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.     

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。     

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。     

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。     

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...     

CD133^＋干细胞移植——肌营养不良治疗的重大突破

进行性假肥大性肌营养不良（DMD）是进行性肌营养不良病的一种最常见类型,主要影响男性X性连锁隐性遗传性肌肉疾病,以缓慢进行性加重的对称性肌无力和肌萎缩为主要特征。大多数病例有明确的家族史。其主要的病理改变是慢性骨骼肌细胞变性、坏死,出现肌细胞萎缩与代偿性增大相嵌分布的典型表现。     

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.     

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。     

DMD的ATP酶组织化学研究

目的观察DMD(Duchenne muscular dystrophy)患者病变肌肉的酶组织化学病理改变,加深对其发病机制和预后的认识.方法对5例Duchenne型肌营养不良症患者进行肌肉活检,标本经处理,分别在HE染色、改良Gomori三色染色以及ATP酶染色(pH9.4和pH4.3)下进行观察,研究病变肌肉酶组织化学的病变特征及其意义.结果在DMD组织中肌纤维大小不等,肌纤维圆形化,炎性细胞浸润,并可见到不透光纤维.在再生肌纤维中可见到IIc型纤维.结论 DMD病变组织中纤维坏死显著,伴有不同程度的再生,故临床症状严重,病程短,预后较差.     

东北地区抗肌营养不良蛋白基因缺失的研究及应用

为了解东北地区Duchenne／Becker型肌营养不良症患者基因缺失的分布及进行产前基因诊断,用12对引物以多重PCR法检测120例DMD／BMD患者,并分析缺失型患者dystrophin基因的断裂点分布及各引物优化组合,并将高危男性胎儿行缺失检测。结果表明,缺失检出率为49．2％,66．4％的断裂点位于内含子44～52内,以内含子50为最多(14．8％),4对外显子引物的优化组合为外显子48、51、45和8,总检出率为41．7％;29例高危胎儿中9例男性胎儿为缺失型,缺失位点与先证者相同。通过首次对我国东北地区DMD／BMD患者筛查缺失发现dystrophin基因缺失主要分布于两个热区内,与国内其他地区比较外显子8附近区域可能是该地区缺失断裂的高发区;内含子44～52高度不稳定,其中内含子44的稳定性要高于中央缺失热区的稳定性,内含子50的不稳定性存在地区及种族差异;引物优化组合为检测患者及产前基因诊断提供了捷径,尤其是对散发家系是可行的并且有其优越性。     

Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

Summary We examined the morphological expression of dystrophin in the intrafusal muscle fibers in skeletal muscle from normal human and Duchenne muscular dystrophy (DMD) patients, using antisera against the N-terminal and C-terminal regions of dystrophin. The intrafusal fibers of normal muscle express dystrophin on their cell surface membrane, but those of DMD muscle do not.Abbreviation DMD Duchenne muscular dystrophy     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin.

Antibodies directed against the amino- and carboxy-terminal regions of dystrophin have been used to characterize 25 Duchenne muscular dystrophy (DMD), two intermediate, and two Becker muscular dystrophy (BMD) patients. Western blot analysis revealed an altered-size (truncated) immunoreactive dystrophin band in 11 of the 25 DMD patients, in one of the two intermediate patients, and in both BMD patients, when immunostained with antiserum raised against the amino terminus of dystrophin. None of the DMD or intermediate patients demonstrated an immunoreactive dystrophin band when immunostained with an antiserum specific for the carboxy terminus of the protein. In contrast, dystrophin was detected in both BMD patients by the antiserum specific for the carboxy terminus. Quantitative studies indicated that the relative abundance of dystrophin in patients with a severe (DMD), intermediate, or mild (BMD) phenotype may overlap, therefore suggesting that differential diagnosis of disease severity based entirely on dystrophin quantitation may be unsatisfactory. Our results suggest that a differential diagnosis between DMD and BMD would benefit from examination of both the N terminus and C terminus of the protein, in addition to measurements of the relative abundance of the protein.     

A Sensitive,Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.     

Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy

Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls.We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials.Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

Cell transplantation and gene therapy in muscular dystrophy.

Duchenne's muscular dystrophy (DMD), which affects 1/3500 live male births, involves a progressive degeneration of skeletal and cardiac muscle, leading to early death. The protein dystrophin is lacking in DMD and present, but defective, in the allelic, less severe, Becker muscular dystrophy and is also missing in the mdx mouse. Experiments on the mdx mouse have suggested two possible therapies for these myopathies. Implantation of normal muscle precursor cells (mpc) into mdx skeletal muscle leads to the conversion of dystrophin-negative fibres to -positive, with consequent improvement in muscle histology. Direct injection of dystrophin cDNA into skeletal or cardiac muscle also gives rise to dystrophin-positive fibres. Although both appear promising, there are a number of questions to be answered and refinements to be made before either technique could be considered possible as treatments for myopathies in man.