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.     

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).     

Dysferlin interacts with affixin (beta-parvin) at the sarcolemma

The dysferlin gene is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin is a sarcolemmal protein that is implicated in calcium-dependent membrane repair. Affixin (beta-parvin) is a novel, integrin-linked kinase-binding protein that is involved in the linkage between integrin and the cytoskeleton. Here we show that affixin is a dysferlin binding protein that colocalizes with dysferlin at the sarcolemma of normal human skeletal muscle. The immunoreactivity of affixin was reduced in sarcolemma of MM and LGMD2B muscles, although the total amount of the affixin protein was normal. Altered immunoreactivity of affixin was also observed in other muscle diseases including LGMD1C, where both affixin and dysferlin showed quite similar changes with a reduction of sarcolemmal staining with or without cytoplasmic accumulations. Colocalization of dysferlin and affixin was confirmed by immunofluorescence analysis using dysferlin-expressing C2 myoblasts. Wild-type and mutant dysferlin colocalized with endogenous affixin. The interaction of dysferlin and affixin was confirmed by immunoprecipitation study using normal human and mouse skeletal muscles. Using immunoprecipitation with deletion mutants of dysferlin, we have identified that C-terminal region of dysferlin is an apparent binding site for affixin. We also found N-terminal calponin homology domain of affixin as a binding site for dysferlin. Our results suggest that affixin may participate in membrane repair with dysferlin.     

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.     

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.     

Muscle protein analysis in the detection of heterozygotes for recessive limb girdle muscular dystrophy type 2B and 2E

The diagnosis of isolated heterozygotes for recessive LGMD is quite difficult because no specific biochemical or protein assays are available, and the molecular analysis is not feasible due to the wide genetic heterogeneity. We investigated a series of definite heterozygotes for different forms of LGMD to determine whether the carrier status will result in a detectable protein defect in muscle biopsy. Definite heterozygotes from 4 families (3 LGMD2B and 1 LGMD2E) underwent quantitative immunoblot analysis of mutant protein in their muscle. While the quantity of beta-sarcoglycan was nearly normal in the LGMD2E carrier, the levels of dysferlin protein were reduced to 50% of controls in the carriers of LGMD2B. We have demonstrated the value of protein analysis in the identification of both familial and isolated LGMD2B heterozygotes, and suggested the use of dysferlin protein testing to select muscle biopsies from suspected carriers for a subsequent mutation analysis. Muscle protein analysis would be used to screen asymptomatic patients who underwent muscle biopsy because of unexplained hyperCKemia.     

Identical dysferlin mutation in limb-girdle muscular dystrophy type 2B and distal myopathy

Limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) are autosomal recessive disorders caused by mutations in the dysferlin gene on chromosome 2p13. The authors studied a large Russian family with both LGMD2B and MM. All affected individuals, as well as one preclinical boy with dystrophic changes on muscle biopsy, were found to be homozygous for a novel dysferlin mutation, TG573/574AT (Val67Asp). This finding supports the view that additional factors (e.g., modifier genes) contribute to the phenotypic expression of causative mutations in dysferlinopathies.     

Limb-girdle muscular dystrophy; update]

Sixteen different forms of limb-girdle muscular dystrophies (LGMDs) have emerged from recent molecular genetic studies, six forms with a dominant trait and ten forms with a recessive trait. Among 1,420 Japanese patients with muscular dystrophy analyzed at NCNP, LGMD is the secondly largest category (19%) following dystrophinopathy (56%). Within LGMDs, the occurrence of LGMD2A (calpainopathy), LGMD2B (dysferlinopathy), and LGMD2C-F (sarcoglycanopathy) is 26%, 18%, and 6.6%, respectively, however, causative genes have not been specified in about 50% of the LGMD patients. LGMD2A patients show atrophy prominent in shoulder and pelvic girdle muscles without calf muscle hypertrophy, and abundant lobulated fibers in muscle biopsy. Four major mutations unique to the Japanese population, have been identified. Pathogenesis attributes to a loss of proteolytic activity of mutant calpain-3. Dysferlin, the defective protein in LGMD2B, is a ferlin family molecule possessing six C2 domains probably mediating the resealing mechanism of the damaged sarcolemma. Mutations in the dysferlin gene result in Miyoshi distal myopathy and distal anterior compartment myopathy other than LGMD2B. Among four sarcoglyconopathies, LGMD2D is the most common form, whereas LGMD2F has not yet been reported. In sarcoglycan-deficient skeletal muscle, matrix metalloproteinases may be involved in the beta-dystroglycan processing which underlies the pathogenesis of sarcoglycanopathy.     

