Quadriceps myopathy: forme fruste of Becker muscular dystrophy

We examined dystrophin, the protein product of the Duchenne muscular dystrophy gene, in muscle biopsy specimens from 4 male patients with quadriceps myopathy, all of whom showed a mild and slowly progressive myopathy confined to the quadriceps muscles. All 4 patients had clear abnormalities of dystrophin, and were diagnosed as having Becker muscular dystrophy by both immunofluorescence and immunoblot examinations; that is, dystrophin of an abnormal molecular mass was visualized in muscle cryosections as "patchy" or discontinuous immunostaining at the surface membrane of the muscle fibers. One patient had a brother who showed widespread myopathic changes consistent with typical Becker muscular dystrophy. We conclude that the syndrome called quadriceps myopathy includes a group of forme fruste Becker muscular dystrophy.     

Dystrophin immunohistochemistry in a symptomatic carrier of becker muscular dystrophy

Summary Immunohistochemical localization of dystrophin was studied in a symptomatic carrier of Becker muscular dystrophy (BMD). Muscle biopsy specimens from a female carrier showed findings compatible with slowly progressive muscular dystrophy by ordinary histochemical examinations. Immunohistochemical study, using an antiserum raised against a synthetic peptide fragment of dystrophin, demonstrated a mixture of staining patterns, including continuous but faint positive fibres, partially disrupted fibres and negative fibres. These findings were identical to those of patients with BMD and appear to differ from previous findings in female carriers of Duchenne muscular dystrophy. This report is the first immunohistochemical study of a symptomatic female proven by molecular genetic analysis to be a carrier of BMD.     

Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies. Part II: clinical protocol

A phase I open clinical study on gene therapy in Duchenne and Becker muscular dystrophy, without direct individual benefit for the patient, is being performed at the Pitié-Salpêtrière Hospital, Paris. The aims of this project are: (a) to determine the tolerance and the safety of the intramuscular administration of dystrophin cDNA and (b) to study the quality of the gene transfer in vivo in human patients affected by Duchenne and Becker muscular dystrophy. This clinical trial is conducted sequentially and includes three cohorts of three patients each. Patients must be at least 15 years of age. Diagnosis of Duchenne and Becker muscular dystrophy was confirmed by molecular analysis of the dystrophin gene and for each patient the abnormal expression of dystrophin was confirmed, in skeletal muscle, with antibodies directed against the deleted part of the dystrophin. This phase I study is scheduled to be completed by the end of 2002.     

Defective dystrophin in Duchenne and Becker dystrophy myotubes in cell culture.

We examined normal and dystrophic human myotubes in cell culture for expression of dystrophin, the protein product of the Duchenne muscular dystrophy locus. Dystrophin levels in developing myotubes detected by Western blotting increased after 24 hours and reached maximum levels after 10 days in fusion medium. We did not detect dystrophin in myotubes cultured from Duchenne myoblasts (7 cases). Myotubes from a Becker muscular dystrophy patient's biopsy produced a lower molecular weight (approximately 408 kd) dystrophin, which was the same size in a whole muscle preparation from the same biopsy. This 408-kd dystrophin was the expected size for this Becker patient whose DNA was deleted for exons 45-48 of the Duchenne gene. This cell culture system will allow a detailed analysis of the effects of potential pharmacologic agents on steady-state dystrophin levels.     

Expression and subcellular localization of dystrophin in skeletal, cardiac and smooth muscles during the human development

Dystrophin, the product of the DMD gene, is present in all muscle types in normal individuals. Its function has yet to be elucidated, but its absence or the presence of a truncated version of the protein is responsible for the appearance of Duchenne and Becker muscular dystrophies. Using monoclonal antibodies raised against distinct regions of the dystrophin protein, we have examined its expression and subcellular distribution during the human development in skeletal and smooth muscles. We show that both dystrophin expression and its association to the plasma membrane take place earlier in cardiac and smooth muscles (8 weeks of gestation) than in skeletal muscle. In skeletal muscle, dystrophin is first detected in the cytoplasm, and progressively localizes to the plasma membrane from 10 weeks onwards. Since we have obtained marked differences in staining when using antibodies against either a central region of the protein or the C-terminal part, we suggest that different fetal and adult dystrophin isoforms are expressed, probably differing in their C-terminal domain. These findings are discussed in the context of the pathology of Duchenne muscular dystrophy.     

