Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal

There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most frequently affected by a deletion includes exons 47-51. The distal region of the dystrophin gene was most frequently affected by the deletion in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.     

Duchenne and Becker muscular dystrophy: a molecular and immunohistochemical approach

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene. We studied 106 patients with a diagnosis of probable DMD/BMD by analyzing 20 exons of the dystrophin gene in their blood and, in some of the cases, by immunohistochemical assays for dystrophin in muscle biopsies. In 71.7% of the patients, deletions were found in at least one of the exons; 68% of these deletions were in the hot-spot 3' region. Deletions were found in 81.5% of the DMD cases and in all the BMD cases. The cases without deletions, which included the only woman in the study with DMD, had dystrophin deficiency. The symptomatic female carriers had no deletions but had abnormal dystrophin distribution in the sarcolemma (discontinuous immunostains). The following diagnoses were made for the remaining cases without deletions with the aid of a muscle biopsy: spinal muscular atrophy, congenital myopathy; sarcoglycan deficiency and unclassified limb-girdle muscular dystrophy. Dystrophin analysis by immunohistochemistry continues to be the most specific method for diagnosis of DMD/BMD and should be used when no exon deletions are found in the dystrophin gene in the blood.     

Molecular pathology of Duchenne and Becker muscular dystrophy

The gene for Duchenne (DMD) and Becker (BMD) types of muscular dystrophy has been isolated by Kunkel's and Worton's groups and shown to be the largest one over known in human, spanning more than 65 exons distributed over 2,500 kb in P21 region of X-chromosome. Fourteen kb cDNA encodes 427 kD cytoskeletal protein "dystrophin", supposed to form an anti-parallel homodimer like alpha-actinin and spectrin. The polyclonal antibodies against the synthetic peptides or fusion proteins predicted from dystrophin cDNA disclosed the complete absence of dystrophin at the surface membrane of both skeletal and cardiac muscles of DMD in marked contrast with the continuous and uniform staining in normal muscles. In manifested carriers, the mosaic expression of dystrophin was observed at the surface membrane of the skeletal muscle. BMD, which is thought to be allelic to DMD, revealed a faint or patchy immunostaining along with the abnormal and/or lower amount of dystrophin. In BMD, there is an intimate connection between the amount of dystrophin and the severity of the clinical course. It should be noted that 5 out of 39 patients with clinical diagnosis of limb-girdle (L-G) muscular dystrophy showed a patchy staining pattern, suggesting BMD not L-G. On the basis of dystrophin discovery, a possible therapeutic trial of DMD is discussed.     

DMD/BMD基因缺失的检测及其临床分析

目的:了解Duchenne/Becker型肌营养不良症(DMD/BMD)致病基因缺失的分布及其与临床病情的关系。方法:应用9对引物多重聚合酶链反应(mPCR)技术对42例DMD/BMD患者进行致病基因检测。结果:21例患者(50.0%)被检出外显子缺失,缺失片段长度各异,其中16例(76.2%)累及中央缺失热区,5例(23.8%)位于5端缺失热区,尤以48号外显子缺失频率最高。结论:多重PCR技术是检测DMD/BMD致病基因缺失的有效方法,该病病情轻重可能与外显子缺失的数量和长度不呈平行关系,而是与缺失类型有关,并受到个体差异的影响。     

Molecular deletion patterns in Turkish Duchenne and Becker muscular dystrophy patients

The dystrophin gene deletion patterns of Duchenne / Becker muscular dystrophy were investigated in 57 DMD, 7 BMD and 1 DMD-BMD intermediate muscular dystrophy patients. Deletions, analyzed by multiplex amplification of selected exons, were observed in 58% (38 cases) of the patients. It was found that exon 48 was the most frequently affected, while exon 44 was the least frequently affected. The number of deleted exons was variable, but single exon deletions were more frequent (41%) than larger deletions in our population and the great majority of deletions began distal to exon 44. The application of PCR to deletion analysis in D /BMD was found to be very useful in delineating the extent of the deletion in most of the cases (82%). It was seen that the frequency of deletion breakpoints in distal part of the dystrophin gene (exons 42–52) was detected in 64% of our cases. In our group, the frequency of deletion breakpoints in the same area of the dystrophin gene was between that of the French and the Finnish patients. The distribution of deletion breakpoints within the dystrophin gene of the Turkish population seems to have some differences from other populations. Deletion breakpoints were found to be clustered mainly in three separate regions covering introns 44, 45 and 50 within the central region of the dystrophin gene. Intron 44 was mostly 5′ breakpoints but it was found not to be involved as 3′ breakpoints. The correlation between phenotype and type of deletion agreed with the reading frame theory except for one DMD case.     

