Search

Background Primary myoblast cell cultures display the phenotypic characteristics and genetic defects of the donor tissue and represent an in vitro model system reflecting the disease pathology. They have been generated only from freshly harvested tissue biopsies. Here, we describe a novel technique to establish myoblast cell cultures from cryopreserved skeletal muscle biopsy tissues that are useful for diagnostic and research purposes. Methods and results This protocol was performed on seven gradually frozen muscle biopsy specimens from various neuromuscular disorders that were stored in dimethylsulfoxide (DMSO)-supplemented freezing media at −80 °C for up to one year. After storage for varying periods of time, primary myoblast cultures were successfully established from all cryopreserved biopsy tissues without any chromosomal abnormality. Desmin immunoreactivity confirmed that the cell cultures contained >90% pure myoblasts. The myoblasts differentiated into multinucleated myotubes successfully. Furthermore, there were no statistically significant differences in cell viability, metabolic activity, population doubling time, and myocyte enhancer factor 2 (MEF2C) expression between cell cultures established from freshly harvested and one year-stored frozen tissue specimens. Conclusions This protocol opens up new horizons for basic research and the pre-clinical studies of novel therapies by using cryopreserved skeletal muscle biopsies stored under suitable conditions in tissue banks.

Background Rhabdomyolysis is a potentially life threatening condition of various etiology. The association between rhabdomyolysis and muscular dystrophies is under-recognized in clinical practice. Objective To identify muscular dystrophies presenting with rhabdomyolysis at onset or as predominant feature. Methods We retrospectively reviewed clinical and laboratory data of patients with a genetically confirmed muscular dystrophy in whom rhabdomyolysis was the presenting or main clinical manifestation. Results Thirteen unrelated patients (males=6; females=7) were identified. Median age at time of rhabdomyolysis was 18years (range, 2–47) and median duration between the first episode of rhabdomyolysis and molecular diagnosis was 2years. Fukutin-related protein ( FKRP ) muscular dystrophy (n=6) was the most common diagnosis, followed by anoctaminopathy-5 (n=3), calpainopathy-3 (n=2) and dystrophinopathy (n=2). Four patients experienced recurrent rhabdomyolysis. Eight patients were asymptomatic and 3 reported myalgia and exercise intolerance prior to the rhabdomyolysis. Exercise (n=6) and fever (n=4) were common triggers; rhabdomyolysis was unprovoked in 3 patients. Twelve patients required hospitalization. Baseline CK levels were elevated in all patients (median 1200IU/L; range, 600–3600). Conclusion Muscular dystrophies can present with rhabdomyolysis; FKRP mutations are particularly frequent in causing such complication. A persistently elevated CK level in patients with rhabdomyolysis warrants consideration for underlying muscular dystrophy.

Introduction Multiple acyl-coenzyme A dehydrogenase deficiency disorder (MADD) is a relatively rare disorders of lipid metabolism. This study aimed to investigate the demographic, clinical, and genetic features of MADD in Iran. Methods Twenty-nine patients with a definite diagnosis of lipid storage myopathy were recruited. All patients were tested for mutation in the ETFDH gene, and 19 had a biallelic mutation in this gene. Results Of 19 patients with definite mutations, 11 (57.9%) were female, and the median age was 31 years. Twelve patients had c.1130 T > C (p.L377P) mutation in exon 10. Two patients had two novel heterozygote pathogenic variants (c.679C > T (p.P227S) in exon 6 and c.814G > A (p.G272R) in exon 7) and two patients had c.1699G > A (p.E567K) in exon 13. Before treatment, the median muscle power was 4.6 (IQR: 4–4.7) that increased to 5 (IQR: 5–5) after treatment (Z = −3.71, p = .000). The median CK was 1848 U/l (IQR: 1014–3473) before treatment, which declined to 188 U/l (IQR: 117–397) after treatment (Z = −3.41, p = .001). Sixteen patients (84.2%) had full recovery after the treatment. The disease onset was earlier (12 years of age; IQR: 6–18) in patients with homozygous c.1130 T > C; p.(L377P) mutation compared to other ETFDH mutations (30 years of age; IQR: 20–35) (p = .00). Discussion MADD has different clinical presentations. As the patients respond favorably to treatment, early diagnosis and treatment may prevent the irreversible complications of the disease.

To investigate the role of the muscular renin-angiotensin system (RAS) in human muscular dystrophy, we used immunohistochemistry and Western blotting to examine the cellular localization of angiotensin-converting enzyme (ACE), the angiotensin II type 1 receptor (AT1) and the angiotensin II type 2 receptor (AT2) in muscle biopsies from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD). In normal muscle, ACE was expressed in vascular endothelial cells and neuromuscular junctions (NMJs), whereas AT1 was immunolocalized to the smooth muscle cells of blood vessels and intramuscular nerve twigs. AT2 was immunolocalized in the smooth muscle cells of blood vessels. These findings suggest that the RAS has a functional role in peripheral nerves and NMJs. ACE and AT1, but AT2 immunoreactivity were increased markedly in dystrophic muscle as compared to controls. ACE and the AT1 were strongly expressed in the cytoplasm and nuclei of regenerating muscle fibers, fibroblasts, and in macrophages infiltrating necrotic fibers. Double immunolabeling revealed that activated fibroblasts in the endomysium and perimysium of DMD and CMD muscle were positive for ACE and AT1. Triple immunolabeling demonstrated that transforming growth factor-beta1 (TGF-beta1) and ACE were colocalized on the cytoplasm of activated fibroblasts in dystrophic muscle. Furthermore, Western blotting showed increases in the expression of AT1 and TGF-beta1 protein in dystrophic muscle, which coincided with our immunohistochemical results. The overexpression of ACE and AT1 in dystrophic muscle would likely result in the increased production of Ang II, which may act on these cells in an autocrine manner via AT1. The activation of AT1 may induce fibrous tissue formation through overexpression of TGF-beta1, which potently activates fibrogenesis and suppresses regeneration. In conclusion, our results imply that the intramuscular RAS-TGF-beta1 pathway is activated in human muscular dystrophy and plays a role at least partly in the pathophysiology of this disease.

