Duchenne肌营养不良（DMD）发病机制及治疗研究进展

Duchenne肌营养不良（Duchenne muscular dystrophy,DMD）为X连锁、隐性、致死性遗传病,其致病基因位于X染色体的Xp21.1-3区,编码抗肌萎缩蛋白dystrophin。随着对该病研究的不断深入,人们从宏观到微观对DMD的再认识不断更新,发现其发病涉及到从基因、胞膜缺陷,到细胞的炎性机制,以及纤维化及肌细胞再生等多个层面。就其细胞及亚细胞水平发病机制及治疗上的进展进行综述。     

Duchenne肌营养不良的基因治疗进展

Ｄｕｃｈｅｎｎｅ肌营养不良（ＤＭＤ）基因治疗的目的是补充骨骼肌和心肌内已经缺失的抗肌萎缩蛋白，本文对ＤＭＤ基因治疗中目的基因的选择，基因靶向，载体的选择，目的基因的运送途径等方面作一综述。     

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。     

太空飞行后秀丽隐杆线虫肌相关基因和蛋白质变化

太空飞行所致的肌萎缩和重力感知的分子机制至今尚不清楚．研究太空飞行对秀丽隐杆线虫（C.elegans）体壁肌细胞结构和功能的影响．经过近15天太空飞行后对其生存率和运动能力进行了观察,并检测了5个重要的肌相关基因的表达和3种蛋白质含量．太空研究是在动物的整体水平进行的,而不是就单个细胞的研究．经历太空飞行后线虫生存率没有明显变化,但运动频率变慢,爬行轨迹也发生了改变,提示线虫运动功能出现障碍,这些数据揭示：微重力下秀丽线虫肌肉发育发生了变化．肌球蛋白A（myosin A）免疫荧光染色观察发现,太空飞行组肌纤维面积缩小,肌细胞致密体（dense－body）荧光亮度下降．这些形态学观察直接提示太空组线虫出现了肌萎缩．但是,肌动蛋白（F-actin）荧光染色显示两组并无明显差别．基因表达水平的分析结果显示,在太空飞行组动物中dys－1表达明显上调,同时hlh－1,myo－3,unc－54和egl—19基因表达下调．抗肌萎缩蛋白（dystrophin,由dys—1编码）是抗肌萎缩蛋白－糖蛋白复合物（DGC）的主要组成成分,而该复合物在微重力下增多,提示肌细胞是为了接受更多的力学刺激以维持细胞内外的力学平衡,所以该复合物在肌细胞的重力感知中起关键作用．基因hlh－1,myo－3,unc－54和egl－19表达下调,说明它们分别从结构和功能两个途径促进了微重力性肌萎缩的发生．最后,Western blot结果提示,太空组线虫体壁肌内肌球蛋白A减少,进一步确证了太空飞行中线虫有肌萎缩发生．     

中国人群中抗肌萎缩蛋白基因突变类型和分布特点及其与临床症状的相关性

假性肥大型进行性肌营养不良症(Duchenne’s muscular dystrophy,DMD)是源于横纹肌的一种X-连锁隐性致死性遗传病,由编码抗肌营养不良蛋白(dystrophin)基因突变所致。为了探讨中国人群中DMD患者的dystrophin基因突变类型和分布特点及其与临床症状的相关性,文章采用Multiplex Ligation-Dependent Probe Amplification(MLPA)方法对720例DMD患者及其母亲和20例正常成年男性进行dystrophin基因分析。结果显示,检出率为64.9%(467/720),54.3%(391/720)的患者为基因缺失;10.6%(76/720)的患者为基因重复。累及Exon45-54缺失突变型占全部缺失型患者的71.9%(281/391);重复突变型累及Exon1-40占全部重复型患者82.9%(63/76);检出的患者中,DMD型和中间型营养不良症(Intermediate muscular dystrophy,IMD)型占90.6%(423/467),Becker型营养不良症(Becker muscular dystrophy,BMD)型占9.4%(44/467)。表明假肥大型肌营养不良症以dystrophin基因缺失突变为主,突变发生在整个基因中非均匀分布,存在突变热区,在缺失和重复的位置和片段长度与肌病的临床症状严重程度之间并不存在简单的相关关系。     

DMD/BMD缺失基因的检测及其表达产物的变化

目的:检测Duchenne/Becker型肌营养不良症(DMD/BMD)患者基因缺失及其表达产物--抗肌营养不良蛋白在肌细胞中的变化,探讨其与临床病情的关系.方法:应用9对引物多重PCR技术对42例DMD/BMD患者进行基因检测;并采用免疫荧光抗体染色技术对5例DMD,2例BMD肌细胞膜上抗肌营养不良蛋白的表达观察分析,以2例正常人的肌组织作为对照.结果:共发现21例外显子缺失,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.5例DMD患者胞膜抗肌营养不良蛋白染色阴性,其中1例未检出基因缺失,但抗肌营养不良蛋白无表达.2例BMD患者染色弱阳性,可见间断斑片状荧光带.结论:DMD/BMD病情轻重可能与基因缺失的数量和片段大小不呈平行关系,而是与外显子的缺失类型有密切关系;基因的表达受个体差异的影响,呈高度的遗传异质性.抗肌营养不良蛋白缺乏或表达异常是造成DMD/BMD表型的病理基础,其临床后果不仅取决于缺失程度,还取决于缺失区域的功能意义.     

