Beyond LGMD1A: myotilin is a component of central core lesions and nemaline rods.

Myotilin is a Z-disc protein that binds alpha-actinin, gamma-filamin and F-actin. The essential role of myotilin in skeletal muscle is highlighted by the recent observation that autosomal dominant limb girdle muscular dystrophy type 1A is caused by mutations in the human myotilin gene. We studied the expression and subcellular distribution of myotilin in nemaline myopathy, central core disease, centronuclear myopathy, and myopathies with tubular aggregates. A prominent myotilin immunostaining of nemaline rods and core lesions was detected in all ten cases of nemaline myopathy and five cases of central core disease. This renders myotilin a sensitive, though non-specific marker for these structural lesions. Western blot analysis did not indicate an increased myotilin expression in nemaline myopathy muscle. However, the analysis indicated upregulation of a 75 kDa immunoreactive band, very weak in control muscle but previously detected in limb girdle muscular dystrophy 1A samples. Our findings indicate that myotilin is a core structural molecule in nemaline rods and central core lesions and suggest modification of myotilin in nemaline myopathy, and further support the notion that myotilin may have a key role in the dynamic molecular events mediating myofibril assembly in normal and diseased human skeletal muscle.

Dysferlin mutations in Japanese Miyoshi myopathy: relationship to phenotype.

OBJECTIVE: To study dysferlin gene mutations and genotype-phenotype correlations in Japanese patients with Miyoshi myopathy (MM). BACKGROUND: MM is an autosomal recessive distal muscular dystrophy that arises from mutations in the dysferlin gene. This gene is also mutated in families with limb girdle muscular dystrophy 2B. METHODS: The authors examined 25 Japanese patients with MM. Genomic DNA was extracted from the peripheral lymphocytes of the patients. The PCR products of each of 55 exons were screened by single strand conformation polymorphism or direct sequencing from the PCR fragments. RESULTS: The authors identified 16 different mutations in 20 patients with MM; 10 were novel. Mutations in Japanese patients are distributed along the entire length of the gene. CONCLUSIONS: Four mutations (C1939G, G3370T, 3746delG, and 4870delT) are relatively more prevalent in this population, accounting for 60% of the mutations in this study. This study revealed that the G3370T mutation was associated with milder forms of MM and the G3510A mutation was associated with a more severe form.

Mutation in the caveolin-3 gene causes a peculiar form of distal myopathy.

The authors describe a patient with sporadic distal myopathy associated with reduced caveolin-3 in muscle fibers in which the muscle atrophy was restricted to the small muscles of the hands and feet. Gene analysis disclosed a heterozygous 80 G-->A substitution in the caveolin-3 gene that was identical to that of reported cases of elevated serum creatine kinase. This patient further demonstrated possible clinical heterogeneity of myopathies with mutations in the caveolin-3 gene.

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.

Tongue atrophy in facioscapulohumeral muscular dystrophy.

Involvement of the lingual muscle is considered one of the exclusion criteria of facioscapulohumeral muscular dystrophy (FSHD). In a series of 151 Japanese patients with 4q35-FSHD, seven patients (4.6%) had tongue atrophy with abnormal MRI findings and typical myogenic patterns of electromyography. All seven patients belong to a group of early-onset FSHD with large gene deletions on chromosome 4q35. Our result suggests that the patients with 4q35-FSHD could have myopathic tongue atrophy.

Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy.

BACKGROUND: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids. OBJECTIVE: To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain. METHODS: Immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCMD and control subjects. RESULTS: The authors found a selective deficiency of highly glycosylated alpha-dystroglycan, but not beta-dystroglycan, on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for alpha-dystroglycan, but were positive for beta-dystroglycan in FCMD in skeletal and cardiac muscles. CONCLUSION: The current findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, alpha-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD.

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.

The product of an oculopharyngeal muscular dystrophy gene, poly(A)-binding protein 2, interacts with SKIP and stimulates muscle-specific gene expression.

Oculopharyngeal muscular dystrophy (OPMD) is caused by short expansions of the GCG trinucleotide repeat encoding the polyalanine tract of the poly(A)-binding protein 2 (PABP2). PABP2 binds to the growing poly(A) tail, stimulating its extension during the polyadenylation process, and limits the length of the newly synthesized poly(A) tail. Whereas PABP2 is expressed ubiquitously, the clinical and pathological features of OPMD patients are restricted to the skeletal muscle. To elucidate the possible role of PABP2 in skeletal muscle, we established the stable C2 cell lines expressing human PABP2. These stable cell lines showed morphologically enhanced myotube formation accompanied by an increased expression of myogenic factors, MyoD and myogenin. In nuclear run-on assay, the transcription rate of the MyoD gene was significantly increased by PABP2 transfection. We found the N-terminal region of PABP2 was responsible for the up-regulation of these myogenic factors. Furthermore, Ski-interacting protein (SKIP) was isolated as a binding protein for PABP2 using the yeast two-hybrid system. The interaction of PABP2 and SKIP was confirmed by glutathione S-transferase-pulldown assay and immunoprecipitation. Confocal laser scanning showed PABP2 was co-localized with SKIP in nuclear speckles. The reporter assays showed that PABP2 co-operated with SKIP to synergistically activate E-box-mediated transcription through MYOD: Moreover, both PABP2 and SKIP were directly associated with MyoD to form a single complex. These findings suggest that PABP2 and SKIP directly control the expression of muscle-specific genes at the transcription level.