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.

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 (theta;=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.

A female patient who fulfilled the diagnostic criteria of Walker-Warburg syndrome had muscle biopsy finding of muscular dystrophy. There was normal expression of merosin (laminin alpha2 chain) and dystrophin and only slightly reduced dystrophin-associated glycoprotein expression. On genetic analysis, she had no specific haplotype, the common mutation of 3kb insertion, or point mutations in the Fukuyama-type congenital muscular dystrophy gene, suggesting that the two diseases are not genetically identical.

Mutations of the calpain 3 gene, an intracellular calcium-activated neutral protease, is one of the causes of limb-girdle muscular dystrophy (LGMD). We examined 14 Japanese patients with sporadic LGMD for calpain 3 mutations, and found four mutations in five patients. Three (R461C, D707G and R147P) were novel missense mutations, and one was a splice-site mutation (801+1g-->a) resulting in skipping of exons 4 and 5. Of the five patients, three patients with homozygous missense mutations showed later onset and slower progression than the other two patients with an exon skipping or mRNA loss of unknown cause. It would appear that the occurrence of calpain 3 gene mutations in sporadic LGMD in Japan may be quite high since all five patients with mutations in this gene were among the 14 patients without apparent family history, an incidence of 36%. These findings also suggest that calpain 3 deficiency occurs in both sporadic and familial LGMD and that direct analysis of the calpain 3 gene may be useful in the definitive diagnosis not only of the 15q-linked familial but also of sporadic cases of LGMD.

A 51-year-old man developed weakness and muscle atrophy in the legs at the age of 41, later followed by choreiform involuntary movements. Neurological and laboratory examinations revealed severe muscle weakness and atrophy, and areflexia in all the extremities, acanthocytosis and an elevated serum creatine kinase level. Together with these findings, the weak expression of Kell blood group antigens and the absence of the Kx antigen led to a definite diagnosis of McLeod syndrome for his condition. Brain magnetic resonance imaging revealed marked atrophy of the head of the caudate nuclei. Although immunocytochemical analysis of dystrophin in muscle specimens from our patient revealed normal staining, we found prominent fiber size variability, central nuclei, and connective tissue proliferation as well as necrotic and regenerating fibers, which are as a whole compatible with the myopathology of muscular dystrophy. Moreover, muscle computerized tomography of the lower extremities revealed the 'selectivity pattern' characteristically reported in muscular dystrophies including Duchenne type muscular dystrophy. The muscular symptoms and pathology in McLeod syndrome have been reported to be mild, but the present case clearly shows that the muscular features in this condition may be much more severe than previously thought.

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder associated with an intellectual deficit which is non-progressive. We obtained localised 1H magnetic resonance spectra from the left frontal lobe and left cerebellum of 15 boys with DMD (mean age 106 months+/-32) and 15 similarly aged control boys (mean age 115 months+/-31); all boys underwent a battery of neuropsychological tests. We found a significant (P0.01) increase in the ratio of choline-containing compounds to N-acetylaspartate (Cho/NA) in the left cerebellum in boys with DMD compared with control boys. There was no change in the creatine/NA ratio and a significant increase (P=0.03) in the Cho/creatine ratio, suggesting that the change in Cho/NA ratio was due to an increase in choline-containing compounds; this increase has been previously observed in the brain of the murine model of DMD, the mdx mouse. No significant changes were observed in spectra obtained from left frontal lobe in DMD compared to controls. We also observed a significant association between Cho/NA in the left cerebellum, and the performance of DMD boys on the Matrix Analogies Test (MAT). The MAT is a test of visuo-spatial ability and non-verbal reasoning which requires neither manual dexterity nor a verbal response for an adequate performance. A comparison of DMD boys whose cerebellar Cho/NA fell within 2 standard deviations of the control norm (0.56+/-0.24) with DMD boys whose cerebellar Cho/NA was outside this range (i.e.0.80) revealed a significant difference in ability on the MAT (P0.05). DMD boys whose Cho/NA ratio is more than two standard deviations higher than controls perform significantly better on the MAT than DMD boys whose Cho/NA ratio is within the normal range. This finding suggests that the observed elevation in Cho/NA and Cho/creatine is not associated with intellectual deficit (as sampled by the MAT), and may represent a compensatory mechanism. The possible interpretations of these metabolic changes are discussed.

We performed ultrastructural studies on nuclear abnormalities in biopsied muscles from seven patients with EDMD, of three non-related families, and two sporadic cases. The diagnosis was based on clinical data and molecular findings. We detected different degrees of abnormalities in the sarcolemmal nuclei ranging from marked condensation of chromatin to complete damage of nuclear components. Other nuclei in the same muscle cell very often appeared normal. The extrusion of nuclear chromatin into sarcoplasm as a consequence of nuclear membrane disintegration was observed in numerous nuclei. All these nuclear changes are considered to be cytological indicators of nuclear dysfunction evoked by emerin deficiency.

