Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin.

Antibodies directed against the amino- and carboxy-terminal regions of dystrophin have been used to characterize 25 Duchenne muscular dystrophy (DMD), two intermediate, and two Becker muscular dystrophy (BMD) patients. Western blot analysis revealed an altered-size (truncated) immunoreactive dystrophin band in 11 of the 25 DMD patients, in one of the two intermediate patients, and in both BMD patients, when immunostained with antiserum raised against the amino terminus of dystrophin. None of the DMD or intermediate patients demonstrated an immunoreactive dystrophin band when immunostained with an antiserum specific for the carboxy terminus of the protein. In contrast, dystrophin was detected in both BMD patients by the antiserum specific for the carboxy terminus. Quantitative studies indicated that the relative abundance of dystrophin in patients with a severe (DMD), intermediate, or mild (BMD) phenotype may overlap, therefore suggesting that differential diagnosis of disease severity based entirely on dystrophin quantitation may be unsatisfactory. Our results suggest that a differential diagnosis between DMD and BMD would benefit from examination of both the N terminus and C terminus of the protein, in addition to measurements of the relative abundance of the protein.     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive genetic disorders resulting from mutations in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central region of the gene. The remaining DMD/BMD cases show no deletions, so they cannot be easily identified by current strategies. In these DMD/BMD families, a linkage analysis that involves DNA markers of the flanking and intragenic dystrophin gene are necessary for carrier and prenatal diagnosis. We analyzed eighteen deletion-prone exons of the gene by a polymerase chain reaction (PCR) in order to characterize the molecular defects of the dystrophin gene in Korean DMD/BMD families. We also performed a linkage analysis to assess the usefulness and application of six short tandem repeat markers for molecular diagnosis in the families. We observed a deletion that eliminated the exon 50. Also, a linkage analysis in the families with six short tandem repeat (STR) markers showed heterozygosity at most of the STR markers. The haplotype analysis was useful for detecting the carrier status. This study will be helpful for a molecular diagnosis of DMD/BMD families in the Korean population.     

Pharmacological treatments for Duchenne and Becker dystrophies

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease affecting 1 boy out of 3500. DMD is due to the lack of a submembranous cytoskeletal protein named dystrophin, leading to the progressive degeneration of skeletal, cardiac and smooth muscle tissue. A milder form of the disease, Becker muscular dystrophy (BMD), is characterised by the presence of a semi-functional truncated dystrophin, or the full-length dystrophin at reduced level. Three different therapeutic approaches are currently under study, gene therapy, cellular therapy and pharmacological therapy. One of the chosen strategies consists of the overexpression of utrophin, a protein 80% homologous with dystrophin, and able to perform similar functions. In this review, we shall consider studies of pharmacological therapy, the aims of which can be classified in three categories: reversal of dystrophic phenotype, dystrophin expression, utrophin overexpression.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.     

假肥大型肌营养不良肌细胞抗肌萎缩蛋白的免疫组化研究

目的研究假肥大型肌营养不良患者肌细胞中抗肌萎缩蛋白(dystrophin)的表达及其诊断意义。方法应用针吸型活检术取121例假肥大型肌营养不良症患者(108例DMD患者,13例BMD患者)的肌组织,采用HE染色观察被检肌肉病理改变,免疫组织化学染色技术检测抗肌营养不良蛋白表达,以正常人肌细胞作为对照。结果正常人肌细胞膜上抗肌营养不良蛋白染色阳性,呈完整环形条带沿肌细胞膜分布;DMD患者肌膜完全无显色;BMD患者染色弱阳性,可见沿肌细胞膜分布的间断表达。结果 应用针吸型活检技术和免疫组化染色法检测抗肌营养不良蛋白,有助于DMD和BMD确诊及鉴别诊断。     

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec

Summary We have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.     

Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls.We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials.Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.     

Analysis of a dystrophin gene deletion by amplification of mRNA isolated from DMD myotubes cultured in vitro

The most frequent causes for the X-linked muscular dystrophy of the allelic Duchenne (DMD) or Becker (BMD) type are partial deletions of the dystrophin gene. These mutations are accompanied either by disrupted or by preserved translational reading frames in mRNAs derived from the deleted genes. As a rule, the reading frame is destroyed in the more severe DMD, whereas it is preserved in the less severe BMD (M. Koenig et al., 1989, Am. J. Hum. Genet. 45, 498-506). We have analyzed in detail a deletion that was detected in a fetus at risk of DMD. The analysis of this mutation included the delineation of the altered subregion in the dystrophin mRNA. mRNA was isolated from myotubes derived from embryonic DMD myoblasts propagated in vitro. This study was based on enzymatic amplification by the polymerase chain reaction (PCR) of dystrophin mRNA and direct sequencing of the amplified cDNA. Exons 47 to 50 were found to be missing in the mRNA. The splicing of exon 46 to exon 51 resulted in a reading frameshift, indicating that this mutation is likely to be responsible for a DMD type of dystrophy. The clinical diagnosis of DMD for a 10-year-old patient in this family was compatible with the "reading frame" assumption.     

