多重连接探针扩增方法在假肥大性肌营养不良产前基因诊断中的应用

假肥大性肌营养不良(Duchenne/Becker muscular dystrophy, DMD/BMD)是一种由于DMD基因突变导致的X连锁隐性致死性遗传病。目前没有有效的治疗方法。为建立一种既可以对携带者进行检测又可以进行产前基因诊断的方法, 文章联合应用多重连接探针扩增技术(Multiplex ligation-dependent probe amplification, MLPA)和短串联重复序列(Short tandem repeats , STR)为遗传标记连锁分析的方法对26例有高风险再生育患儿的假肥大性肌营养不良家系的孕妇通过羊水穿刺进行产前基因诊断。26例进行产前基因诊断的羊水标本中有7例诊断为男性患儿, 4例诊断为女性携带者。MLPA可以作为筛查DMD基因缺失和重复突变的首选方法。联合应用MLPA和STR连锁分析, 可以提高假肥大性肌营养不良的产前基因诊断率。     

杜氏肌营养不良症(DMD)及其动物模型研究进展

杜氏肌营养不良症（Duchenne muscular dystrophy,DMD）属于X连锁隐性遗传病.DMD基因是人类最大基因,突变机制复杂.随着分子生物学的研究进展,对DMD的基因和其编码的抗肌萎缩蛋白（dystrophin）及抗肌萎缩蛋白相关蛋白（utrophin）的认识不断深入.本文就DMD的病理学特点,Dys基因结构、表达、功能,DMD突变及其相关检测技术,DMD实验动物模型及相关治疗的研究进展进行综述.     

应用PCR技术对DMD患者进行缺失检测与产前基因诊断

运用聚合酶链式反应(polymerasechainreaction,PCR)技术对3个Duchenne型肌营养不良症(DMD)家系中的患者进行dystrophin基因内9个外显子缺失检测,在2个家系中检测到外显子45、48、51缺失,同时运用PCR技术扩增位于dystrophin基因内内含子短串联重复序列,对非缺失型DMD家系进行了产前诊断,胎儿为正常女性.dystrophin基因外显子缺失检测方法快速、敏感、准确,可在临床推广中应用;短串联重复序列(STR)多态性分析方法可用于DMD家系的产前基因诊断和携带者检出.     

一例X染色体结构重排导致DMD疾病

假肥大型肌营养不良(Duchenne/Becker muscular dystrophy,DMD/BMD)是一种最常见的进行性肌营养不良疾病，呈X-连锁隐性遗传，主要由DMD基因的缺失、重复及点突变所致，极少数病例是由于染色体结构重排破坏了DMD基因而引起疾病的发生。本文报告了1例经多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)和下一代测序检测后原因未明的、具有典型症状的DMD患者。采用核型分析、FISH分析及三代测序、 Sanger测序综合分析发现，患者存在母源性的X染色体臂间倒位(Chr.X:g.[31939463–31939465del; 31939466–131765063 inv; 131765064–131765067del])半合子变异。由于该变异破坏了DMD基因和HS6ST2基因，因此推测该变异是患者发病的遗传学病因。患者表现肌无力等典型的DMD症状，没有明显的Paganini-Miozzo综合征相关症状。本病例的明确诊断，提示结构重排破坏DMD基因也是导致DMD重要原因之一；常规遗传学...     

窖蛋白-3及其与人类肌肉疾病的关系

窖蛋白-3(caveolin-3,Cav-3)是整合在窖上的肌细胞特异性蛋白质。人cav-3基因定位于3p25,其主要突变包括跨膜区的错义突变及支架区的染色体微缺失,所导致的表现型包括肢带型肌营养不良(1imb-girdle muscular dystrophy-1C,LGMD-1C)、杜氏肌营养不良(Duchenne muscular dystrophy,DMD)、自发性和家族性高CK血症(hyper CKemia,HCK)、末端肌病(distal myopathy,DM)和波形肌肉疾病(rippling muscle disease．RMD)等。Cav-3不仅与肌肉营养不良症相关,也是维持心脏正常功能的必要因素。     

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.     

两个DMD cDNA片段在进行性肌营养不良症基因诊断中的应用

本文使用了缺失热点区的两个DMD cDNA片段1b-2a及8为探针检测Duc-henne型及Becker型肌营养不良(DMD/BMD)患者的基因缺失。在34例不相关患者中分别检测到5例及8例基因片段缺失,缺失检测率分别为14.7％及23.5％,总检出率为38.2％。结果表明,中国肌营养不良患者的基因缺失也不是随机分布的,主要集中于基因中心附近,其次在基因5′侧。     