Dystrophin gene was analysed in 32 unrelated DMD families (46 subjects: 32 index cases and 14 sibs) for the presence of deletions by mPCR for 27 exons and cDNA probes for the entire gene. Deletions were identified in 32 patients (25 index cases and seven sibs) from 25 families. The concordance between the clinical phenotype and 'reading frame' hypothesis was observed in 24 (75%) cases. Of these, nine patients were wheelchair bound between 8-12 years of age, nine (age range 5-10 years) showed progressive difficulty in walking and six (age range 1.6-4 years) had onset of muscle weakness. One patient (CH), who was wheelchair bound at 12 years, the effect of mutation on the ORF could not be ascertained due to the presence of a junction fragment. Seven patients had inframe deletions of which four were wheelchair bound by the age of 13 years, and three (age range 5-7 years) although, ambulatory had difficulty in walking. There were eight patients who showed no deletion, of which four became wheelchair bound by the age of 12 years, four, though still ambulatory, were unable to run and tired easily. Correlation between phenotype and genotype of these DMD patients demonstrates that genetic studies of lymphocyte DNA may not always reflect the situation in the tissue involved in dystrophin, i.e. muscle. We describe a common dystrophin gene polymorphism in the Indian population with cDNA 11-14 that alters the Hind III restriction sites. Novel RFLPs were observed in 26 patients and their family members. Whether this is a polymorphism or, related to the diseased phenotype needs confirmation.

A new homozygous alpha-sarcoglycan (adhalin) gene mutation was found in a Japanese patient with severe childhood autosomal recessive muscular dystrophy (SCARMD). Muscle biopsy specimens from the patient showed marked reduction but not complete deficiency of alpha-sarcoglycan. The sequence of part of exon 3 of the alpha-sarcoglycan gene exhibited a cytosine to thymidine substitution at nucleotide position 220. Since the same mutation was not found in 100 normal control samples, this new alpha-sarcoglycan gene mutation is not a polymorphism but is presumed to be responsible for the marked reduction of alpha-sarcoglycan in skeletal muscle. Most patients with homozygous alpha-sarcoglycan gene mutation were reported to show complete alpha-sarcoglycan deficiency. Present case showed the homozygous missense mutation of alpha-sarcoglycan and associated with incomplete alpha-sarcoglycan deficiency and severe clinical phenotype.

Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a ‘blind’ study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature α- and β-dystroglycan proteins. The β-dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processing of dystroglycan. © 1997 Elsevier Science B.V. All rights reserved.

Fifteen persons from two consecutive generations of one family affected with facio-scapulo-humeral muscular dystrophy (FSHD) were clinically and neurophysiologically examined. Diagnostic muscle biopsies were obtained from two members. Linkage analysis showed that all four affected members of the family inherit the same 4q35 haplotype giving a lod score of z = +1.44. Six family members were examined by ECG at rest and under stress, by two-dimensional echocardiography, and by cardiac Thallium-201 single-photon-emission computed tomography (Tl-201-SPECT) under dobutamine stress and at rest. Abnormal reduced Tl-201 uptake in cardiac SPECT was only found in the affected members of the family. Therefore we suggest that cardiac Tl-201-SPECT abnormalities in FSHD reflect cardiomyogenic changes in this type of muscular disease.

We have reported the reduction of the B1 subunit of laminin and that of heparan sulfate proteoglycan (HSPG) in two Japanese patients with adhalin deficiency. We here investigated immunohistochemically the expression of cell adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), neural cell adhesion molecule (NCAM), and CD44 (HCAM), in four Japanese patients with adhalin deficiency, compared to other types of muscular dystrophy. We found that NCAM was upregulated in a fair number of muscle fibers, regardless of the type of muscular dystrophy. ICAM-1 was detected on the rare muscle cell membrane in all patients. CD44 was barely detected on the muscle cell membrane in adhalin deficiency, in contrast to the strong expression of CD44 which was observed in other types of muscular dystrophy. These findings suggest that a different degenerative or regenerative process is involved in adhalin deficiency compared to other types of muscular dystrophy.

alpha-Sarcoglycan (adhalin), a 50-kDa component of the dystrophin-associated complex of proteins, participates in the stabilization of the myofiber plasma membrane in the membrane cytoskeleton. Deficiencies of alpha-sarcoglycan cause a subset of childhood-onset muscular dystrophy (SCARMD) cases. However, secondary deficiencies of alpha-sarcoglycan are common. To begin to establish the rates of false positives (secondary deficiencies), we used immunofluorescence to screen 30 Italian dystrophin-normal muscular dystrophy patient biopsies and identified 4 patients with partial alpha-sarcoglycan deficiency and 2 patients with complete deficiency. The entire alpha-sarcoglycan gene was screened for mutations using RT-PCR and SSCP of messenger RNA isolated from muscle biopsies in each of the six patients. Aberrant SSCP conformers and novel mutations were found only in the two complete immunohistochemical deficient patients. One patient was homozygous for a R34H amino acid substitution, while the other was a compound heterozygote (R77C, D97G). These three missense mutations, with additional mutations we and others have previously described, are all localized in the extracellular domain of alpha-sarcoglycan, and most result in the loss or gain of a positively charged amino acid. These data have strong implications for structure/function maps of the alpha-sarcoglycan molecule. Our results suggest that most patients showing partial alpha-sarcoglycan deficiency exhibit this as a secondary consequence of genetically distinct disorders. In support of this, we show biochemical data indicating that secondary deficiency patients show decreased immunostaining with antibodies directed against alpha-sarcoglycan, while having nearly normal quantities of alpha-sarcoglycan protein on immunoblot. This data also suggests that approximately 5% of childhood-onset dystrophin-normal muscular dystrophy patients will show a primary alpha-sarcoglycan deficiency.

We describe a late-onset autosomal dominant limb girdle myopathy, associated with dilated cardiomyopathy and mental deterioration. In two affected members of the pedigree with histochemical (ragged-red and cytocrome c oxidase — negative fibers) and ultrastructural abnormalities of muscle mitochondria, in vivo muscle phosphorus MR spectroscopy disclosed a slow rate of phosphocreatine resynthesis after exercise. Brain phosphorus MR spectroscopy revealed a defect of the energy metabolism in the two patients and in a third asymptomatic member, as shown by a significantly low phosphocreatine, increased ADP and decreased phosphorylation potential. Molecular analysis of muscle mitochondrial DNA failed to reveal any known mutation, including multiple deletions of the mtDNA which have been associated with some autosomal dominant mitochondrial diseases. The multisystem clinical involvement, the presence of ragged-red fibers and the alterations revealed by in vivo brain and muscle 31 P-MRS suggest that this limb-girdle syndrome represents an unusual phenotype of mitochondrial cytopathy.