A simple explanation for a case of incompatibility with the reading frame theory in Duchenne muscular dystrophy: failure to detect an aberrant restriction fragment in Southern blot analysis.

According to the translational reading frame theory, Duchenne muscular dystrophy (DMD) patients harbor out-of-frame deletion mutations in the dystrophin gene. We identified a Japanese DMD case who appeared to have an in-frame deletion of exons 46-54 that was disclosed by Southern blot analysis using a dystrophin cDNA as a probe. Analysis of dystrophin mRNA in skeletal muscle revealed the presence of an out-of-frame deletion of exons 46-53. In agreement with this result, the region encompassing exon 54 could be amplified from genomic DNA by polymerase chain reaction (PCR). Furthermore, re-analysis by Southern blot using an exon specific probe disclosed that a HindIII fragment containing exon 54 was present at aberrant size, leading to the incorrect conclusion that exon 54 had been deleted. Thus, this particular DMD case does not constitute an exception to the reading frame theory.     

肌营养不良蛋白表达对肌营养不良症的诊断意义

目的：探讨肌营养不良蛋白在肌营养不良症患者肌组织中表达的意义。方法：运用免疫组化法对12例Duchenne型肌营养不良症（DMD）患者及5例Becker型肌营养不良症（BMD）患者的肌组织中肌营养不良蛋白的表达进行分析。并用6例非神经肌肉疾病患者的肌组织作为对照。结果：对照组6例肌组织标本中均可见肌营养不良蛋白表达,其阳性染色勾画出肌细胞的边界,胸及胞浆呈阴性。在DMD中有10例（83．33％）肌细胞膜肌营养不良蛋白不表达。BMD中3例（60）可见沿肌细胞膜分的不连续斑片状弱阳性染色。结论：肌营养不良蛋白的缺失或异常表达,是DMD／BMD型较为特异的改变。运用免疫组化法检测患者肌组织中肌营养不良蛋白的表达,可为DMD／BMD型的病理诊断提供特异指标。     

Re-evaluation of reading frame-shift hypothesis in Duchenne and Becker muscular dystrophy

The reading frame hypothesis has been proposed to explain the molecular basis of two allelic forms of muscular dystrophies, Duchenne/Becker muscular dystrophy (D/BMD). To evaluate the hypothesis in Indian D/BMD patients, we analyzed deletion of dystrophin exons in 147 DMD and 19 BMD patients. Our studies showed deviation of more than 30% from the reading frame hypothesis in DMD patients (47/147). The present results implicate a need to reevaluate the reading frame hypothesis.     

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.     

A screening for dystrophin gene deletions in Japanese patients with Duchenne/Becker muscular dystrophy by the multiplex polymerase chain reaction.

A new screening method involving the multiplex polymerase chain reaction was developed to detect dystrophin gene deletions in Japanese patients with Duchenne and Becker muscular dystrophy (DMD/BMD). Eleven exonic regions including deletion "hot spots" were analyzed. Gene deletions were found in 33% of 92 unrelated Japanese patients, mainly in the central portion (exons 43-52) and at the 5' end (exons 1-17). This is a useful laboratory test for the rapid genetic diagnosis of DMD/BMD.     

