Pattern of skeletal muscle involvement in primary dysferlinopathies: a whole‐body 3.0‐T magnetic resonance imaging study

Objectives and methods – Mutations in the gene encoding dysferlin cause limb girdle muscular dystrophy type 2B (LGMD2B), distal Miyoshi myopathy (MM), and a rare form of distal anterior compartment myopathy. To study the correlations between clinical manifestations and muscle imaging changes we conducted a 3.0‐T magnetic resonance imaging (MRI) study in six German patients with primary dysferlinopathies defined by absence of dysferlin expression in muscle (MM, n = 3; LGMD2B, n = 2; hyperCKemia without clinical symptoms, n = 1). Results – Patients with manifest myopathy had widespread muscular pathology. In analogy to previous imaging studies, we confirmed an involvement of the anterior and posterior thigh compartments and a predominant involvement of posterior lower legs. However, our whole‐body MRI study further provided evidence of signal alterations in the glutei, erector spinae and shoulder girdle muscles. Correlation of clinical findings with imaging demonstrated the potential of MRI to detect subclinical muscle pathology. Conclusions – Whole‐body 3.0‐T MRI is a non‐invasive method to demonstrate various degrees of skeletal muscle alterations and disease progression in muscular dystrophies. Furthermore, whole‐body high‐field MRI may serve as a helpful diagnostic tool in differentiating primary dysferlinopathies from other forms of LGMD and distal myopathies.     

Developmental and tissue-specific regulation of a novel dysferlin isoform

Dysferlin plays an essential role in the muscle repair machinery, and its deficiency is associated with limb-girdle muscular dystrophy type 2B and with two different distal myopathies (Miyoshi myopathy and distal anterior compartment myopathy). Our aims were to characterize the pattern of dysferlin expression during myogenic cell differentiation and to assess possible differentially spliced isoforms of the DYSF gene. Human primary myogenic cells express a splice variant of dysferlin mRNA lacking exon 17 (Delta17), together with full-length dysferlin mRNA. Real-time polymerase chain reaction analysis of human myoblasts, myotubes, and normal skeletal muscle showed that Delta17 expression inversely correlates with muscle differentiation. Indeed, Delta17 is progressively replaced by the wild type as myoblast fusion proceeds, and it disappears in adult skeletal muscle. Conversely, Delta17 is the predominant dysferlin variant in mature peripheral nerve. Our findings suggest that the two proteins play different roles in myogenic cell differentiation and that dysferlin function in peripheral nerve might be accomplished by this novel isoform.     

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, annexin A1, and mitsugumin 53 are upregulated in muscular dystrophy and localize to longitudinal tubules of the T-system with stretch

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.     

Progress and challenges in diagnosis of dysferlinopathy

Dysferlin‐deficient limb girdle muscular dystrophy type 2B, distal Miyoshi myopathy, and other less frequent phenotypes are a group of recessive disorders called dysferlinopathies. They are characterized by wide clinical heterogeneity. To diagnose dysferlinopathy, a clinical neuromuscular workup, including electrophysiological and muscle imaging investigations, is essential to support subsequent laboratory testing. Increased serum creatine kinase levels, distal or proximal muscle weakness, and myalgia with onset in the second or third decades are the main clinical features of the disease. In muscle biopsies, severe dysferlin deficiency by immunoblot or its abnormal localization by immunohistochemistry are the gold standard, as they have a high diagnostic value. Dysferlin testing on monocytes is a valuable alternative to muscle immunoblotting. Molecular techniques for gene mutation detection, such as next generation sequencing, have improved the genetic diagnosis, which is crucial for treatment and genetic counselling. Muscle Nerve 54 : 821–835, 2016     

A novel, blood-based diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy.

Limb girdle muscular dystrophy 2B and Miyoshi myopathy were recently found to be allelic disorders arising from defects in the dysferlin gene. We have developed a new diagnostic assay for limb girdle muscular dystrophy 2B and Miyoshi myopathy, which screens for dysferlin expression in blood using a commercially available monoclonal antibody. Unlike current methods that require muscle biopsy for immunodiagnosis, the new method is simple and entails a significantly less invasive procedure for tissue sampling. Moreover, it overcomes some of the problems associated with the handling and storage of muscle specimens. In our analysis of 12 patients with limb girdle muscular dystrophy 2B or Miyoshi myopathy, the findings obtained using the new assay are fully consistent with the results from muscle immunodiagnosis.     

Positional cloning of the gene for Miyoshi myopathy and limb-girdle muscular dystrophy]

Miyoshi myopathy (MM) is autosomal recessive distal muscular dystrophy that we have mapped to chromosome 2 p13. We constructed a 3 Mb P 1-derived artificial chromosome contig spanning the MM candidate region. Using this and new polymorphic markers within it, we recently identified a novel, full-length 6.9 kb muscle cDNA, whose corresponding protein we designated "dysferlin" (Nature Genet, 1998: 20: 31-36). We described eighteen mutations in the dysferlin gene with MM or limb-girdle muscular dystrophy type 2 B (LGMD 2 B). Most are predicted to block translation of dysferlin protein. In some cases, corresponding Western immunoblotting reveals absence of dysferlin in muscle biopsies. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb-girdle dystrophy, distal myopathy with anterior tibial onset).     