We have investigated protein expression and genotype in 59 Becker muscular dystrophy (BMD) patients. The aim was to identify possible causes of the marked variability in phenotype in patients with similar deletions/mutations. The patients were examined neurologically and functionally and underwent Manual Muscle Testing. Dystrophin expression was analysed by immunohistochemistry and western blot using antibodies against six different segments of the protein. DNA mutations were investigated by PCR amplification of 30 exons. Based on dystrophin expression at the sarcolemma, two groups of patients were identified: group A (29 patients) with the classic patchy distribution of dystrophin and group B (30 patients) with absence or reduction of one or more dystrophin portions and variable, although mostly normal, expression of the other portions of the protein. Dystrophin molecular weight was normal or slightly reduced in group A and was variably reduced, generally conspicuously so, in group B. The quantity of dystrophin expressed varied markedly in both groups. The pattern of immunohistochemical staining in group B patients correlated with milder clinical phenotype, suggesting that small dystrophin molecules lacking a portion in the N-terminus or in the rod domain, are more functional than proteins with normal or slightly reduced molecular weight that display the BMD-typical patchy distribution at the sarcolemma.

The differential diagnosis between autosomal recessive limb-girdle (LGMD) and X-linked Becker muscular dystrophy (BMD) is very important for genetic counseling. It has been hypothesized that all BMD patients would have dystrophin alterations and dystrophin analysis could identify the Xp21 MD. Qualitatively abnormal dystrophin is easily detectable, but it is generally associated with in-frame DNA deletions or duplications. In patients with no detectable DNA deletions, in which X-linked inheritance cannot be proved, dystrophin quantification is still the only available test for differential diagnosis. In order to assess the accuracy of dystrophin quantification test in delineating Becker patients, we analyzed dystrophin abundance in BMD patients with a positive history of X-linked inheritance and no DNA detectable mutation, as compared to patients from families with LGMD. We observed that patients from 2 among the 5 BMD families have nearly normal dystrophin, while alteration in dystrophin content was observed in patients from 2 among the 7 LGMD families studied (probably as a secondary effect of alteration in the whole dystrophin-glycoproteins complex). These results suggest that dystrophin quantification, as an isolated test is not helpful for differential diagnosis between BMD and LGMD.

Dystrophin is normally localized in smooth muscle fibers of various organs in experimental animals, and it has been shown to be defective in the smooth muscle fibers of the mdx mouse, including the myoepithelial cell layer of the sweat glands. We investigated dystrophin localization, using three antisera raised against different domains of skeletal muscle type of dystrophin, in the smooth muscle structures of the skin, using immunohistochemical methods with monoclonal antibodies against dystrophin, in 24 patients with various neuromuscular diseases, and in a normal control. Skin biopsy showed a strong dystrophin reaction in the arrector pili muscles and in the myoepithelial cells of the sweat glands of patients with congenital muscular dystrophy, polymyositis, distal myopathy, putative Duchenne muscular dystrophy carriers, myoglobinuria, neurogenic atrophy and in a normal control. A faint positive dystrophin reaction was seen in four patients with Becker muscular dystrophy, whereas it was absent in 3 patients with Duchenne muscular dystrophy. As our data suggest that immunohistochemical dystrophin expression in smooth muscle structures of the skin is similar to that observed in striated muscle, skin biopsy may represent an alternative way to ascertain dystrophin deficiency.

To better define limb-girdle muscular dystrophy (LGMD), we examined 58 patients clinically and pathologically who fulfilled the criteria for LGMD and had normal dystrophin expression in their muscle biopsies. Only 27.6% of patients had evidence of inheritance. The onset of disease varied from 2 to 58 years of age, averaging 17.2 years. The disease progression also differed from patient to patient. In addition to evidence of muscle fiber necrosis and regeneration, in all muscle biopsies there were fibers with architectural changes of disorganized intermyofibrillar networks including moth-eaten (100%), lobulated (40%), whorled (17%) and targetoid (8%) fibers. The lobulated fibers which have never been reported in Duchenne muscular dystrophy (DMD) were seen in the advanced stages of LGMD, although the significance of such fibers remains unknown. On immunohistochemical examination, dystrophin-associated proteins (DAPs) and laminin were normally expressed along the surface membrane of muscle fibers, including the lobulated fibers.

Immunocytochemical studies on serial sections of muscles from 19 patients with Duchenne muscular dystrophy (DMD) were done using seven kinds of antidystrophin antibodies that span dystrophin. Fifteen of the patients were screened for intragenic deletions by the multiplex polymerase chain reaction (PCR); 7 were also tested, using Southern blots. Dystrophin-positive fibers were detected in 10 of the 19 patients, occurring with a frequency of 0.06-75.7%, as clusters or as single fibers in certain muscle bundles. In 7 of the 10 patients, the fibers stained with all antibodies from the N- through the C-terminal region. However, in one patient, there were dystrophin-positive fibers that stained with an N-terminal antibody (DYS-3) and an antibody specific to the rod region (DYS-1), but not with C-terminal antibody (DYS-2). In 2 patients, there were two kinds of fibers: one that did not stain with DYS-1 or DYS-2 and another that stained with all the antibodies used. Single-fiber PCR analysis in 2 patients showed that the genotype of dystrophin-positive fibers differed from that of dystrophin-negative fibers. These results suggest that the majority of dystrophin-positive fibers are of the same clonal origin, but that some are derived from plural reverse mutations, each with a different translation-frame-repairing modality and somatic mosaicism.