Molecular pathology of Duchenne and Becker muscular dystrophy]

Duchenne and Becker muscular dystrophies (DMD and BMD) are two allelic recessive X-linked disorders. Molecular deletions of various regions of the dystrophin gene are the main mutations detected in DMD and BMD patients. Molecular study of DMD and BMD DNA are instrumental to understand the pathological molecular mechanisms and the function of the protein. We describe here dystrophin and its interaction with a glycoprotein complex and we then focus on two particular patients with partial deletions of the dystrophin gene: 1) a typical Becker patient, who shows an intragenic deletion disrupting the reading frame. We describe in this case alternative splicings restoring the reading frame, which might explain the mild clinical phenotype of this patient, 2) a deletion of the distal part of the DMD gene coding for the carboxyterminal domain of the dystrophin in a young patient. The normal localization of dystrophin at the inner face of the plasma membrane in the muscle of this patient suggests that the last domain of this protein is not sufficient to anchor dystrophin at the membrane.     

DNA deletions in mild and severe Becker muscular dystrophy

Summary The DNA of 33 patients diagnosed as suffering from Becker muscular dystrophy (BMD) has been probed with cloned DNA sequences from Xp21, known to reveal DNA deletions in patients suffering from the more severe Duchenne muscular dystrophy (DMD). Two BMD cases showed clear deletions. A third case gave aberrant band sizes, which further analysis showed to be caused by a small deletion. This suggests that deletions in DXS164 occur approximately as frequently in BMD as they do in DMD. Of the two cases showing large deletions, one is at the severe end of the Becker clinical spectrum, whilst the other is a classical Becker-type dystrophy. The fact that loci defined by probes commonly deleted in classical DMD patients are also deleted in BMD patients of varying severity is strong additional evidence that these disorders are allelic, and further justifies the use of probes with defined linkage relationships to DMD also being used for counselling in BMD families.     

A small deletion in the Duchenne/Becker muscular dystrophy locus —a functionally important region?

Summary A DNA deletion in a patient with Becker muscular dystrophy (BMD) has been delineated by restriction endonuclease mapping. The deletion is unusually small, removing six kilobases (kb) of DNA distal to pERT 87-1 (DXS164). This region has previously been shown to contain an exon of a candidate gene which, when defective, causes Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy. Removal of this exon and surrounding DNA is apparently sufficient, in this case, to cause a BMD phenotype. The occurrence of this deletion in DXS164 would appear to confirm that this region is part of the BMD locus. Many DMD patients have deletions in and around this region, adding further evidence for the allelic nature of the two disorders. This fortuitous deletion may identify a functionally important domain of the protein product in terms of the severity of phenotype manifested.     

Current knowledge of dystrophin and dystrophin-associated proteins in the retina

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Creatine kinase, cell membrane and Duchenne muscular dystrophy

In 1958 Professor Setsuro Ebashi found that serum creatine kinase activity is increased in patients suffering from various muscular dystrophies, especially Duchenne muscular dystrophy (DMD). He and others proposed that creatine kinase passes through the cell membrane as it is released from DMD muscle fibers.Since then, it has been found that dystrophin and dystrophin-associated proteins are connected to several other components, including the basal lamina and subsarcolemmal cytoskeletal networks on the cell membrane, while dystrophin anchors these dystrophin-associated proteins to the actin filaments inside the muscle cell. In DMD muscle, dystrophin has been found to be absent and dystroglycans and sarcoglycans decreased. However, how creatine kinase molecules can pass through the DMD muscle cell membrane still remains unanswered.On the basis of recent findings on the structure of the protein layers which sandwich the lipid bilayer of muscle cell membranes, this essay stresses the importance of these lipid bilayers in protecting creatine kinase release from protoplasma in normal muscle. It further indicates the possibility that the absence of dystrophin in DMD muscle during muscle contraction may result in temporal damage to the lipid bilayer.     

Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1

Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking α-dystrobrevin (Dtna^−/−), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna^−/− mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.     

Analysis of deletions in the dystrophin gene in patients with Duchenne muscular dystrophy in the Bashkir Republic]

The deletion spectrum and distribution of deletion breakpoints (DBs) in 36 patients with Duchenne muscular dystrophy (DMD) from 33 families and in three patients with Becker muscular dystrophy (BMD) from one family from Bashkortostan were studied by amplifying 20 exons of the dystrophin gene by multiplex polymerase chain reaction (mPCR). Eight out of 34 unrelated DMD (BMD) patients (23.2%) were shown to carry a deletion varying in size from one to seven exons. Most DBs (15 out of 16, 93.7%) were in the distal region of the gene, commonly between exons 44-45, 45-47, and 50-52. Thus, high-polymorphic intergenic markers located in introns 44 (STR 44), 45 (STR 45), 49 (STR 49), and 50 (STR 50) can be used for indirect or direct carrier detection among women closely related to DMD patients that carry a deletion with DB located between exons 44-45, 45-47, and 50-52. Prenatal diagnosis of DMD is also possible in these families.