DMD基因突变检测技术的回顾与展望

DMD是一类发病率较高的X连锁隐性遗传病。DMD基因突变机制复杂,探索更直接,简便,准确的基因突变检测技术是目前对DMD研究的热点。随着分子生物学的发展,DMD基因诊断技术取得了一定的进展,本探讨了各项技术的优势与缺陷,并展望了生物芯片等新技术的应用前景。     

DMD基因突变检测技术的回顾与展望

DMD是一类发病率较高的 X连锁隐性遗传病。DMD基因突变机制复杂 ,探索更直接、简便、准确的基因突变检测技术是目前对 DMD研究的热点。随着分子生物学的发展 ,DMD基因诊断技术取得了一定的进展 ,本文探讨了各项技术的优势与缺陷 ,并展望了生物芯片等新技术的应用前景     

Dystrophin相关蛋白-Utrophin的研究进展

近年来,DMD基因诊断技术已取得了很大进展,可是DMD基因治疗的研究却仍举步唯艰.随着基因治疗DMD模型mdx鼠研究的进一步深入,使DMD基因治疗迈上了一个新台阶,而Dystrophin相关蛋白-Utrophin的发现,则更为DMD基因治疗开拓了广阔的新前景.     

DMD基因突变类型及其研究方法

ＤＭＤ基因位于Ｘｐ２１．１－２１．３，由７９个外显子及其相应的内含子约２ ３００ｋｂ组成，１３９７４ｋｂ的ｍＲＮＡ中 １１ ３ｋｂ的序列为３ ６８５个氨基酸残基的抗肌营养不良蛋白（ｄｙｓｔｒｏｐｈｉｎ ｐｒｏｔｅｉｎ）编码。随着Ｓｏｕｔｈｅ颍钣〖：透髦郑校茫壹际醯囊冢模停幕蛏弦逊⑾至耍担爸郑遥疲蹋校保持郑樱裕遥ǎ螅瑁铮颍? ｔａｎｄｅｎ ｒｅｐｅａｔ），８种碱基置换，同时探明了ＤＭＤ基因     

应用PCR技术对DMD患者基因缺失及携带者检测

本文采用１８对引物，Ａ、Ｇ两套ＰＣＲ多重反应体系，对我院３７例进行性肌营养不良（ＤＭＤ）患者进行基因缺失检测，其中１９例患者有不同区域的缺失。其缺失区域售在４５－５２号外显子，最高峰在４８号外显子，与国外文献报道基本一致。另外应用６对重复双核苷酸序列多态性引物采用ＰＣＲ方法对４个非缺失型家系，进行扩增片段长度多态性分析发现，所有４个家系，均具有２个以上多态性标高。对这些家系通过连锁分析，可对非缺失     

DMD基因杂合金携带者基因诊断技术研究进展

正常等位基因的存在和干扰，为ＤＭＤ基因杂合子携带者的基因诊断设置了重重的障碍。但是近年来一系列以ＰＣＲ为核心的基因诊断技术开始出现，从而基本上解决了长期以来甚为棘手的ＤＭＤ杂合子携带者基因诊断的难题，本文将系统地综述ＤＭＤ基因杂合金携带者基因诊断技术研究的历史和现状。     

微卫星DNA序列与DMD／BMD的基因诊断

在人类基因组ＤＮＡ中，存在着大量的微卫星序列，这些微卫星ＤＮＡ序列具有高度多态性，是重要的遗传标记，在基因连锁分析中具有重要的作用。目前，在ｄｙｓｔｒｏｐｈｉｎ基因５＇及３＇端及基因内部，发现了多个ＣＡ：ＴＧ重复序列，具有高度的多态性，并且均可用ＰＣＲ方法进行检测；应用这些位点进行单体型分析，将大大提高非缺失型ＤＭＤ／ＢＭＤ家系中携带者的基因诊断以及产前基因诊断的可靠性及效率。     