We examined 19 muscle biopsies from 14 patients with autosomal recessive distal muscular dystrophy (DisMD) histochemically and immunohistochemically to characterize the histologic features of this disease and to determine whether dystrophin and dystrophin-associated proteins (DAPs) are normally present in the muscles of patients with this disorder. The common histologic features in DisMD were active muscle fiber necrosis and regeneration with variation in fiber size, predominantly in the gastrocnemius muscle. There were occasional disorganizations of the intermyofibrillar network such as moth-caten appearance, lobulated, whorled and targetoid fibers. In one half of the patients, small angular fibers and scattered rimmed vacuoles were also found. On immunohistochemical examination, dystrophin, DAPs, spectrin and laminin were normally expressed along the surface membrane of muscle fibers, even in the advanced stages of the disease. In contrast, dystrophin was absent and DAPs reduced in the sarcolemma of Duchenne muscular dystrophy (DMD) muscles. The overall histochemical features in DisMD were similar to those seen in DMD, though dystrophin and DAPs were normally expressed even in severely affected gastrocnemius muscle. A defect in an as yet unidentified protein rather than in DAPs and dystrophin is probably responsible for the muscle fiber necrosis in DisMD.

We studied a family with late-onset (fifth or sixth decade) or asymptomatic hereditary myopathy of the anterior tibial muscle. The occurrence of the disease in two successive generations pointed out an autosomal dominant pattern of inheritance. The initial symptom was uni- or bilateral foot drop resembling peroneal paresis. Surprisingly many of the diagnosed patients were asymptomatic and considered themselves healthy whether there was any foot drop or not. The anterior tibial muscles were atrophic in patients with foot drop but the long toe extensors were usually and the short ones were always spared. Apparently the toe extensors could relieve the foot drop symptom. As shown by computed tomography there was often an early uni- or bilateral involvement of the semimembranosus muscle in males. The proband showed also a late involvement of the femoral biceps and the minor gluteal muscles. The muscles of the upper extremity were spared. The anterior tibial muscles had a characteristic myopathic alteration with rimmed vacuoles in histopathological study. This picture was most evident in latent cases without atrophy of the anterior tibial muscle, but with distinctly abnormal EMG of that muscle. Non-affected muscles showed only slight non-specific histopathological changes. We suggest that this disease is a new mild variety of autosomal dominant distal myopathy with rimmed vacuoles.

Morphological, morphometrical, histoenzymological, immunocytochemical and biochemical analysis were performed on muscle biopsies taken from patients suffering from tunisian autosomal recessive Duchenne-like muscular dystrophy (TDLMD) selected both by Duchenne-like clinical criteria and by the presence of normal dystrophin. Data were compared to that obtained from DMD biopsies characterized by the absence of dystrophin. The distribution of myosin heavy chain isoforms, desmin, vimentin and titin were determined in type I and type II muscle fibers. The protein pattern appeared to be less affected in TDLMD than in DMD biopsies. The regenerating fibers were mainly but not exclusively type IIC; a noticeable percentage of both type I and type II fibers coexpressed fast and slow MHC isoforms in TDLMD. This percentage was lower than in DMD. The expression of embryonic, fetal, and fast/slow myosin isoforms in type IIC fibers in TDLMD and DMD suggest different fiber type transformations in these two diseases.

To address potential involvement of muscle basal lamina and membrane cytoskeleton proteins in the etiology of non-dystrophinopathy muscular dystrophies, we examined the immunostaining intensity and distribution of laminin subunits (A, B1, B2 and M), type IV collagen, dystrophin and spectrin in skeletal muscle biopsies from 64 myopathic patients (17 Fukuyama congenital muscular dystrophy: FCMD, 13 congenital muscular dystrophy unrelated to FCMD: other CMD, 16 Duchenne muscular dystrophy: DMD, and 18 other neuromuscular diseases. In FCMD muscle, we found a significant reduction of laminin M (merosin; a striated muscle specific basal lamina-associated protein) with approximately 26% of levels seen in controls by quantitative immunofluorescence. Other CMD and DMD muscles showed less dramatic reductions (78%, 80%, respectively). The localization of laminin M was also abnormal in FCMD muscle. Laminin B1 and B2 showed abnormalities similar to those observed with laminin M, but were less marked. Laminin A was only detected in rare regenerating fibers in control biopsies, whereas it was seen around most muscle fibers in FCMD patients, and in dystrophin deficient muscle fibers from DMD patients and its carrier. Staining intensity of type IV collagen in FCMD muscle was not significantly different from the other diseases. These findings may implicate a primary or central role for the basal lamina in FCMD muscle.

In Duchenne muscular dystrophy, the progression of the disease is always severe and predictable, while in Becker dystrophy there is a wide variability (intra and inter familial) in the severity of the phenotype. We report here a family in which the proband, who is currently 15 years old, is showing a severe DMD progression, while his affected maternal uncle, aged 29, has a more benign course, compatible with BMD. No DNA deletion was detected in both patients. Dystrophin analysis through immunofluorescence and western blotting showed a negative pattern in the youngest patient and a positive one in the oldest. Apparently, this is the first report on intrafamilial variability in dystrophin abundance correlated with a difference in the severity of the phenotype.