应用多重连接依赖性探针扩增定量技术检测假肥大型肌营养不良重复突变及携带状态

目的 应用多重连接依赖性探针扩增(MLPA)技术对假肥大型肌营养不良(DMD及BMD)患者及其家系成员进行dystrophin基因分析,探讨MLPA定量技术在本病重复突变及携带者检测中的优势.方法 以355例DMD及BMD患者、46名缺失型患者之母和8名重复型患者之母为研究对象,应用MLPA技术对dystrophin基因全长外显子进行分析,对于单一外显子缺失的样本采用PCR及测序进行验证.结果 经MLPA分析,全部355例患者中190例为dystrophin基因缺失型患者,在其余非缺失型患者中检测出34例重复型突变.此外,在46名缺失型患者的母亲中发现了28名携带者,在8名重复型患者的母亲中发现了6名携带者,两组患者母亲携带者频率差异无统计学意义.经过测序验证,在1例单一外显子缺失的患者中发现17号外显子存在AGGGAACAGATCCTGGTAAAGCA小片段缺失.结论 与传统的定量方法相比,MLPA定量技术可对DMD及BMD患者全长外显子区域同时进行缺失、重复分析,并能对患者家系成员的携带状态进行判定.此外,MLPA检测结果受模板DNA的浓度及纯度影响较小.     

Genetic abnormalities in Duchenne and Becker dystrophies: clinical correlations

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two allelic forms of an X-linked muscle disorder exhibiting phenotypic heterogeneity. We studied 49 individuals clinically diagnosed as having classic DMD, female DMD, mild DMD "outliers," and BMD. The patients' DNA was analyzed and alterations detected were correlated with particular phenotypes. We found that 14 of 32 classic DMD patients have an internal deletion in the same, relatively small, region of the gene; therefore this region may undergo deletions at a higher rate than the remainder of the gene. We could detect no alterations in the DNA in the remaining 18 patients. Selected patients from both groups failed to show muscle dystrophin. Seven of 11 patients with a mild DMD or BMD phenotype showed deletions at the 5' end of the gene. The other 4 patients failed to show deletions. Three of the patients with both a mild phenotype and a deletion at the 5' end had normal or low amounts of a dystrophin of smaller molecular weight. Patients with classic DMD who had a detectable deletion had a milder clinical course than those without. Contrary to a previous report, no patient in the population of clinically precisely defined DMD boys showed a deletion at the 5' end; thus, the outlier and BMD patients may be genetically different from boys with classic DMD. This correlation may be of diagnostic and prognostic significance.     

Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis.

We developed a method for the detection of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers. The method is based on the quantitative analysis of the products of standard multiplex polymerase chain reaction (PCR) from 18 different exons of the dystrophin gene, and is designated "QM-PCR." We detected deletions of one or more exons by standard multiplex PCR in DMD/BMD patients in 14 of 18 families examined (77.7%). The same deletions were readily demonstrated by QM-PCR in nine of 14 mothers (64.3%) and in another six of 22 possible carriers in these families. In five families where deletions were detectable in DMD/BMD patients, the mothers did not exhibit any deletions in their peripheral blood (35.7%). We obtained evidence for germinal mosaicism in at least two of these families and confirmed carrier identification by haplotype analysis using CA repeat polymorphisms at the 5' and 3' ends of the dystrophin gene. Furthermore, analysis of 17 coded DNA samples from normal females and obligatory carriers by QM-PCR showed that this technique could directly identify carriers of deletions in any of 18 different exons of the dystrophin gene. Its application in combination with existing techniques is expected to significantly improve the accuracy of carrier diagnosis in many families, and it may also be applicable to families in which pedigree and polymorphism information is insufficient for carrier diagnosis.     

Molecular analysis of the duchenne muscular dystrophy gene in patients with becker muscular dystrophy presenting with dilated cardiomyopathy

Molecular analysis of the Duchenne muscular dystrophy (DMD) gene was performed on 4 unrelated patients with Becker muscular dystrophy (BMD) presenting with dilated cardiomyopathy. Two patients with a deletion involving exon 1 were quite unique in that they developed fatal myocardial involvement in their teens, despite the absence of significant muscular weakness. The deletion found in these patients comprised the 3′-end of exon 1 and the greater part of intron 1. Two other patients with a deletion of exon 47 showed progressive muscular atrophy and weakness; they were considered to be typical BMD in both clinical features and the type of gene deletion. We speculate that a deletion around exon 1 may severely damage the expression and/or the function of dystrophin selectively in cardiac muscle, but not in skeletal muscle. © 1993 John Wiley & Sons, Inc.     