模拟微重力下秀丽隐杆线虫的力学感知和肌萎缩的发生机制——太空飞行线虫试验地面平行对照研究部分

目前,微重力导致肌萎缩的分子机制尚不清楚,重力感知是该事件发生的关键环节．为了回答这一问题,在此之前首先实施了太空线虫试验,这部分结果已经在本刊报道过．而本次研究主要是在地面上建立了模拟微重力环境,观察处理后秀丽隐杆线虫（C.elegans）体壁肌细胞结构和功能的变化,一方面用于验证太空试验,同时比较两种处理结果的异同,以便于评价地面模拟微重力的有效性．经过14天19．5h旋转模拟微重力处理后,对线虫生存率和运动能力进行了观察,并检测了几个重要的肌相关基因表达和蛋白质水平．模拟微重力下线虫生存率没有明显变化,但运动频率显著下降,爬行轨迹也发生了轻微改变,运动幅度降低,提示线虫运动功能出现障碍．从形态学上观察发现：肌球蛋白A（myosin A）免疫荧光染色显示模拟微重力组肌纤维面积缩小,而肌细胞致密体（dense-body）染色可见荧光亮度下降．这些结果直接提示模拟微重力使线虫出现了肌萎缩．随后Western blotting试验结果揭示,模拟微重力组线虫体壁肌的主要结构蛋白——myosin A含量减少,进一步确证了微重力性肌萎缩发生．在基因水平,旋转后抗肌萎缩蛋白基因（dys-1）表达明显上升,而hlh-1,unc-54,myo-3和egl-19的mRNA水平均下调,提示dys-1在骨骼肌感知和传导力学信息方面有重要作用,而hlh-1,unc-54,myo-3和egl-19则分别从结构和功能两个途径促进了微重力性肌萎缩的发生和发展．本次试验所得到的结果同太空飞行试验结果十分相似,一方面强化了太空试验结论,另一方面说明在地面上模拟微重力对生物体进行研究是有效可行的,将有助于提高太空试验的质量．     

α-抗肌萎缩相关糖蛋白病脑结构畸形的研究进展

α-抗肌萎缩相关糖蛋白病(α-dystroglycanopathy,α-DGP)是由于抗肌萎缩相关糖蛋白(dys-troglycan,DG)缺陷或翻译后糖基化修饰不足导致的一组常染色体隐性遗传病,具有高度临床和遗传异质性.目前已发现至少18种致病基因,每种基因型可导致多种表型,从起病早、症状重的先天性肌营养不良(con...     

α-抗肌萎缩相关糖蛋白病研究进展

抗肌萎缩相关糖蛋白病(α-dystroglycanopathy,α-DGP)是由于α-抗肌萎缩相关糖蛋白(α-dystroglycan,α-DG)的O-连接糖基化缺陷导致的一组常染色体隐性遗传病,临床表现为肌营养不良、脑和眼发育畸形,具有明显的临床和遗传异质性。基于临床诊断现多把它们分为7种不同表型,但互相之间有交叉重叠,病情轻重程度不等,严重影响患者的智力运动发育。目前已知α-DGP致病基因有19个,应用常规一代测序逐个筛查候选基因花费高且耗时长,阳性率很低,不能满足临床上基因诊断需求。目前已知多数致病基因的表达产物作为糖基转移酶参与α-DG的糖基化过程,但部分致病基因的功能目前仍未完全明确,α-DGP具体的发病机制仍未完全阐明。本综述主要总结α-DGP在临床特点、致病基因、α-DG糖链结构、发病机制及诊断治疗等方面的研究进展。     

运动神经元生存蛋白SMN与Sm蛋白的结合作用

脊髓性肌萎缩症（spinal muscular atrophy,SMA）是一类与运动神经元存活基因（survival of motor neurons gene,SMN gene）突变有关的神经系统变性疾病,而SMN基因的转录产物即为SMN蛋白（survival of motorneurons protein,SMN protein）。SMN蛋白与多种蛋白结合后发挥作用,如SMN-Sm蛋白的相互作用在富含尿嘧啶的小核核糖核蛋白体（uridine—richsmallribonucleo—proteins,UsnRNPs）转运装配中有重要意义。SMN蛋白是通过其Tudor结构域与剪接体sm蛋白的二甲基化修饰的富含精氨酸一氨基乙酸域（ar—ginineandglycine—rich,RG）结合。     

Novel mutations of dystrophin gene in DMD patients detected by rapid scanning in biplex exons DHPLC analysis

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.     

Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database

ABSTRACT: BACKGROUND: Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. Methods and results We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION: This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.     

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.     

Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi children

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene (DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe. In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion regions (HFDR’s) in the DMD gene mutations in KSA.     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.     

Different mosaicism frequencies for proximal and distal Duchenne muscular dystrophy (DMD) mutations indicate difference in etiology and recurrence risk.

In about 65% of the cases of Duchenne muscular dystrophy (DMD) a partial gene deletion or duplication in the dystrophin gene can be detected. These mutations are clustered at two hot spots: 30% at the hot spot in the proximal part of the gene and about 70% at a more distal hot spot. Unexpectedly we observed a higher frequency of proximal gene rearrangements among proved "germ line" mosaic cases. Of the 24 mosaic cases we are aware of, 19 (79%) have a proximal mutation, while only 5 (21%) have a distal mutation. This finding indicates that the mutations at the two hot spots in the dystrophin gene differ in origin. Independent support for the different mosaicism frequency was found by comparing the mutation spectra observed in isolated cases of DMD and familial cases of DMD. In a large two-center study of 473 patients from Brazil and the Netherlands, we detected a significant difference in the deletion distribution of isolated (proximal:distal ratio 1:3) and familial cases (ratio 1:1). We conclude from these data that proximal deletions most likely occur early in embryonic development, causing them to have a higher chance of becoming familial, while distal deletions occur later and have a higher chance of causing only isolated cases. Finally, our findings have important consequences for the calculation of recurrence-risk estimates according to the site of the deletion: a "proximal" new mutant has an increased recurrence risk of approximately 30%, and a "distal" new mutant has a decreased recurrence risk of approximately 4%.     

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR

Summary About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.