Cytosolic pH and phosphorus metabolite ratios in skeletal muscle were measured by 31P magnetic resonance spectroscopy in patients with Duchenne muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD) and in Duchenne/Becker carriers. In resting dystrophin-deficient muscle, there was a decrease in phosphocreatine (PCr) and increase in orthophosphate (Pi) relative to ATP, and an increase in calculated free [ADP]. Phosphomonester and phosphodiester were also increased relative to ATP. These changes were largest in DMD, smaller in BMD and small or absent in carriers. Cytosolic pH was increased substantially in DMD, moderately in BMD and slightly but significantly in gastrocnemius of carriers. Raised intracellular pH thus appears to be the most characteristic abnormality in dystrophin-deficient muscle. Responses to erobic exercise were studied in the forearm muscle flexor digitorum superficialis of carriers. PCr depletion during exercise was greater than normal but the fall in pH was disproportionately small, resulting in increased [ADP]. This is likely to result either from reduced anaerobic glycogenolysis to lactic acid or from increased proton efflux (as is seen in mitochondrial myopathy). Detailed analysis suggests: (1) at the start of exercise, calculated lactic acid production was increased, as was the rate of PCr depletion, suggesting that there was no absolute defect of glycogenolysis. (2) At the start of recovery, calculated proton efflux was not increased, although as the pH at the end of exercise was higher than in controls and proton efflux is normally pH-dependent, an up-regulation of proton efflux cannot be excluded. (3) Recovery of PCr, Pi and ADP after exercise were not impaired, suggesting that mitochondrial function is normal.

We report a male patient with exercise-induced focal myalgia in the leg muscles. Dystrophin immunostaining of a biopsied muscle specimen from the patient showed the absence of or only faint immunoreactivity in 20% of the muscle fibers. The patient was diagnosed as having Becker muscular dystrophy. The myalgia was intractable and did not respond to non-steroidal anti-inflammatory drugs. The patient was placed on prednisone and found to be sensitive to it. Although he had recurrences of the symptom during tapering of the steroid, slower tapering over one year was tolerated. Steroid treatment may be useful for other Becker muscular dystrophy patients with myalgia.

Skeletal muscle fibrosis is present in the diaphragm of the mdx mouse, a model for Duchenne dystrophy. In both the mouse and human, dystrophic muscle exhibits pronounced increases in NF-kappa B signaling. Various inhibitors of this pathway, such as pyrrolidine dithiocarbamate (PDTC) and ursodeoxycholic acid (UDCA), have been shown to have beneficial effects on dystrophic (mdx) muscle. The present study characterizes the development of fibrosis in the mdx musculature, and determines the fibrolytic efficacy of PDTC and UDCA. The results indicate that collagen accumulation and the expression of fibrogenic (TGF-beta1) and fibrolytic (MMP-9) mediators are dependent on muscle origin in both nondystrophic and mdx mice. Excessive collagen accumulation is observed in the mdx respiratory musculature prior to substantial muscle degeneration and cellular infiltration, and is associated with dystrophic increases in the expression of TGF-beta1 with no corresponding increases in MMP-9 expression. Treatment with PDTC or UDCA did not influence collagen deposition or TGF-beta1 expression in the mdx respiratory musculature. These results indicate that dystrophic increases in collagen are the result of NF-kappaB-independent signaling abnormalities, and that efforts to reduce excessive collagen accumulation will require treatments to more specifically reduce TGF-beta1 signaling or enhance the expression and/or activity of matrix metalloproteases.

Late adult onset distal myopathies usually show vacuolar degeneration as a characteristic feature in muscle pathology. In this study vacuolar degeneration was not present in 12 patients with late adult onset distal myopathy. All patients were members of a large kindred, with 26 patients showing this new form of distal leg myopathy. Additionally, a severely disabling proximal muscular dystrophy appeared in eight other members of the large consanguineous kindred. Muscle biopsies were obtained from clinically affected muscles, and from clinically unaffected muscles in patients with distal myopathy. For comparison specimens from various muscles of patients with severe proximal dystrophy were also studied. Histopathological changes correlating to muscular dystrophy were extensive in all muscles studied in patients with proximal dystrophy, and in tibial anterior muscles in patients with distal myopathy. Mild myopathic changes, mainly increased internal nuclei in muscle fibers, were detected in clinically unaffected muscles in the distal myopathy. The spectrum of findings is compatible with the hypothesis of previous clinical and genetic studies, indicating that the severe proximal dystrophy could be a homozygous manifestation of the dominantly inherited gene of the distal tibial muscle dystrophy.

We present here a unique expression of dystrophin on biopsied muscle from 2 siblings with Becker muscular dystrophy (BMD). They had neither muscle weakness nor atrophy. Clustered dystrophin-deficient fibers were constituted to regenerating basophilic fibers (mainly type 2C fiber) based on histochemical stainings. We speculate that the developmental delay in the expression of dystrophin is a characteristic finding in regenerating fibers from asymptomatic and young BMD patients, such as the siblings in this report.

3',5'-Cyclic nucleotide phosphodiesterase (PDE) is known to play an important role in the regulation of cyclic nucleotide levels in various tissues including the muscle. Previous studies have estimated the level of this enzyme in several neuromuscular disorders but the results have been variable. Moreover, there was no attempt made to correlate the enzyme levels with the levels of calcium and calmodulin, both of which regulate diverse biological processes including muscle contraction. In the present study we have estimated phosphodiesterase in the muscle of normal controls as well as patients with myotonic (MyD) and Duchenne muscular dystrophy (DMD) and amyotrophic lateral sclerosis (ALS). PDE was found to be increased significantly in all of the diseased muscles as compared to controls (P less than 0.01). But the increase could be coupled with an increase in calcium and calmodulin only in Duchenne dystrophic muscle.

Muscles from Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) patients were analysed using monoclonal and polyclonal antibodies raised against different regions of the dystrophin molecule. On blot, two of the antibodies detected a protein of Mr 400K in muscle extracts from all patients, including a BMD patient with a deletion which spanned more than 40% of the central rod domain of the Xp21 encoded dystrophin. Immunocytochemical labelling of tissue sections from the same patients showed that the same two antibodies labelled a protein at the surface membrane of smooth muscle fibers in blood vessels of both BMD and DMD muscles. Thus we have demonstrated a 400K blood vessel-associated protein, which is immunologically homologous with dystrophin, for at least two epitopes from the carboxy terminal and the central rod domains must be encoded by another gene than the dystrophin gene.

