Sequence and predicted structural comparisons have highlighted the high degree of similarity of dysferlin and FER1L3, which have sequences corresponding to six C2 domains and which share more than 60% amino acid sequence identity.

Amino Acid Sequence↗

PubMed2003

Clinical, morphological and immunological evaluation of six patients with dysferlin deficiency.

Limb girdle muscular dystrophy (LGMD) type 2B and distal Miyoshi myopathy (MM) are caused by mutations in a recently discovered mammalian gene coding for a skeletal muscle protein called dysferlin. The protein is normally expressed at the skeletal muscle level and absent or reduced in affected patients. We selected a clinically heterogeneous population of Italian myopathic patients with clinical evidence of myopathy and/or hyperCKemia, EMG myopathic pattern, and no alterations of the dystrophin-sarcoglycan complex. Calpain, merosin, emerin and caveolin were also tested and found normal in all patients. Dysferlin immunohistochemical and Western blot analyses allowed us to identify six patients with dysferlin deficiency: one with distal myopathy, four with limb girdle myopathy and one with hyperCKemia. No apoptosis was found in any of the six muscle specimens, although expression of the pro-apoptotic Fas antigen was mildly increased in two cases. Inflammatory reactions were present in two of the six cases, but we found no evidence of immune-mediated processes.

Adolescent↗

PubMed2001

Phenotypic variation in a large Japanese family with Miyoshi myopathy with nonsense mutation in exon 19 of dysferlin gene.

Miyoshi myopathy, an autosomal recessive muscular dystrophy involving distal muscles, is caused by dysferlin mutations. We present clinical and genetic studies of two men and six women, aged 25-83 years, from a Japanese family with consanguineous marriages. Onset was between ages 17 and 59 years. Six of the patients had muscle involvement typical of Miyoshi myopathy, one initially had severe proximal muscle involvement, and one had scapuloperoneal-type muscle involvement. Three patients showed steppage gait. Genetic linkage analysis identified a maximum lod score of 3.34 (θ=0.00) at marker D2S292 in 2p13. Analysis of dysferlin revealed the mutation G2090T (Glu573Stop) in exon 19 in all affected patients. This is the largest Japanese family with Miyoshi myopathy showing intrafamilial phenotypic variation and sharing a common mutation in dysferlin.

Adult↗

PubMed2001

Dysferlinopathy (LGMD2B): a 23-year follow-up study of 10 patients homozygous for the same frameshifting dysferlin mutations.

The limb-girdle muscular dystrophies are a group of inherited neuromuscular disorders which are clinically and genetically heterogeneous. We have been able to carry out a follow-up study on 10 patients from a large Palestinian family with a confirmed mutation in the dysferlin gene. These patients have been followed for more than 23 years since the onset of the disease. They all had normal developmental milestones. The onset of the disease was usually in the second decade, more rarely in the third and fourth decades. The first symptoms were difficulty with running and climbing stairs. Patients showed a distinct type of gait due to the unique pattern of muscle involvement which was characterised by early involvement of the posterior muscle compartment of the thighs and legs (hamstrings, adductors, gastrocnemius and soleus). The shoulder and upper limb musculature became involved later, especially supra and infraspinatus and biceps. In the early stages of disease these patients may clinically show only proximal lower limb-girdle muscle weakness; however, the use of muscle imaging techniques were very important, always detecting in these patients also distal lower limb muscle involvement, so that the pattern of muscle involvement found in dysferlin deficiency may not strictly conform to the definition of limb-girdle muscular dystrophy. The pattern of muscular dystrophy is essentially uniform and has clearly distinct features (involving mainly the initial pattern of muscle involvement and the mode of gait) which differ significantly from the well reported clinical features associated with sarcoglycanopathy, calpainopathy and Miyoshi myopathy.

Adolescent↗