Protein and gene analyses of dysferlinopathy in a large group of Japanese muscular dystrophy patients

Mutations in the dysferlin gene cause muscular dystrophies called dysferlinopathy, which include limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM). To clarify the frequency, clinicopathological and genetic features of dysferlinopathy in Japan, we performed protein and gene analyses of dysferlin. We examined a total of 107 unrelated Japanese patients, including 53 unclassified LGMD, 28 MM and 26 other neuromuscular disorders (ONMD). Expression of dysferlin protein was observed using immunohistochemistry (IHC) and mini-multiplex Western blotting (MMW), and mutation analysis was performed. We found a deficiency of dysferlin protein by both IHC and MMW in 19% of LGMD and 75% of MM patients, and mutations in the dysferlin gene were identified in this group alone. 19% of dysferlin-deficient patients had 3370G-->T missense mutation and 16% had 1939C-->G nonsense mutation. The patients with homozygous 3370G-->T mutation showed milder clinical phenotypes. Twenty-five percent of MM muscles had normal dysferlin protein contents that suggested the genetic heterogeneity of this disease. Altered immunolocalization of dysferlin was observed in not only primary dysferlinopathy, but also in the several diseased muscles with normal protein contents. This result implies the necessity of other protein(s) for proper membrane localization of dysferlin, or some roles of dysferlin in the cytoplasmic region.     

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.     

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.     

Painful enlargement of the calf muscles in limb girdle muscular dystrophy type 2B (LGMD2B) with a novel compound heterozygous mutation in DYSF

Limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi Myopathy are caused by mutations in the dysferlin gene. The phenotype of these allelic disease variants can vary considerably. We report on an adolescent female with a severe and rapidly progressing clinical course of LGMD2B which has been suggested by the muscle histopathology and Western blot and proven by mutation analysis in the Dysferlin gene. We detected a novel compound heterozygous mutation of which one affects the extracellular part of the protein. This is the first report on a mutation in this region of dysferlin and might explain the unusual phenotype of the patient.     

应用免疫印迹法诊断肢带型肌营养不良2A型

目的 应用免疫印迹法(Western blot)诊断肢带型肌营养不良2A型(LGMD2A)患者并与LGMD2B型相鉴别.方法 收集我院诊治的4例LGMD2型患者的临床、病理及生化检验资料.取肌肉活体组织行组织化学和免疫组织化学染色,用Western blot分析dysferlin蛋白及calpain-3蛋白的表达.结果 LGMD2A与2B型患者的临床症状相似;免疫组织化学染色显示所有患者均出现不同程度的dysferlin缺失.但Western blot揭示:LGMD2A型患者calpain-3蛋白完全缺失,dysferlin蛋白部分缺失;而2B型患者则相反.结论 用Western blot检测calpain-3蛋白可在dysfedin蛋白表达缺失的LGMD患者中鉴别出2A型患者,该方法对临床辅助诊断LGMD2A有很好的价值.     

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.     

DYSF mutation analysis in a group of Chinese patients with dysferlinopathy

Objective

Dysferlinopathies belong to heterogeneous group of autosomal recessive muscular disorders caused by mutations in the gene encoding dysferlin. The classifications of the dysferlinopathies mainly include limb-girdle muscular dystrophy 2B (LGMD2B) with predominantly proximal weakness, Miyoshi myopathy (MM) with calf muscle weakness and atrophy, and distal myopathy with anterior tibial onset (DMAT) with tibialis muscle atrophy. We describe the genetic character of dysferlinopathies in a group of Chinese patients.

Methods

DYSF mutations screening were done after muscle biopsy and immunohistochemical staining.

Results

Eight patients showed an absence or drastic decrease of dysferlin expression in biopsied muscle. We identified 6 different mutations, including one nonsense mutation, two insertion mutation, two deletion mutations and one splice site mutation. Five of them were novel mutations.

Conclusion

We described 8 Chinese patients with dysferlinopathy (four had a distal phenotype of MM; one had a phenotype of DMAT and three presented with LGMD2B). It is the first report of genetic confirmed DMAT in China. Mutations c.3112C>T and c.1045dup, may be recurrent mutations in China.     