Unraveling the mysteries of Duchenne and Becker muscular dystrophy

Through a process that has come to be known as reverse genetics, the gene and gene product involved in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) have been identified. The DMD/BMD gene is over 2 million base pairs in size and over 50% of DMD/BMD patients harbor submicroscopic deletions for portions of the gene. The gene product, named dystrophin, is 400 Kd in size. Dystrophin is present in skeletal, cardiac, and smooth muscles, as well as brain. The protein is absent or altered in DMD/BMD patient muscle. The normal function of dystrophin and the reasons why its alteration results in the DMD/BMD phenotypes are presently unknown. The discoveries to date, however, provide a starting point for investigating the fundamental pathogenetic mechanisms involved in DMD/BMD.     

Dystrophin as a diagnostic marker in Duchenne and Becker muscular dystrophy. Correlation of immunofluorescence and western blot

Dystrophin is the gene product of the Duchenne (DMD) and Becker (BMD) muscular dystrophy gene locus on the short arm of the X chromosome. Complete lack of dystrophin is pathognomonic for DMD and variable changes of the molecule may be observed in the milder allelic form of BMD. In the present study the two methods available for dystrophin assessment, immunofluorescence detections on cryosections (IF) and Western blotting (WB) were systematically compared using polyclonal and monoclonal antibodies to various regions along the dystrophin molecule. A total of 95 patients with DMD or BMD were investigated including two female patients. Dystrophin assessment revealed abnormal abundance and/or distribution in all 95 patients with DMD or BMD. Only trace amounts of dystrophin were detected in 29% of the DMD patients and complete lack of dystrophin was found in 71%. In two females with DMD but with normal karyotype single dystrophin-positive fibres were found among more than 90% negative fibres. Out of 26 patients with BMD 19 (73%) had a dystrophin molecule of abnormal molecular weight. The results of IF were largely compatible with those from WB but differences were also observed, e.g. one barely symptomatic BMD patient with dystrophin of increased molecular weight showed normal IF. Out of four carriers of BMD three showed evidence of reduced dystrophin immunostaining in some muscle fibres. In 20 other patients limb girdle muscualar dystrophy with "Duchenne-like" or "Becker-like" phenotype was suspected because dystrophin showed normal abundance and distribution. Focal discontinuity of muscle cell-surface dystrophin staining was observed in one patient with a congenital, autosomal recessive muscular dystrophy and in one out of five patients with polymyositis/dermatomyositis. The study emphasizes the need for, and value of, dystrophin assessment in every case of suspected BMD or DMD.     

Preservation of the C-terminus of dystrophin molecule in the skeletal muscle from Becker muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disorder of muscle in children. The DMD gene product, "dystrophin", is absent from DMD, while the allelic disease, Becker muscular dystrophy (BMD), exhibits dystrophin of abnormal size and/or quantity. But we are still uncertain about the scenario that internally deleted (or duplicated) dystrophin in BMD possesses its carboxy (C)-terminal region, and severely truncated dystrophin in DMD does not. Here we use a new monoclonal antibody directed against an peptide in the C-terminal end of the dystrophin molecule to show that the C-terminus is preserved in 30 BMD and 24 control skeletal muscles but not in 21 DMD specimens. This result, taken together with data on deletions of the dystrophin gene, emphasizes both the diagnostic and biological importance of the C-terminal domain which is required for proper function and stability of dystrophin, and substantiates the validity of the reading frame hypothesis for DMD versus BMD deletions on a biochemical level.     

Analysis of dystrophin gene deletions by multiplex PCR in eastern India

The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD) is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR) to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63%) showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e, between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.     

Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families – Detection of carrier status in symptomatic and asymptomatic female relatives

A comprehensive study of the Hungarian Duchenne/Becker muscular dystrophy (DMD/BMD) families is presented. Deletions in the hot spots regions were identified by multiplex PCR, whereas rare mutations were detected by Southern blot and multiplex ligation-dependent probe amplification (MLPA) techniques. DMD/BMD disease was confirmed and exact deletion borders were determined in 19 out of 135 affected males using multiplex PCR. Additional exons involved as well as rare exon deletions were identified by MLPA in 71 male patients, whereas duplications were observed in seven patients. In two DMD patients, the entire dystrophin gene and adjacent genes were deleted. Out of the 95 female relatives, 41 proved to be carriers, including three manifesting carrier females. Using MLPA method, a large portion of the Hungarian DMD/BMD patients and their female relatives were exactly genotyped. For the first time, the incidence and prevalence of asymptomatic and symptomatic female carriers in Hungary was estimated.