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.     

Variable reduction of caveolin-3 in patients with LGMD2B/MM

Abstract. Mutations in the human dysferlin gene (DYSF) cause autosomal recessive muscular dystrophies characterized by degeneration and weakness of proximal and/or distal muscles: limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). Recently, an interaction between caveolin-3 and dysferlin in normal and dystrophic muscle (primary caveolin-3 deficiency; LGMD1C) was shown. In this study, clinical,morphological and genetic analysis was carried out in four independent LGMD2B/MM patients. All patients presented with an adult-onset, slowly progressive muscular dystrophy with variable involvement of proximal and distal muscles. We found complete lack of dysferlin in the four LGMD2B/MM patients. Secondary reduction of caveolin-3 was detected in three out of the four patients. Regular caveolae were detected along the basal lamina in two patients by electron microscopy. We provide further evidence that dysferlin and caveolin-3 interact in human skeletal muscle. It remains to be elucidated whether the loss of this interaction contributes to pathogenic events in muscular dystrophy.M. C. Walter and C. Braun contributed equally.     

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.     

微量标本Western blot在诊断肢带型肌营养不良2A型中的应用

目的 探讨微量标本Western blot在诊断肢带型肌营养不良2A型中的应用.方法 对73例以肢体近端肌肉受累为首要临床表现的进行性肌营养不良患者行开放式骨骼肌活体组织检查,标本行组织化学染色以及抗dystrophin-N、C、R,α、β、γ、δ-sarcoglycan,dysferlin,caveolin-3单克隆抗体免疫组织化学染色.对其中已经除外dystrophin、sarcoglycans、dysferlin、caveolin-3蛋白异常的29例患者再行骨骼肌Western blot抗calpain-3、caveolin-3单克隆抗体免疫反应.结果 共有10例患者被确诊为肢带型肌营养不良2A型,其临床特点均为肢体近端肌无力起病,血清肌酸激酶不同程度升高,肌电图呈肌源性改变.组织化学染色见肌纤维大小不一,可见不同程度肌纤维变性、坏死和再生,结缔组织增生,6例见分叶状肌纤维;免疫组织化学染色见肌纤维中dystrophin、sarcoglycan、dysferlin、caveolin-3蛋白均正常表达.Western blot发现该10例患者相对分子质量94 000条带(calpain-3)与Duchenne/Becker型肌营养不良对照相比,呈完全(8例)或部分(2例)缺失;30 000区域附加条带均呈弱表达,22 000区域条带(caveolin-3)均正常表达.结论 骨骼肌微量标本Western blot是诊断LGMD2A的有效方法,适用于临床LGMD的分型诊断.     

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.     

Distal myopathies

Distal myopathies are a heterogeneous group of genetic disorders characterized clinically by progressive muscular weakness and atrophy beginning in the hands or feet, and pathologically by myopathic changes in skeletal muscles. Five distinct distal myopathies are identified, among them four have been recently defined by their gene and causative mutations. They are classified according to age at onset, mode of inheritance, and muscle groups initially involved into the following: Laing myopathy (infancy onset, autosomal dominant inheritance, onset in anterior compartment of legs) caused by mutations in a myosin gene (MYH7) on chromosome 14q; Nonaka myopathy (early adult onset, autosomal recessive inheritance, onset in anterior compartment of legs), identical to quadriceps-sparing familial inclusion myopathy, caused by mutations in the GNE gene on chromosome 9p-q; Miyoshi myopathy (early adult onset, autosomal recessive inheritance, onset in posterior compartment of legs) caused by mutations in the dysferlin gene on chromosome 2p; Welander myopathy (late adult onset, autosomal dominant inheritance, onset in hands) linked to chromosome 2p; Udd/Markesbery-Griggs myopathy (late adult onset, autosomal dominant inheritance, onset in anterior compartment of legs) caused by mutations in the titin gene on chromosome 2q. Except for Miyoshi myopathy, which has a striking elevated serum creatine kinase level and the typical findings of muscular dystrophy, most of the distal myopathies have normal or midly elevated creatine kinase levels and share the common pathologic feature of rimmed vacuoles.     

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.     

Dysferlinopathy in the Jews of the Caucasus: a frequent mutation in the dysferlin gene

Dysferlin encoding gene (DYS) is mutated in the autosomal recessive disorders Miyoshi myopathy, Limb Girdle Muscular Dystrophy type 2B (LGMD2B) and distal anterior compartment myopathy, causing dysferlin deficiency in muscle biopsy. Three ethnic clusters have previously been described in Dysferlinopathy: the Libyan Jewish population originating in the area of Tripoli, Italian and Spanish populations. We report another cluster of this muscular dystrophy in Israel among Jews of the Caucasus region. A genomic analysis of the dysferlin coding sequence performed in patients from this ethnic group, who demonstrated an absence of dysferlin expression in muscle biopsy, revealed a homozygous frameshift mutation of G deletion at codon 927 (2779delG) predicting a truncated protein and a complete loss of functional protein. The possible existence of a founder effect is strengthened by our finding of a 4% carrier frequency in this community. These findings are important for genetic counseling and also enable a molecular diagnosis of LGMD2B in Jews of the Caucasus region.     