Immunohistochemical staining of dystrophin on formalin-fixed paraffin-embedded sections in Duchenne/Becker muscular dystrophy and manifesting carriers of Duchenne muscular dystrophy

We succeeded in immunostaining of monoclonal anti-dystrophin antibodies on formalin-fixed and paraffin-embedded muscle sections from patients with Duchenne muscular dystrophy, patients with Becker muscular dystrophy, and manifesting carriers of Duchenne muscular dystrophy using Catalyzed Signal Amplification(TM) system. The Catalyzed Signal Amplification system is an extremely sensitive immunohistochemistry staining procedure based on the peroxidase-catalyzed deposition of a biotinylated phenolic compound. We used three mouse monoclonal antibodies: DYS1, DYS2, and DYS3. Muscle sections were treated using the Target Retrieval Solution(TM) and the Catalyzed Signal Amplification system. In control patients, DYS1 and DYS2 were stained at the sarcolemma, but DYS3 remained unstained. In Duchenne muscular dystrophy patients, DYS1 and DYS2 staining were undetected. In Becker muscular dystrophy patients, the immunolabeling of DYSI and DYS2 were weak and discontinuous. In manifesting carriers of Duchenne muscular dystrophy, DYS1 and DYS2 staining showed a mosaic pattern of dystrophin-positive fibers and dystrophin-negative fibers. DYS1 and DYS2 staining patterns of this study are similar to those of frozen sections using conventional methods previously reported. In cases from whom frozen muscle sections cannot be obtained, immunohistochemical dystrophin analysis using the Catalyzed Signal Amplification system will be beneficial for the diagnosis and the screening of neuromuscular diseases.     

Improved diagnosis of Becker muscular dystrophy by dystrophin testing

We assessed the quantity (relative cellular abundance) and quality (approximate molecular weight) of dystrophin in muscle biopsies from 97 patients with a diagnosis of possible Becker muscular dystrophy. Fifty-four (all male) had dystrophin abnormalities and were deemed to have true Becker muscular dystrophy. The other 43 patients (14 female, 29 male) had no detectable dystrophin abnormalities. Of the dystrophin-verified Becker dystrophy patients, 35% (19/54) had a family history consistent with X-linked recessive inheritance. On the other hand, none of the 43 patients with apparently normal dystrophin had a clear X-linked family history, suggesting that few of these 43 actually had a form of Becker dystrophy. The data suggest that of all patients with a clinical picture consistent with Becker dystrophy but no family history, about 60% will be true Becker patients. The correlation of both the biochemical and clinical data suggests that Duchenne/Becker dystrophy can be divided into 4 clinically useful categories: Duchenne dystrophy (wheelchair at about age 11 years; dystrophin quantity less than 3% of normal); severe Becker dystrophy (wheelchair age 13 to 20 years; dystrophin 3% to 10%); and moderate/mild Becker dystrophy (wheelchair greater than 20 years; dystrophin quantity greater than or equal to 20%). Given the observed clinical variability of Becker dystrophy, it appears that dystrophin analysis is required for accurately distinguishing between Becker dystrophy and clinically similar autosomal recessive myopathies.     

Abnormal dystrophin expression in patients with limb girdle syndromes

Clinical differential diagnosis between Becker muscular dystrophy (BMD) and limb gridle muscular dystrophy (LGMD) may be difficult because the BMD clinical phenotype tends to overlap with other limb girdle syndromes, especially with LGMD. Therefore we studied the expression of dystrophin, the protein product of the Becker and Duchenne muscular dystrophy gene, in muscle biopsy specimens of 30 patients (18 males, of whom 15 represented spradic cases, and 12 females) diagnosed as having LGMD according to traditional clinical, electrophysiological and histological criteria. For dystrophin analysis, six different monoclonal antibodies directed against different epitopes of the dystrophin molecule were used. Immunocytochemically, five of the 30 LGMD patients (17%) showed abnormal dystrophin staining patterns diagnostic of BMD. Western blotting in these five patients, all sporadic cases, showed dystrophin of reduced size and/or abundance. Analysis of blood or muscle DNA using multiplex polymerase chain reaction revealed deletions in the dystrophin gene in three of the five. Thus, 5 of 15 (33%) sporadic male patients previously thought to have LGMD were identified as having BMD.     