DMD基因治疗中AAV载体优化的研究进展

杜氏肌营养不良症(Duchenne muscular dystrophy, DMD)是一种由抗肌萎缩蛋白(dystrophin)编码基因突变引起的进行性肌肉萎缩疾病,机体无法产生正常功能的dystrophin,最后由呼吸肌或心肌衰竭引发成年早期死亡。全身系统性基因治疗是最大程度治疗DMD的最有效方法。腺相关病毒(adeno-associated virus vector, AAV)是当前极具应用前景的基因治疗载体,在多种遗传性疾病的临床治疗中取得了前所未有的成功。然而,针对DMD的AAV载体基因治疗仍面临巨大挑战,包括无法容纳dystrophin全长编码序列,载体的肌肉靶向性不足且大量滞留在肝脏,AAV在体内大幅降解严重降低转导效率,机体对AAV衣壳蛋白产生免疫反应,AAV规模化制备的实施难度,以及安全性风险等。AAV载体优化旨在利用基因工程技术改变其相关特性以定制适用于DMD基因治疗的最佳载体。本文综述了AAV载体优化的方向及策略,以期跨越DMD基因治疗的障碍。     

女性Duchenne/Becker型肌营养不良症携带者发病机理的研究进展

Duchenne/Becker型肌营养不良(DMD/BMD)是一类常见的X连锁隐性遗传病,多见于男性患者,女性携带者一般不发病,因为女性体内会发生随机的X染色体失活,而使体内呈现镶嵌型。目前,越来越多的文献报道DMD/BMD女性携带者发病的病例,其症状有轻有重,但发病机制尚不明了,大多数研究认为与X染色体的偏斜失活有关,即携带DMD突变的X染色体异常活化,使正常DMD基因弱或无表达,从而无法生成正常功能的dystrophin蛋白,表现为DMD/BMD。本文主要综述了X偏斜失活与DMD女性携带者发病相关性的研究进展。     

Dystrophin基因3-7号外显子缺失后下游新翻译区启动与BMD关系的生物信息学分析

目的:研究dystrophin基因3-7号外显子缺失后下游新翻译区启动与贝克氏肌营养不良(Becker muscular dystrophy,BMD)的关系及可能机制.方法:用生物信息学方法对dystrophin基因3-7号外显子缺失后可能的启动子、开放阅读框、翻译起始位点、蛋白疏水性性质改变及重要结构域进行分析.结果:3-7号外显子缺失后,起始于正常肌肉启动子的转录体,其翻译阅读框提前终止,但在8号外显子内可能启动一个新的翻译阅读框;内含子2或7里可能含有类似启动子的元件,也可能导致8号外显子内翻译区的启动.新阅读框编码的蛋白仍然保留有重要的功能区,患者可以表现为BMD.结论:dystrophin基因3-7号外显子缺失后下游新翻译区仍有存在启动的可能,患者可以表现为BMD表型.     

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.     

Proportion and pattern of dystrophin gene deletions in North Indian Duchenne and Becker muscular dystrophy patients

Population-based variations in frequency and distribution of dystrophin gene deletions have been recognized in Duchenne/Becker (DMD/BMD) muscular dystrophy patients. In the present study, DNA samples from 121 unrelated DMD/BMD patients from North India were analyzed for deletional studies with multiplex PCR and Southern hybridization. A total of 88 (73%) patients showed intragenic deletions in the dystrophin gene. The observed proportion of gene deletions is relatively high, particularly compared with that of Asian counterparts. However, the distribution of breakpoints across the gene does not show significant variations. Received: 5 June 1996 / Revised: 4 September 1996     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchenne muscular dystrophy

To compare the frequency and distribution of rearrangements in the dystrophin gene in Duchenne muscular dystrophy (DMD) between Japanese DMD patients and those in North America and Europe, Southern blot analyses of the dystrophin gene were carried out in 88 probands classified as DMD. Gene rearrangements were found in 61 (69%) subjects, and they were composed of partial gene deletions in 53 (60%) probands and partial duplications in 7 (8%) probands. A total deletion of the gene was found in 1 (1%) patient. Among 53 patients with deletions, 34 (64%) had breakpoints between introns 44 and 52 and 7 (13%) had breakpoints between introns 2 and 11. Both the frequency and the distribution of gene rearrangements found in this study were similar to those reported in North America and Europe. These data suggest that there are no ethnic or racial differences in the frequency and distribution of rearrangements thought to be caused by similar mechanisms in the dystrophin gene in all human racial groupings.     

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec

Summary We have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.     

Detection of gene deletions in Chinese patients with Duchenne/Becker muscular dystrophy using CDNA probes and the polymerase chain reaction method.

One hundred thirty-eight patients with Duchenne/Becker muscular dystrophy (DMD/BMD) were screened with complete cDNA probes and the multiplex polymerase chain reaction (mPCR) amplification of 18 pairs of oligonucleotide primers. Eighty-six deletions and 4 duplications were detected, the deletion frequency being 62.3%. Eighty-two deletions were detected with the two sets of primers described by Chamberlain et al. and Beggs et al, which was 95.4% of deletions detected by complete cDNA probes. Consistent with the deletion locations described previously, the deletions of dystrophin gene in Chinese individuals are clustered mainly in two high-frequency deletion regions of exons 44-52 (68.6%) of 3' side of the gene central regions and exons 1-19 (26.7%) in the 5' side. The distribution of deletions in dystrophin gene is associated with the phenotype of DMD/BMD. In the 25 cases with in-frame deletions, 15 deletions located in the region of exons 2-47 were milder BMD and intermediate patients, as the location of deletions was not the important region of the dystrophin gene.     

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.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.