The childhood muscular dystrophies: making order out of chaos

New discoveries have dramatically changed the way we approach and think about patients with childhood muscular dystrophies. An aura of order and organization seems to be at hand for a group of diseases which previously seemed endlessly heterogeneous. We have learned that young boys and girls with proximal muscle weakness, large calves and elevated serum CK may have any one of a number of closely connected disorders which affect a complex of interacting proteins of the dystrophin-glycoprotein complex. This complex links the intracellular cytoskeleton to the extracellular matrix. Patients with Duchenne and Becker dystrophies lack dystrophin, while some of the limb girdle muscular dystrophies (an archaic term) are deficient in sarcoglycans and other proteins. The concept of interrelated disorders extends to the previously orphaned distal muscular dystrophies, or distal myopathies, as they are often called. A surprise finding is that the C. elegans protein, dysferlin, is conserved and expressed in man. We know little of the function of this protein in human primates, but its loss in muscle has brought seemingly disparate disorders together, since both a form of LGMD (2B) and distal myopathy (Miyoshi myopathy) are deficient in this same gene product. The congenital muscular dystrophies are also well-entrenched in our expanding concepts of orderliness of disease. The defect in the laminin-alpha2 chain, a direct ligand to the dystrophin-glycoprotein complex, causes a form of muscular dystrophy which affects infants. Another variant of congenital muscular dystrophy is deficient the integrin alpha7, an important laminin receptor. Finally, in Fukuyama congenital muscular dystrophy, the deficient fukutin gene product may also be linked to the basal lamina, permitting overmigration of neuronal cells which lead to micropolygyria in the brain, and at the same time cause basal lamina defects in the extracellular matrix of skeletal muscle, which leads to muscular dystrophy. As we approach the millennium, those of us who have seen the transition from the pre-molecular to the molecular era of myology know that we leave behind a great legacy of chaos (no great loss), replaced by a foundation for conceptual organization which will serve to establish new roots for research as well as for the enriched practice of medicine. The future looks bright for our field and our patients!     

Evaluation of commercial dysferlin antibodies on canine,mouse and human skeletal muscle

Immunostaining of muscle biopsy cryosections is a powerful tool for identifying protein deficiencies. For dysferlin, a protein associated with limb-girdle muscular dystrophy and Miyoshi myopathy, weak immunostaining of normal muscle has been a problem in reliably identifying dysferlin deficiency in human patients or dystrophic animals. Here we use skeletal muscle cryosections from dog, mouse and human to test several dysferlin antibodies under different conditions of fixation, and without fixation. NCL-Hamlet antibody (mouse monoclonal), following fixation in acetone/methanol, provided the strongest and most reliable staining in sections of human muscle as well as of dog and mouse muscle. Unlike animal tissue, unfixed human muscle also gave strong and reliable staining. NCL-Hamlet 2 gave good staining in all species. Epitomics (rabbit monoclonal) antibody gave good staining of all muscles, and did not stain muscle of dysferlin-deficient mice. However, it strongly stained muscle sarcolemma of patients with dysferlin deficiency, making the antibody less useful. Abcam antibody gave weak staining, and Santa Cruz antibodies did not immunostain muscle dysferlin in any species tested. NCL-Hamlet antibody was optimal for immunoblotting in all species. Use of select antibodies for immunostaining and immunoblotting, and optimization of immunostaining methods, should increase the sensitivity of detecting dysferlin deficiency in skeletal muscle.     

Phenotypic study in 40 patients with dysferlin gene mutations: high frequency of atypical phenotypes

OBJECTIVE: To describe the phenotypic spectrum of dysferlin (DYSF) gene mutations (which cause dysferlinopathies, autosomal recessive muscular dystrophies) in patients with a dysferlin protein deficiency. DESIGN: Clinical, biological, and pathological data from 40 patients were reviewed. The diagnosis of dysferlinopathy was based on the absence or strong reduction of dysferlin in muscle, and confirmed by mutational screening of the DYSF gene. SETTING: Two French neuromuscular diseases centers (in Paris and Marseilles). RESULTS: Two main dysferlinopathy phenotypes are well recognized: Miyoshi myopathy and limb-girdle muscular dystrophy type 2B. Typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B were found in 20 (50%) patients only. Unusual phenotypes included a mixed phenotype, referred to as "proximodistal," combining distal and proximal onset in 14 (35%) patients, pseudometabolic myopathy in 4 (10%), and asymptomatic hyperCKemia (an increased serum creatine kinase level) in 2 (5%). The disease may worsen rapidly, and 10 (25%) patients were initially misdiagnosed as having polymyositis. We suggest a relationship between proximodistal phenotype, inflammation, and severity. CONCLUSION: In addition to typical Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, dysferlinopathies are a clinically heterogeneous group of disorders ranging from asymptomatism to severe functional disability.