Two types of progressive muscular dystrophy occur in Tunisian children. The first type is characterized by normal dystrophin assays and affects girls and boys in an autosomal recessive pattern of inheritance. The second type has the features of the typical Duchenne muscular dystrophy (DMD) and has abnormal dystrophin. Between 1974 and 1986, 77 patients with Duchenne muscular dystrophy were examined, 66 were biopsied. Among affected siblings and within family kindreds, we observed both clinical and histopathological variability. However, there was a close correlation between the clinical condition and the biopsy findings in each case, allowing accurate prediction of the patient's course and probable duration of the disease.

To investigate the role of tissue inhibitors of metalloproteinases (TIMPs) in muscular dystrophy, we examined the expression of TIMP-1 using plasma and biopsied muscle from patients with various muscular dystrophies by ELISA, immunohistochemistry, and Western blot analysis. TIMP-1 immunolocalization was also studied in mouse models of muscular dystrophy. Plasma TIMP-1 was elevated and correlated with TGF-β1 in Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD), but not in Becker muscular dystrophy. In dystrophic human muscles, TIMP-1 was immunopositive in the regenerating and non-regenerating muscle fibers, and interstitial cells that consist of activated fibroblasts and macrophages. TIMP-1 immunoreactivity was also closely associated with TGF-β1. Western blot analysis showed elevated TIMP-1 protein in muscles in DMD. The semiquantitative analysis of TIMP-1 staining intensity and tissue fibrosis showed that TIMP-1 immunoreactivity is closely associated with the extent of tissue fibrosis in human and mouse dystrophic muscles. In conclusion, the present study implied that the TGF-β1-TIMP-1 pathway is activated in dystrophic muscles and the overexpression of TIMP-1 may result in increased deposition of extracellular matrix leading to tissue fibrosis.

Dystrophin-deficient muscle exhibits substantial increases in nuclear NF-kappaB activation. To examine potential mechanisms for this enhanced activation, the present study employs conventional Western blot techniques to provide the first determination of the relative expression of NF-kappaB signaling molecules in adult nondystrophic and dystrophic (mdx) skeletal muscle. The results indicate that dystrophic muscle is characterized by increases in the whole cell expression of IkappaB-alpha, p65, p50, RelB, p100, p52, IKK, and TRAF-3. The proportion of phosphorylated IkappaB-alpha, p65, NIK, and IKKbeta, and the level of cytosolic IkappaB-alpha, were also increased in the mdx diaphragm. Proteasomal inhibition using MG-132 increased the proportion of phosphorylated IkappaB-alpha in nondystrophic diaphragm, but did not significantly increase this proportion in the mdx diaphragm. This result suggests that phosphorylated IkappaB-alpha accumulates in dystrophic cytosol because the rate of IkappaB-alpha degradation is lower than the effective rate of IkappaB-alpha synthesis and phosphorylation. Dystrophic increases in SUMO-1 (small ubiquitin modifier-1) protein and in Akt activation were also observed. The results indicate that increases in nuclear p65 activation in dystrophic muscle are not produced solely by increases in the activity of IkappaB-alpha kinase (IKK), but are due primarily to increases in the expression of p65 and other NF-kappaB signaling components.

Using immunocytochemical methods, we examined the intensity and distribution of dystrophin and spectrin immunostaining of skeletal muscles from 51 congenital muscular dystrophy (CMD) patients including 36 Fukuyama congenital muscular dystrophy (FCMD) and 15 non-FCMD (other CMD). 17 age-matched spinal muscular atrophy (SMA) and 5 Duchenne muscular dystrophy (DMD) patient biopsies were studied as controls. All 15 non-FCMD and SMA patients showed normal localization of dystrophin at the surface membrane of each muscle fiber which was undetectable in DMD. In contrast, 34 of 36 FCMD patients exhibited an unusual immunostaining pattern with occasional (17–43%; mean = 28) negative or abnormally immunoreacted (partially deficient, fluffy or intense) fibers for dystrophin. Dystrophin was absent in 2 of 36 patients having a clinical diagnosis of FCMD, and intragenic deletion of the DMD gene was detected in one. Spectrin, a membrane cytoskeletal protein related to dystrophin, also showed an increased number of abnormally immunostained fibers in FCMD (25%), but not so high in age-matched DMD (9%) or SMA patient muscle (0%). Thus, our results suggested the presence of intrinsic factor(s) that produce abnormality of the plasma membrane of FCMD muscle.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, was studied in needle biopsy samples taken from the quadriceps muscle of 15 asymptomatic carriers of DMD (13 adults and 2 young girls) and one symptomatic adult carrier. Antibodies to N- and C-terminal regions of dystrophin were used for both Western blot analysis and immunocytochemistry and a monoclonal antibody to beta-spectrin used to assess membrane integrity. All asymptomatic adult carriers showed some abnormality in dystrophin immunostaining but very few negative fibres were present. A clear mosaic of dystrophin positive and negative fibres was seen only in the adult symptomatic carrier and the two young girls. On a Western blot, all carriers studied had dystrophin of normal molecular weight, but most had reduced abundance. In adult carriers, the amount of dystrophin relative to normal controls varied, but it was unrelated to age, serum creatine kinase (CK) levels or to the degree of pathology. Carriers with normal CK showed abnormalities in dystrophin expression. The dystrophin immunoblotting profile of the 2 young girls was very similar to that of their mothers, but the mosaic pattern of immunostaining was not apparent in the older carriers. In conclusion, dystrophin immunostaining and Western blot analysis of biopsy samples from asymptomatic carriers is often abnormal and they may be useful additional aids for establishing carrier status, particularly in younger girls.