Massive muscle cell degeneration in the early stage of merosin-deficient congenital muscular dystrophy.

Primary merosin-deficient congenital muscular dystrophy (CMD) is a severe form of congenital muscular disorder which is caused by mutations in the laminin alpha2 chain gene (LAMA2). The disease is characterized by marked dystrophic changes in skeletal muscles during early infancy, while little is known about the pathological process of the muscle fiber degeneration. Here, we report the immunohistochemical analysis of skeletal muscle in ten patients with primary merosin-deficient CMD using a panel of molecular markers for skeletal muscle proteins, cellular necrosis, and apoptosis. In the youngest patient (a 52 day old baby), prominent massive muscle cell degeneration occurred in association with the deposition of the C5-9 complement membrane attack complex (MAC). Most of the MAC-positive muscle fibers showed a severely deranged immunoreaction to dystrophin, dystroglycans, and other sarcolemmal proteins. In addition, we found scattered positive signals for apoptosis. Similar but milder changes were also observed in six other patients younger than 1 year. In the patients older than 3 years, muscle fibers positive for MAC and apoptotic signals were barely detectable. These findings imply that massive muscle fiber degeneration occurs in the very early stage of merosin-deficient CMD and may contribute to the severe dystrophic changes in muscle from early infancy.

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.

Interaction between emerin and nuclear lamins.

Emerin is an inner nuclear membrane protein that is involved in X-linked recessive Emery-Dreifuss muscular dystrophy (X-EDMD). Although the function of this protein is still unknown, we revealed that C-terminus transmembrane domain-truncated emerin (amino acid 1-225) binds to lamin A with higher affinity than lamin C. Screening for the emerin binding protein and immunoprecipitation analysis showed that lamin A binds to emerin specifically. We also used the yeast two-hybrid system to clarify that this interaction requires the top half of the tail domain (amino acid 384-566) of lamin A. Lamin A and lamin C are alternative splicing products of the lamin A/C gene that is responsible for autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD). These results indicate that the emerin-lamin interaction requires the tail domains of lamin A and lamin C. The data also suggest that the lamin A-specific region (amino acids 567-664) plays some indirect role in the difference in emerin-binding capacity between lamin A and lamin C. This is the first report that refers the difference between lamin A and lamin C in the interaction with emerin. These data also suggest that lamin A is important for nuclear membrane integrity.

[Recent advances in limb-girdle muscular dystrophy research].

In our laboratory, limb-girdle muscular dystrophy (LGMD) accounted for 20% of all patients with muscular dystrophy. To determine the incidence of various forms of LGMD phenotypes, we looked for mutations in the calpain 3 gene and, for deficiencies in dysferlin and sarcoglycan by immunohistochemical studies with specific antibodies on muscle biopsies from patients with probable autosomal recessive inheritance (LGMD2), which were mostly sporadic cases of LGMD. Fourteen of 276 (5%) patients examined had sarcoglycan complex deficiency (sarcoglycanopathy) and 21 of 80 (26%) had mutations in the calpain 3 gene. Although we have not performed gene analysis in all patients, 10 of 64 (15%) patients examined had no apparent immunoreactivity against the dysferlin antibody. Thus, approximately 46% of LGMD2 patients had the above 3 distinct disorders, but in 54% the causative defects remain unknown.

[Exercise training improves activities of daily living in elderly patients with congestive heart failure].

Exercise training which is one of the multidisciplinary interventions for elderly patients with congestive heart failure, plays an important role for improving the quality of life and reducing the re-admission rate of these patients. We assessed the validity of exercise training for the improvement of patient's skeletal muscle functions and activities of daily living along with monitoring cardiac functions. Exercise training programs were performed in 12 patients with congestive heart failure (New York Heart Association class III or IV), including 5 with valvular disease, 4 with dilated cardiomyopathy and 3 with ischemic cardiomyopathy (mean 79 +/- 9 years). All patients were admitted because of exacerbation of congestive heart failure and were treated conventionally. The exercise training program was started after stabilization of their cardiac condition. The medication was not changed during the training period. After exercise training programs, the cardio-thoracic ratio decreased from 63.8 +/- 7.9% to 60.1 +/- 6.9% (p < 0.01), ejection fraction on echocardiography increased from 47.4 +/- 18.2% to 56.0 +/- 17.5% (p < 0.01), and brain natriuretic peptide decreased from 404.8 +/- 267.5 pg/ml to 313.6 +/- 239.5 pg/ml (p < 0.05). The quadriceps muscle power increased from 0.77 +/- 0.36 Nm/kg to 0.97 +/- 0.41 Nm/kg (p < 0.01). The maximum walking distance on flat surface increased from 149 +/- 164 m to 456 +/- 394 m (p < 0.05). In most patients, the activities of daily living, especially mobility, improved. Appropriate exercise training for the elderly patients with congestive heart failure improves activities of daily living and also reduces the amount of required care by the patients.