135例Duchenne型肌营养不良症DMD基因缺失分析

目的：杜氏肌营养不良（DMD）患者基因缺失检测。方法：对135个患者,应用12对引物,PCR多重反应体系,聚丙烯酰胺凝胶电泳进行基因分析研究,设正常和空白对照,检测结果再以二重PCR进行验证。结果:其中54例患者有不同区域的缺失,基因缺失检出率为40%。结论：其缺失区域集中在45～53号外显子,最高峰在48号外显子。     

Duchenne肌营养不良（DMD）的无创伤性产前基因诊断

目的：对DMD进行无创伤性产前基因诊断。方法：采用孕妇外周血,ZFY基因及DMD基因多重PCR反应体系,对3例进行性肌营养不良DMD患者的母亲再次怀孕时的胎儿进行无创伤性产前基因诊断。结果：1例女性胎儿,两例男性胎儿,男性胎儿中1例与其先证者哥哥同为外显子48缺少,另1例同为基因突变型。结论：无创伤性前前基因诊断X－连锁隐性遗传病DMD是可行的。     

反转录与套式PCR技术在DMD／BMD杂合子基因诊断…

以外周血为标本提取细胞质总ＲＮＡ，应用ＤＭＤ基因的反转录多聚酶链反应技术（反转录ＰＣＲ，ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ，ＲＴ－ＰＣＲ）检测了１０个ＤＭＤ／ＢＭＤ家系。结果在４个家系中发现６个患者获得较短的ＰＣＲ片段，其中在２个家系中发现部分女性同时获得正常大小和相应较瓣ＰＣＲ片段，而得到了满意的患者和杂合子携带者的诊断。该法有利于ＤＭＤ／ＢＭＤ患者、尤其是杂合子携带者的确诊，为产前基因     

Erratum: A novel splice site mutation (3157+1G>T) in the dystrophin gene causing total exon skipping and DMD phenotype

Erratum: An error was printed in the original version of this article in the Comments section, paragraph 2, relating to the size of exon 22 and the RT‐PCR product size described as resulting from the mutation 3157+1G>T. The paragraph should read: “We report a case of a 5 year old DMD patient with a novel splice site mutation affecting the GT dinucleotide splice donor of exon 22. The RT‐PCR analysis with primer sets spanning dystrophin exons 17‐25 amplified no normal size fragment (1251 bp), but a product shorter by 146 bp (the length of exon 22). Direct sequencing of the faster migrating fragment revealed total skipping of exon 22.”     

Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene

Straightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations.     

A novel insertional mutation of a single base in exon 12 of the dystrophin gene

A new point mutation in exon 12 of the dystrophin gene was identified in a DMD patient using multiple SSCP analysis, which allows the simultaneous study of several exons. The mutation is an A insertion at position 1580 of the cDNA sequence, leading to a stop codon in the translational reading frame. This mutation was not observed in a sample of 70 DMD patients.     

Occurrence of two different intragenic deletions in two male relatives affected with Duchenne muscular dystrophy

The occurrence of 2 different intragenic deletions (exons 10–44 and exon 45, respectively) is reported in 2 male relatives affected with Duchenne muscular dystrophy, both showing the same haplotype for DNA markers not included in the deleted segment. The 2 different deletions seem to have occurred independently in the same X chromosome. This finding, together with other reports, suggests possibly an increased predisposition to mutations within the DMD locus in some families. Therefore, when dealing with prenatal diagnosis, the investigation on fetal DNA cannot be restricted only to the region in which a mutation was previously identified in the family. © 1994 Wiley-Liss, Inc.     

DMD and BMD in the same family due to two distinct mutations

We report on a family with a boy affected by Duchenne muscular dystrophy (DMD) and an asymptomatic cousin with a Beckertype dystrophin abnormality, diagnosed by chance. Dystrophin gene analysis showed that these conditions were caused by two distinct deletions with breakpoints in different exons. In Xp21 families, DNA analysis and dystrophin testing of asymptomatic males with high CK plasma levels might detect different dystrophin mutations in separate haplotypes as in our family, although we stress there should be clear clinical or familial indications for such testing. © 1995 Wiley-Liss, Inc.     

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.     