It has been reported that immunofluorescent labelling of dystrophin in muscle from patients with Becker muscular dystrophy (BMD) is invariably patchy or discontinuous. This observation has led to the suggestion that BMD dystrophin molecules, which are usually smaller than normal due to the presence of "in frame" gene deletions, cannot be assembled into a complete lattice network under the plasma membrane and instead form isolated patches. Our experience with immunoperoxidase labelling of BMD muscle indicates that complete gaps in the reaction around fibres are uncommon. We have therefore compared immunofluorescence and immunoperoxidase labelling patterns on sets of serial sections from 6 BMD patients using a monoclonal antibody to dystrophin. No difference was detected between the two types of label used: the incidence of discontinuous labelling was rare in both cases. We suggest that significantly different patterns of dystrophin labelling may be obtained using different primary antibodies, and that caution needs to be exercised in extrapolating models of structure/function relationships from observations of antibody binding patterns.

Normal skeletal muscle does not express class I MHC antigens. In contrast, these antigens are strongly expressed at the periphery of muscle fibres in patients with juvenile dermatomyositis (JDM) and Duchenne muscular dystrophy (DMD). Interferons can induce the expression of class I antigens, but in this study interferon-gamma could not be detected in JDM muscle biopsy specimens using four different immunocytochemical techniques. However, an infiltrate of mononuclear cells capable of synthesising interferons was present in biopsies from JDM and DMD patients. The predominant cell types detected in both diseases were macrophages and T lymphocytes, these two cell types comprising more than 80% of the infiltrating mononuclear cells. A striking predominance of CD4+ helper/inducer T cells was observed. The majority of these cells expressed class II MHC antigens and were, therefore, considered to be activated. Additional evidence for the functional activity of CD4+ T cells was derived from the finding that it was this population of cells from JDM biopsies which proliferated in response to interleukin-2 in vitro. T cell subsets in peripheral blood were also investigated in JDM and DMD patients. Only in the case of JDM were any significant differences from normal observed, where a significant reduction in the number of peripheral blood CD8+ T cells resulted in an elevation of CD4+/CD/8+ ratios. The role of CD4+ T cells and aberrant class I MHC antigen expression in mediating muscle damage in JDM and DMD is discussed.

The lectin Dolichos biflorus agglutinin has been used to identify mast cells in normal skeletal muscle and to investigate changes in their number in a wide range of human neuromuscular diseases and in rat muscle damaged by the local anaesthetic bupivacaine. Few mast cells were found in the perimysium of normal skeletal muscle but numbers were increased in human muscle biopsies which showed necrosis and regeneration or fibrosis. In bupivacaine-induced muscle damage, increased mast cell counts occurred during the necrotic phase and particularly during the phase of active regeneration. In addition, increased numbers of mast cells were observed in the underlying histologically normal muscle. These results show that mast cells are influenced by pathological changes in skeletal muscle and, in view of the known functions of mast cells in other tissues, it is possible that they are capable of modulating disease processes in muscle.

Antibodies directed against neural cell adhesive molecules (NCAM) and in particular a monoclonal antibody recognizing polysialylated isoforms, were used to characterize the expression of these molecules in normal and diseased human muscles. Normal subjects as well as patients with inflammatory, dystrophic and denervating diseases were examined. By immunohistochemistry the main observations were (1) satellite cells expressed the non-sialylated form of NCAMs; (2) regenerative fibers strikingly expressed NCAMs and their sialylated isoforms both on membranes and in the cytoplasm; (3) in denervated muscles, fibers in atrophic groups and some fibers in acute denervation expressed NCAMs on their membrane but not the highly sialylated form; (4) finally, some fibers in myotonic dystrophy and fibers with rimmed vacuoles also expressed NCAMs. Biochemical approaches, using enzymes such as endoglycosidase N and phosphatidylinositol phospholipase C combined with immunoblot analysis allowed visualization of the nature of the expressed isoforms. We have shown that non activated cells, i.e. satellite cells and denervated fibers do not express polysialylated NCAMs. This post-translational modification may be only observed in activated or regenerating fibers. This would parallel the sequence of NCAM expression occurring in normal myogenic pathways.

Cosegregation of facioscapulohumeral muscular dystrophy (FSHD) and familial adenomatous polyposis (FAP) has been described in two small families. The gene for FAP is located on the long arm of chromosome 5. We studied two large Dutch families with FSHD and found no evidence for linkage with gene markers closely linked to FAP. These results strongly suggest that the FSHD gene segregating in the Dutch families is not localized close to the FAP locus on chromosome 5.

Cultured muscle cells from Duchenne muscular dystrophy (DMD) patients show altered growth from the mononucleated stage: abnormal morphology, decreased adhesiveness, reduced number of population doublings and delayed fusion. On the basis of these findings, a study was undertaken to observe cell shape and surface morphology by scanning electron microscopy and to define the immunocytochemical localization of 4 basal lamina components (type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan (HSPG]. Eight DMD muscle cultures with fusion indices higher than 65% were compared to muscle cultures from 10 age-matched controls. The following results were noted for the dystrophic muscle cells: (1) the cell surface was smooth with a few slender cell processes and anchorage extensions; (2) distribution of type IV collagen and laminin was heterogenous, with large patches (type IV collagen) or a reticulum (laminin); (3) in contrast, fibronectin and HSPG levels were clearly decreased. These molecules did not form a network but rather were arranged in thick filaments and patches. Cell surface morphology may be related to the decreases in fibronectin and HSPG, which could reflect a more general decrease in basal lamina. Such findings could explain the low adhesiveness of the cells from dystrophic cultures and the delayed fusion of myoblasts. Although these abnormalities were maximally expressed after myoblast fusion, they were already present in mononucleated cells and their connection with the primary defect in DMD, i.e., lack of dystrophin, must now be clarified.