A homologue of dystrophin is expressed at the blood vessel membrane of DMD and BMD patients: immunological evidence.

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.     

Duchenne muscular dystrophy manifesting carriers

Seven unrelated women were manifesting carriers of Duchenne muscular dystrophy. A manifesting carrier of Duchenne muscular dystrophy is defined as a female with a history of Duchenne muscular dystrophy in her pedigree who has symptomatic weakness. All were characterized by slowly progressive weakness that began in the second or third decade of life. Asymmetric weakness was present in only three of the seven patients. Serum creatine kinase values were elevated in all patients and none had an electrocardiogram indicating ventricular hypertrophy. The electromyogram and muscle biopsy specimens were reported as myopathic in all patients studied. In the absence of a male relative with Duchenne muscular dystrophy, clinical distinction from cases of autosomal recessive limb girdle muscular dystrophy may not be possible. The development of new techniques in molecular genetics should allow precise identification of manifesting carriers of Duchenne muscular dystrophy in the near future.     

Treatment with antisense oligonucleotides in Duchenne's disease

In this paper I review the results of the treatments directed to modify the mRNA of dystrophin with the goal of converting the severe Duchenne type to the milder Becker muscular dystrophy. Antisense oligomers potential to modify Duchenne muscular dystrophy (DMD) gene expression and therapeutic strategies to induce ribosomal read-through of nonsense mutations (PTC124) are described. They are an important advance in the treatment of DMD, so far unspecific. Significant expression of new dystrophin is observed in biopsies of peripheral muscle, although the functional improvement is not so encouraging. New modification of chemistries are expected to improve the liberation, broad distribution in muscles, as well as their efficacy and safety enough to allow a positive chronic treatment of DMD.     

Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy

Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease.     

Dystrophin-related protein in skeletal muscles in neuromuscular disorders: immunohistochemical study

Summary Thirty-four biopsied muscles of Duchenne, Becker and congenital muscular dystrophy, congenital myotonic dystrophy and amyotrophic lateral sclerosis were exmined by an immunocytochemical method with an anti-dystrophin-related protein (DRP) antibody. Strongly positive immunoreaction to DRP at the neuromuscular junctions in all biopsied specimens and faint reaction on the surface membrane of atrophic fibers in amyotrophic lateral sclerosis suggest that DRP is an anchor protein of the acetylcholine receptor. Additionally, the surface membrane of muscle fibers of Duchenne muscular dystrophy was positively stained. DRP is, therefore, thought to be expressed to compensate for dystrophin deficiency in these muscle fibers.Supported in part by a grant-in-aid from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Heterogeneity of dystrophin expression in patients with Duchenne and Becker muscular dystrophy

Summary This report documents the results of an integrated biochemical and immunocytochemical investigation into the expression of dystrophin (the protein product of the Duchenne muscular dystrophy gene) in muscle biopsies from 226 patients. It is the first study in which dystrophin has been analysed on blots and on tissue sections in such a large number of patients using the same (monoclonal) antibody. The 140 patients with Xp21 muscular dystrophy who were included in this study represent a continuous spectrum of disease severity and this range was reflected in the heterogeneity of dystrophin expression which was observed with respect to abundance, size and the pattern of tissue localisation. Approximately 40% of biopsies obtained from patients diagnosed as having Duchenne muscular, dystrophy (DMD) contained isolated clearly positive fibres and a further 20% had very weak labelling on a large number of fibres. Biopsies from patients with Becker muscular dystrophy (BMD) showed labelling patterns which varied from weak labelling on the majority of fibres to clear labelling on all fibres. Typically, however, there was inter-and intra-fibre variation in labelling intensity. Approximately 85% of the 52 BMD and 54 DMD patients who had unequivocal labelling on blots demonstrated a protein of abnormal size. The remaining 15% had a protein of normal size but reduced abundance. Overall, the estimated abundance of dystrophin correlated well with clinical assessments of the disease severity expressed in patients: We conclude that dystrophin analysis is an essential and dependable technique for the differential diagnosis of patients with Xp21 muscular dystrophy.Supported by the University of Newcastle-upon-Tyne Research Committee, the Muscular Dystropy Group of Great Britain and the Medical Research Council     