Mutation rates in the dystrophin gene: a hotspot of mutation at a CpG dinucleotide

An analysis of mutations was performed in 141 Duchenne muscular dystrophy (DMD) patients previously found to be negative for large deletions by standard multiplex PCR assays. Comprehensive mutation scanning of all coding exons, adjacent intronic splice regions, and promoter sequences was performed by DOVAM-S, a robotically enhanced, high throughput method that detects essentially all point mutations. Samples negative for point mutations were further analyzed for duplications by multiplex amplifiable probe hybridization (MAPH). Presumptive causative mutations were detected in 90% of the patients (70% protein truncating point mutations, 13% duplications, and 7% deletions not detected by the standard multiplex screening method). A total of 40 of the mutations are putatively novel. Most duplications involve multiple exons with an average and median size of about 160 and 153 kb, respectively. This is the first analysis of the absolute and relative rates of point mutations in the dystrophin gene. Relative to microdeletions (0.68 x 10(-9) per bp per generation), transitions at CpG dinucleotides are enhanced 150-fold while complex indels, the least common mutation type, are less frequent than microdeletions by a factor of five. The frequency of microdeletions and microinsertions at mononucleotide repeats increases exponentially with length. When compared to the well-studied human factor IX gene (F9), the results are similar, with two exceptions: a hotspot of mutation in the dystrophin gene (c.8713C>T/p.R2905X) at a CpG dinucleotide and an altered size distribution of microdeletions. The hotspot reflects a difference in the underlying pattern of mutation, while the altered size distribution of microdeletions reflects certain abundant sequence motifs within the dystrophin coding sequence (relative to factor IX).     

Sensitivity and frequencies of dystrophin gene mutations in Thai DMD/BMD patients as detected by multiplex PCR

Background: Duchenne muscular dystrophy (DMD), a lethal X-linked disease affecting 1 in 3500 male births, and its more benign variant, Becker muscular dystrophy (BMD), are caused by mutations in the dystrophin gene. Because of its large size, analysing the whole gene is impractical. Methods have been developed to detect the commonest mutations i.e. the deletions of the exons. Although these tests are highly specific, their sensitivity is inherently limited by the prevalence of deletions, which differs among different populations. Methods: We reviewed our database for the detection of Dystrophin gene mutation by means of 31-exon multiplex PCR in Thai males, diagnosed clinically and biochemically with DMD or BMD from July 1994 to November 2006. One index patient was chosen from each family for statistical analysis. The overall sensitivity of the test, the number of fragment deleted, and the deletion frequency of each fragment were calculated, along with their 95% confidence intervals (C.I.). Results: We found deletions in 99 out of the 202 index patients (49%; Bayesian 95% C.I. = 42%–56%). 51% of these had deletion in only one of the 31 exons tested, while the patient with the most extensive deletions had 14 exons deleted. The mean number of deleted exons were 2.84 (BC_a bootstrap 95% C.I. = 2.37–3.48), or 5.02 (3.81–6.85) if all the untested exons adjacent to the confirmed deleted exons were assumed to be deleted. The region spanning exons 44-52 was the most frequently deleted. These were similar to those reported in the Japanese. Conclusion: The multiplex PCR detected deletions only in about half of the Thai patients. The diseases therefore should not be excluded solely on the negative result if DMD/BMD is strongly suspected.     

反转录与套式PCR技术在DMD／BMD杂合子基因诊断中的应用

以外周血为标本提取细胞质总ＲＮＡ，应用ＤＭＤ基因的反转录多聚酶链反应技术（反转录ＰＣＲ，ｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎＰＣＲ，ＲＴ－ＰＣＲ）检测了１０个ＤＭＤ／ＢＭＤ家系。结果在４个家系中发现６个患者获得较短的ＰＣＲ片段，其中在２个家系中发现部分女性同时获得正常大小和相应较短的ＰＣＲ片段，而得到了满意的患者和杂合子携带者的诊断。该法有利于ＤＭＤ／ＢＭＤ患者、尤其是杂合子携带者的确诊，为产前基因诊断和遗传咨询提供了科学的依据。