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.     

Dysferlin mutation analysis in a group of Italian patients with limb-girdle muscular dystrophy and Miyoshi myopathy

Mutations in the dysferlin gene (DYSF) on chromosome 2p13 cause distinct phenotypes of muscular dystrophy: limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy, which are known by the term 'dysferlinopathy'. We performed mutation analyses of DYSF in 14 Italian patients from 10 unrelated families with a deficiency of dysferlin protein below 20% of the value in normal controls by immunoblotting analysis. We identified 11 different mutations, including eight missense and three deletion mutations. Nine of them were novel mutations. We also identified a unique 6-bp insertion polymorphism within the coding region of DYSF in 15% of Italian population, which was not observed in East Asian populations. The correlation between clinical phenotype and the gene mutations was unclear, which suggested the role of additional genetic and epigenetic factors in modifying clinical symptoms.     

Dysferlin肌病的临床和病理特点分析(附6例报道)

目的:探讨dysferlin肌病的临床和病理特点。方法:对9例患者的肌肉病理标本进行组织化学和免疫组化染色检查,并对肌肉组织进一步行dysferlin蛋白的Westernblot分析。结果:9例患者中确诊6例为dysferlin肌病,病理表现均为肌源性损害,无边缘空泡,其中2例患者的病理分析有炎细胞浸润。根据临床表现特点,6例患者中有3例为Miyoshi肌病(MM),2例为肢带型肌营养不良2B型(LGMD2B),1例为远端前群肌病(DACM)。结论:Dysferlin肌病在肌电图上为肌源性损害、肌酶显著升高,其临床表现多样,dysferlin免疫组化染色联合Westernblot有着重要诊断意义。     

Challenges for the genetic screening in dysferlin deficiency--report of an instructive case and review of the literature

The homozygous or compound heterozygous mutation of the alleles of DYSF gene causes dysferlinopathy resulting in limb girdle muscular dystrophy Type 2B (LGMD 2B) or Miyoshi myopathy. However, patients with only 1 (heterozygous) mutation on 1 allele are increasingly recognized. Based on the Leiden database (www.dmd.nl) among 257 different mutations resulting in dysferlin-deficient muscular dystrophy, pathogenic mutations were detected only on 1 allele in 45 cases, while the exons of the other allele did not show any pathological alterations. The relatively high number of these so-called heterozygous cases raises the question if present routine molecular techniques are sufficient alone for confirming the diagnosis of dysferlin deficiency. In fact, the heterogenous genetic background of the disease makes it impossible to make the correct diagnosis without Western blot of the muscle dysferlin. This paper presents the clinical, myopathological and molecular genetic results of a 30-year-old male with dysferlinopathy as an instructive case. The cDNA sequencing of the dysferlin gene revealed a single C5302T heterozygous mutation resulting in Arg1768Trp exchange. The paper highlights the importance of the protein analysis in the diagnosis of dysferlin deficiency, discusses the difficulties of the complete genomic analysis of the dysferlin gene alterations and the possible etiopathogenetic role of the noncoding DNA sequence of the dysferlin gene in dysferlin deficiencies.     

A novel mutation in the DYSF gene in a patient with a presumed inflammatory myopathy

Dysferlinopathy, a progressive muscular dystrophy, results from mutations in the Dysferlin gene (DYSF, MIM*603009). Traditional diagnosis relies on the reduction or absence of dysferlin. However, altered dysferlin has been observed in other myopathies, leading to a precise diagnosis through molecular genetics. In this study, we report a patient who was previously misdiagnosed as inflammatory myopathy based on routine clinicopathological examinations alone. However, muscle biopsy specimens were analyzed further by immunohistochemistry of muscular dystrophy‐related proteins, and gene‐targeted next generation sequencing (NGS) was used to correctly identify muscular dystrophy. DNA was sequenced with NGS and the detected mutation was verified by Sanger sequencing. Our targeted NGS found a novel missense mutation (c.5392G > A) in the DYSF gene, allowing correct diagnosis of LGMD2B in our patient. We discovered of a novel missense mutation in the DYSF gene and have broadened the DYSF mutation spectrum, which may be correlated in patients with presumed dysferlinopathy, especially when lymphocytic infiltration is observed.