应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

应用多重PCR和MLPA技术检测DMD患者和携带者的基因突变及产前诊断

目的 应用多重PCR和多重连接依赖探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测Duchenne/Becker肌营养不良症(Duchenne/Becker muscular dystrophy,DMD/BMD)患者、携带者并应用于产前诊断.方法 首先采用多重PCR对临床诊断为DMD/BMD的患者检测DMD基因的26个外显子,未查到缺失突变者和可能的携带者采用MLPA检测全部79个外显子是否有缺失或重复突变.对产前诊断病例,用PCR法检测缺失突变,用MLPA法检测重复突变.结果 多重PCR对22例患者的DMD基因的26个外显子检测.13例有缺失突变.未查到常见缺失突变的9例患者经MLPA检测DMD基因的全部79个外显子,3例为重复突变、1例为单个第18外显子缺失、其他5例未查到缺失和重复突变.16例携带者中,3例有家族史,其中2例检出突变;13例为检测到突变的散发病例患儿的母亲,有8例检测到突变.产前诊断9个胎儿(其中双胎1例),2例胎儿有突变,引产后核实无误;7例胎儿未检测到突变,现均已分娩.结论 多重PCR可检出92.86%的缺失突变并可用于缺失突变的产前诊断,因其简便、可靠、价廉可作为临床上DMD/BMD基因诊断的初选.MLPA可用于多重PCR未检测到缺失突变的患者及携带者的检查.     

Detection of carriers of Duchenne and Becker muscular dystrophy using the fluorescence in situ hybridization method

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the dystrophin gene. A large intragenic deletion has been described in about 65% of DMD/BMD patients. Mothers of affected males are DMD/BMD carriers in two thirds of the cases. Routine deletions detection in DMD/BMD males is performed using multiplex polymerase chain reaction (mPCR), RT-PCR with a protein truncation test (PTT) or using Southern blotting. In females the deletions detection is complicated by the presence of a normal gene copy on the second X-chromosome. We are presenting the diagnostic strategy using FISH for the deletions detection in the dystrophin gene of female DMD/BMD carriers. We have used a set of six cosmid probes for the detection of the most frequently deleted areas of the dystrophin gene from the Department of Human Genetics, Leiden University Medical Center. We have examined 14 mothers of DMD/BMD males with a deletion in the dystrophin gene identified using mPCR. Four mothers of affected males have been diagnosed as carriers of a deletion in the dystrophin gene. We have revealed no deletion mutations in the exons examined in a control group of four healthy females. No discrepancy has been found between the FISH analysis results and the results of mPCR. Our results indicate that FISH is an effective and direct method for the identification of DMD/BMD carriers and we suggest this method as a method of a first choice in the identification of DMD/BMD carriers.     

Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.     

Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.     

Alternative splicing of dystrophin mRNA complicates carrier determination: report of a DMD family.

Carrier determination is important for genetic counselling in DMD/BMD families. The detection of altered PCR amplified dystrophin mRNA fragments owing to deletions, insertions, or point mutations has increased the possibilities of carrier determination. However, problems may occur because of alternative splicing events. Here we present a family with a DMD patient characterised by a deletion of exons 45 to 54. At the mRNA level we detected a corresponding altered fragment which served for carrier determination. The mother and the sister of the patient showed the same altered dystrophin mRNA fragment as the patient and are therefore carriers. In the mother two additional altered dystrophin mRNA fragments were detectable, obviously resulting from alternative splicing in the normal allele. The grandmother and two other related females of the patient possess only the normal mRNA fragment. In a further female we detected an altered fragment owing to an mRNA deletion of exon 44. This fragment is created either by alternative splicing or a new mutation. Therefore, the carrier status of this female is still ambiguous indicating problems in carrier determination by the method of dystrophin mRNA analysis.     

Comparative analysis of PCR-deletion detection and immunohistochemistry in Brazilian Duchenne and Becker muscular dystrophy patients

We studied 48 patients with dystrophinopathies (29 Duchenne muscular dystrophy (DMD), 13 Becker muscular dystrophy (BMD), four possible carriers, one female with DMD, and one intermediate form, using polymerase chain reaction (PCR) analysis of muscle tissue for 20 exons and compared them with immunohistochemistry studies for dystrophin. Of these, 42 (87.5%) showed at least one intragenic deletion. Most of them (47.45%) involved exons 2 to 20. All BMD patients presented deletions on the dystrophin gene. The 29 patients with DMD showed abnormal dystrophin in immunohistochemistry studies, some with total absence (17/29), others with residual (3/29), and the remaining with scattered positive fiber (9/29). The majority of the 13 patients with BMD had abnormal immunohistochemistry studies with diffuse reduction in the majority of muscle fibers (10/13), a few with patch discontinuation in the sarcolemma (2/13), and one normal (1/13). The immunohistochemistry exam for dystrophin is still the gold-standard method for DMD/BMD diagnosis. An ethnic difference, the analysis of several exons, the sample size, and the use of muscle tissue could explain this high frequency of deletions in the dystrophin gene found in our cases.     

Identification of carriers of Duchenne/Becker muscular dystrophy by a novel method based on detection of junction fragments in the dystrophin gene.

We developed a Southern blotting based method that uses rare cutting restriction endonucleases and electrophoresis of single stranded DNA to detect junction fragments resulting from the rearranged dystrophin gene. By conventional Southern blot hybridisation, no junction fragments were detected in 27 unrelated patients with Duchenne (DMD) or Becker (BMD) muscular dystrophy, who had 20 deletions and seven duplications in the dystrophin gene. With our new method, junction fragments were detected in 21 of these 27 patients. When the junction fragments were used as markers, five carriers were unequivocally diagnosed among six females from two families of DMD/ BMD patients. This novel method allows simple and definitive identification of carriers with risk factors for DMD/BMD without using quantitative Southern blot hybridisation.     

Integrated study of 100 patients with Xp21 linked muscular dystrophy using clinical, genetic, immunochemical, and histopathological data. Part 2. Correlations within individual patients.

This report is the second part of a trilogy from a multidisciplinary study which was undertaken to record the relationships between clinical severity and dystrophin gene and protein expression. The aim in part 2 was to correlate the effect of gene deletions on protein expression in individual patients with well defined clinical phenotypes. Among the DMD patients, most of the deletions/duplications disrupted the open reading frame, but three patients had in frame deletions. Some of the intermediate D/BMD patients had mutations which were frameshifting while others were in frame. All of the deletions/duplications in the BMD patients maintained the open reading frame and 25/26 deletions in typical BMD group 5 started with exon 45. The deletion of single exon 44 was the most common mutation in patients from groups 1 to 3. Dystrophin was detected in sections and blots from 58% of the DMD patients with a size that was compatible with synthesis from mRNA in which the reading frame had been restored. Certain deletions were particularly associated with the occurrence of limited dystrophin synthesis in DMD patients. For example, 9/11 DMD patients missing single exons had some detectable dystrophin labelling compared with 10/24 who had deletions affecting more than one exon. All patients missing single exon 44 or 45 had some dystrophin. Deletions starting or finishing with exons 3 or 51 (8/9) cases were usually associated with dystrophin synthesis whereas those starting or finishing with exons 46 or 52 (11/11) were not. Formal IQ assessments (verbal, performance, and full scores) were available for 47 patients. Mean IQ score among the DMD patients was 83 and no clear relationship was found between gene mutations and IQ. The mutations in patients with a particularly severe deficit of verbal IQ were spread throughout the gene.     

Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn). Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR) to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.     

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.