The serum nitric oxide levels in patients with Duchenne muscular dystrophy

Nitric oxide is formed in skeletal muscle by the neuronal type nitric oxide synthase and the signalling function of dystrophin and related compounds are in part mediated by nitric oxide. Duchenne muscular dystrophy, mdx mice and patients with Becker dystrophy demonstrated neuronal type nitric oxide synthase deficiency in muscle biopsy specimens. We have intended to find out whether the plasma nitric oxide levels show any abnormality in patients with Duchenne muscular dystrophy. Serum NO levels of Duchenne patients (4.191+/-2.82 micromol/l) were significantly lower than those of the control (39.53+/-19.43 micromol/l) and cerebral palsy (77.84+/-21.70 micromol/l) groups.     

Manifesting carrier of Becker muscular dystrophy (BMD): clinical and recombinant DNA studies

We studied a Becker muscular dystrophy (BMD) family with a manifesting carrier. Proximal muscle weakness, pseudohypertrophy of the calves, significantly elevated serum creatine kinase and dystrophic alterations in the muscle biopsy were the characteristic phenotypical features of this manifesting carrier. The recombinant DNA study showed a recombinant chromosome with a crossover between pERT 87-8 and pERT J-Bir in the manifesting carrier. However, the proximal part of the short arm of her X chromosome was identical to a non-manifesting carrier (her sister) and to her affected brother. For this reason, we assumed the BMD mutation was proximal to the crossover. The dystrophin cDNA probes showed no deletion of DMD/BMD gene.     

Expression of dystrophin-associated glycoproteins and utrophin in carriers of Duchenne muscular dystrophy

The expression of dystrophin, the dystrophin-associated proteins and utrophin has been studied immunocytochemically in three young, manifesting carriers of Duchenne muscular dystrophy, aged 3, 5 and 12 yrs, one adult manifesting carrier, aged 60 yrs, and one presumptive carrier with a raised serum creatine kinase, aged 24 yrs, the mother of the 5-yr-old manifesting carrier. The manifesting carriers had variable degrees of weakness; the presumptive carrier had no weakness. Morphological abnormalities were also variable and were most marked in the young manifesting carriers. The three young manifesting carriers and the presumptive carrier had a mosaic pattern of dystrophin-positive and dystrophin-negative fibres. All the dystrophin-associated proteins were reduced in the dystrophin-deficient fibres, giving a similar mosaic pattern to dystrophin. Expression of dystrophin and the dystrophin-associated proteins was normal in the adult manifesting carrier. Utrophin was detected on the sarcolemma of fibres both with and without dystrophin and the dystrophin-associated proteins. Thus, dystrophin and utrophin are co-expressed in several fibres in carriers. The results emphasize the close association between dystrophin and the glycoprotein complex and their role in the pathogenesis of muscle damage. In addition, the presence of utrophin in fibres with greatly reduced glycoproteins suggests that very little of the glycoprotein complex may be required to anchor the amount of utrophin expressed at the sarcolemma in these particular cases.     

Duchenne and Becker muscular dystrophies: an Indian update on genetics and rehabilitation

The application of molecular diagnostic techniques has greatly improved the diagnosis, carrier detection, prenatal testing and genetic counseling for families with Duchenne and Becker muscular dystrophy (D/BMD) in India. The prediction of Duchenne muscular dystrophy (DMD) patients to have out-framed deletions and Becker's muscular dystrophy (BMD) patients to have in-frame deletions of dystrophin gene holds well in the vast majority of cases. Mutation detection is obviously critical for diagnosis but it may also be important for future therapeutic purposes. These factors underscore the need for earlier referral, genetic counseling and provision of support and rehabilitation services which are the main priorities for psychosocial assessment and intervention at medical and social levels.