Nuclear envelope proteins and associated diseases.

There is a growing body of evidence in favour of the presence of human diseases caused by mutations in genes that encode the nuclear envelope proteins emerin and lamin A/C (lamin A and C are alternatively spliced variants of the same gene). Emerin deficiency results in X-linked Emery-Dreifuss muscular dystrophy (EDMD). Lamin A/C mutations cause the autosomal-dominant form of EDMD, limb-girdle muscular dystrophy with atrioventricular conduction disturbances (type 1B), hypertrophic cardiomyopathy and Dunnigan-type familial partial lipodystrophy. In the targeted mouse model of lamin A gene deficiency, loss of lamin A/C is associated with mislocalization of emerin. Thus, one plausible pathomechanism for EDMD, limb-girdle muscular dystrophy type 1B, hypertrophic cardiomyopathy and familial partial lipodystrophy is the presence of specific abnormalities of the nuclear envelope. Therefore, a group of markedly heterogeneous disorders can be classified as 'nuclear envelopathies'. The present review summarizes recent findings on nuclear envelope proteins and diseases.

Developmental expression of myotonic dystrophy protein kinase in brain and its relevance to clinical phenotype.

To investigate the pathophysiologic role of myotonic dystrophy protein kinase (DMPK) in the brain in myotonic dystrophy (MD), the developmental characteristics of DMPK immunoreactivity in the central nervous system and its alteration with disease were studied. Eleven patients' brain with MD (5 congenital form, 6 adult form) were examined by immunohistochemistry using a specific antibody against synthetic DMPK peptides, antipeptide DM1, and compared with 30 control brains, including 16 age-matched controls. In controls, DM1-immunoreactive neurons appeared in the early fetal frontal cortex and cerebellar granule cell layer, persisting through 29 weeks of gestation and then disappearing. In contrast, immunoreactive neurons continued to persist in the cerebral cortex and cerebellar granule cell layer of MD patients. When we counted DM1-immunoreactive neurons, the increase over controls was greater in the congenital form of MD than in the adult form, and was greater in the cerebrum than in the cerebellum in both forms of MD. DM1 immunostaining was predominantly nuclear, mirroring Western blotting of subcellular fractions. Differences in DM1 expression related to development and to the two forms of MD may be closely related to the pathogenesis of mental retardation in this disease.

Muscular dystrophy.

Muscular dystrophy is a group of genetically determined muscular disorders marked by progressive wasting and weakness of the skeletal muscle, but which often affect cardiac and smooth muscles or other tissues. The patterns of inheritance are either dominant or recessive although the gene may be defective because of a new mutation. Growing evidence revealed the marked heterogeneity of the muscle disorders, and considerable numbers of Japanese scientists and physicians have contributed to the research progress in muscular dystrophy. Among these the discovery of an increased serum creatine kinase activity in muscular dystrophy opened the way for the most reliable laboratory test for muscular dystrophy in 1959, and subsequently accelerated progress in a broad range of research areas in medicine. Progress in modern genetics and molecular pathology provided another breakthrough in muscular dystrophy research and, in 1987, dystrophin was identified, a deficiency of which causes DMD. The present review article highlights contributions of Japanese scientists to muscular dystrophy research.

Nuclear accumulation of expanded PABP2 gene product in oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by (GCG) repeat expansions in exon 1 of the poly(A) binding protein 2 gene (PABP2). To elucidate the molecular mechanism underlying the disease, we raised an antiserum against a synthetic peptide fragment predicted from PABP2 cDNA. The peptide corresponded to amino acids 271-291 where a cluster of posttranslational arginine methylation occurs. We examined the subcellular localization of PABP2 in muscle specimens from five patients with OPMD, 14 patients with various neuromuscular disorders, and three normal controls. All Japanese patients with OPMD have been shown to have expanded (GCG)(8, 9, or 11) mutations in PABP2, as well as intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm. None of 50 separate Japanese control individuals were shown to have expanded (GCG) repeat in PABP2. Positive immunoreaction for polyclonal PABP2 was confined to the intranuclear aggregates of muscle fibers exclusively in patients with OPMD. Frequency of the nuclei positive for PABP2 (2%) was similar to that of ITFI detected by electron microscopy (2.5%). There was no apparent relationship between the frequency of PABP2-positive intranuclear aggregates and the severity of muscle fiber damage. In contrast, nuclear immunoreaction was not detected in any samples from normal controls or from other neuromuscular diseases. These results suggest the presence of molecular modification of the product of expanded (GCG) repeat in PABP2, since the synthetic antigen peptide may not recognize a highly dimethylated cluster of arginine residues of the native PABP2, but may recognize the mutated form. Nuclear accumulation of expanded PABP2 product implies a causative role for ITFI.

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A.

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.