Exon Deletion Patterns of the Dystrophin Gene in 82 Vietnamese Duchenne/Becker Muscular Dystrophy Patients

Abstract

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited muscle diseases caused by mutations in the dystrophin gene. The reading frame rule explains the genotype-phenotype relationship in DMD/BMD. In Vietnam, extensive mutation analysis has never been conducted in DMD/BMD. Here, 152 Vietnamese muscular dystrophy patients were examined for dystrophin exon deletion by amplifying 19 deletion-prone exons and deletion ends were confirmed by dystrophin cDNA analysis if necessary. The result was that 82 (54%) patients were found to have exon deletions, thus confirming exact deletion ends. A further result was that 37 patterns of deletion were classified. Deletions of exons 45–50 and 49–52 were the most common patterns identified, numbering six cases each (7.3%). The reading frame rule explained the genotype-phenotype relationship, but not five (6.1%) DMD cases. Each of five patients had deletions of exons 11–27 in common. The applicability of the therapy producing semifunctional in frame mRNA in DMD by inducing skipping of a single exon was examined. Induction of exon 51 skipping was ranked at top priority, since 16 (27%) patients were predicted to have semifunctional mRNA skipping. Exons 45 and 53 were the next ranked, with 12 (20%) and 11 (18%) patients, respectively. The largest deletion database of the dystrophin gene, established in Vietnamese DMD/BMD patients, disclosed a strong indication for exon-skipping therapy.     

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.     

Relatively low proportion of dystrophin gene deletions in Israeli Duchenne and Becker muscular dystrophy patients

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55–65%). Seventy-eight percent of the deletions were confined to exons 44–52, half of these to exons 44–45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. © 1994 Wiley-Liss, Inc.     

Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy

We have developed a preimplantation genetic diagnosis (PGD) strategy for Duchenne muscular dystrophy (DMD) allowing the simultaneous amplification of four exons (6, 8, 28 and 32) of the dystrophin gene together with ZFX/ZFY genes for gender determination. Preliminary experiments were carried out on 215 single lymphocytes from male and female individuals. Amplification rates ranged from 90.2% for exon 6 to 96.7% for exons 8 and 32. At least four of the five sequences were successfully amplified in 95.8% of single cells, and sexing was possible in 98.5%. This 5-plex assay was found to be robust enough to be used in a PGD clinical procedure and was therefore applied to a family whose female partner was a heterozygous carrier of a large deletion extending from exon 21 to exon 34 of the dystrophin gene. We have thus analysed two exons located in the deleted region of the gene, two non-deleted exons used as intrasample controls, and ZFX/ZFY genes. Cleavage stage embryo biopsy followed by PCR resulted in transfer of three unaffected embryos. The advantage of the present approach is to identify and subsequently transfer unaffected male embryos in addition to female embryos, and is now applicable to all families displaying a deletion involving at least one of these exons.     

应用荧光原位杂交技术诊断基因缺失型进行性假肥大性肌营养不良携带者

目的 建立应用荧光原位杂交(fluorescent in situ hybridization，FISH)方法检查进行性假肥大性肌营养不良(Duchenne／Becker muscular dystrophy，DMD／BMD))患者家系中女性亲属是否为携带者的方法。方法 采用多重聚合酶链反应对19例DMD／BMI)先证者进行基因诊断，从中筛选出两例缺失dystrophin基因外显子46的患者，其中l例有阳性家族史，另l例为散发病例，采用双色FISH对其女性亲属进行携带者的检查。结果 在有阳性家族史的1例患者的家系中检出4例携带者；在另一散发病例的家系中检出1例所缺失基因片段的体细胞嵌合体。结论 与多重PCR相结合，应用双色FISH检出基因缺失型DMD／BMD携带者是一个切实可行的诊断方法，对于所缺失基因片段的体细胞嵌合体的诊断是FISH方法的一个突出的优点，这对DMD／BMD家系的遗传咨询以及产前诊断指征的确立具有重要意义。     

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.     

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.     

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.     

假肥大型肌营养不良症基因诊断及遗传分析

目的对假肥大型肌营养不良症（DMD／BMD）患者进行基因诊断并对家系进行遗传分析,以提高对DMD／BMD的基因诊断水平及有效的遗传咨询。方法对40例DMD／BMD患者应用18对引物多重PCR技术进行Dystrophin基因缺失诊断,收集完整家系资料进行遗传分析以判断致病基因携带者及评估风险。结果40例DMD／BMD患者基因诊断有27例至少存在一个外显子片段缺失（67．5％）,13例未检测到缺失（32．5％）。通过对家系的遗传分析判断出致病基因携带者。结论多重PCR作为一种简便快速的诊断方法可对DMD／BMD患者进行基因诊断;对风险家系进行遗传分析、判断致病基因携带者以进行有效的遗传咨询,进而控制遗传病。     

Mutation and haplotype analysis for Duchenne muscular dystrophy by single cell multiple displacement amplification

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder with mutational heterogeneity. The scarcity of DNA from single cells in preimplantation genetic diagnosis (PGD) for DMD limits comprehensive genetic testing. Multiple displacement amplification (MDA) is reported to generate large amounts of template and give the most complete coverage and unbiased amplification to date. Here, we developed mutation and haplotype analysis in conjunction with gender determination on MDA products of single cells providing a generic approach that widens availability of PGD for female carriers with varied mutations. MDA amplified with 98.5% success for single lymphocytes and 94.2% success for single blastomeres, which was evaluated on 60 lymphocytes and 40 blastomeres. A total of six commonly mutant exons, eight short tandem repeat markers within dystrophin gene and amelogenin were incorporated into subsequent singleplex PCR assays. The mean allele dropout rate was 9.0% for single lymphocytes and 25.5% for single blastomeres. None of the blank controls gave a positive signal. Genotyping of each pedigree for three families provided 2-3 fully informative alleles per dystrophin haplotype besides specific mutant exons and amelogenin. We suggest that this approach is reliable to identify non-carrier female embryos other than unaffected male embryos and reduce the risk of misdiagnosis.     

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.     

Duchenne肌营养不良症患者及携带者的基因缺失和重复突变检测

目的利用多重连接依赖探针PCR扩增技术检测Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）患者及其可能的女性携带者的dystrophin基因的缺失、重复突变。方法利用多重连接依赖探针PCR扩增对32例DMD患者及其27个可能的女性携带者的dystrophin基因缺失、重复进行检测。结果32个先证者中，共检测出了24例DMI）患者具有一个或多个外显子的缺失，l例DMD患者具有重复突变，l例患者为第19外显子的无义突变（R768X），6例没有检测出缺失、重复突变的先证者可能是点突变所致。17个先证者的18位女性亲属具有和先证者相同的缺失、重复突变。结论多重连接依赖探针PCR扩增技术可用于检测DMD基因的缺失、重复突变，可以检测DMD基因女性携带者的基因杂合情况，在检测DMD基因缺失和重复方面，具有一定的应用价值。     

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.