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。     

免疫荧光检测抗肌萎缩蛋白诊断肌营养不良症的临床应用

目的 采用免疫荧光技术对Duchenne型肌营养不良症（Duchenne muscular dystrophy,DMD),Becker型肌营养不良症（Becker muscular dystrophy,BMD)，面肩肱型肌营养不良症（facioscapulohumeral muscular dystrophy,FSHD)以及神经性肌萎缩患者骨骼肌细胞膜的dystrophin蛋白进行检测，为临床诊断、分类肌营养不良症提供简便的实验方法。方法 对47例患者选择3种dystrophin 的鼠抗单克隆抗体、羊抗和兔抗多克隆抗体，分别进行免疫荧光技术检测。结果 16例DMD患者均为阴性染色；11例BMD患者为弱阳性染色；10例FSHD和10例神经性肌萎缩患者均为阳性染色。结论 检测肌营养不良症患者骨骼肌膜dystrophin蛋白，有助于肌营养不良症的临床诊断和分型。     

非缺失型DMD/BMD家系中基因携带者的短串联重复序列多态性连锁分析

目的 准确检出非缺失型杜氏／贝氏进行性肌营养不良症（Duchenne/Becker muscular dystrophy,DMD/BMD)家系中女性携带者，为产前基因诊断提供信息。方法 利用dystrophin基因中5个短串联重复序列多态位点（STR－44，STR－45，STR－49，STR－50和5'-DysⅡ）的特异寡核苷酸引物进行PCR扩增，经聚丙烯酰烯酰胺凝胶电泳分离，DNA片段的多态性用连锁分析其家系中各成员的基因单体型。结果 连锁分析了8个家中26名女性亲属基因型，检出16名女性DMD／BMD携带者和10名非携带者。结论 5个短串联重复序列多态位点的联合连锁分析，可较准确而简便地检出DMD／BMD家系中的女性携带者。     

应用多个微卫星DNA位点进行Duchenne/ Becker肌营养不良症携带者的检测

目的　筛查和确定 Duchenne/ Becker肌营养不良症 ( Duchenne/ Becker muscular dystrophy,DMD/ BMD)家系的女性成员中的致病基因携带者与正常者 ,为进一步行产前诊断或植入前遗传学诊断提供信息。方法　用 PCR方法对 dystrophin基因第 4 4、4 5、4 9和 5 0内含子以及 5′DMD 的短串联重复序列( short tandem repeats,STR)扩增 ,然后进行基因扫描、软件分析 ,对 4个 DMD/ BMD家系中 2 7个成员的这 5个微卫星 DNA位点的多态性进行连锁分析。结果　在 4个家系 17名女性成员中 (有 1例女性 DMD患者 ) ,系谱和 STR多态性分析结果均符合的 DMD基因肯定携带者有 6名 ;单纯根据 STR多态性连锁分析结果确诊为 DMD基因携带者的女性成员有 5名 ,确诊为正常女性成员有 5名。在这 5个 STR位点中 ,最具多态性的位点是 STR- 4 9,多态性最少的位点是 STR- 5 0。结论　短串联重复序列多态性结合基因扫描能快速、准确、客观地检出 Duchenne/ Becker型肌营养不良症的女性携带者     

克隆缺失连接片段——一种基于PCR技术的缺失型假肥大型肌营养不良症的携带者检测

目的 依据dystrophin基因缺失后断端重接可形成一段变异的DNA序列,提出一种利用缺失连接片段进行缺失型假肥大型肌营养不良症携带者检测的新方法.方法 实验以来自广东省肇庆地区的一个Becker型肌营养不良(Becket muscular dystrophy,BMD)家系为研究对象,其中2例确诊的男性BMD患者,3例待诊的女性携带者,1例待诊的人工流产绒毛.先证者经外显子PCR检测确定第3～5外显子缺失,随后采用PCR步移法在相应内含子设计引物定位断裂点的位点,最后利用靠近断裂点设计的引物直接对家系的6例基因组DNA进行缺失连接片段的PCR扩增和测序.结果 6例基因组DNA均扩增出阳性的产物片段且连接片段的测序序列完全一致,绒毛的性别诊断结果为女性,可以确诊本家系中的3个女性和流产绒毛均为缺失型BMD携带者.结论 作者成功地将整个家系患者和携带者的缺失连接片段进行克隆和测序分析,实现了利用缺失连接片段对缺失型假肥大型肌营养不良症携带者进行准确基因诊断的设想,同时对在产前诊断上的应用前景进行了探讨.     

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。     

进行性肌营养不良患者视网膜眼电图表型与临床分型及基因型的关系

目的 研究进行性肌营养不良（Duchenne/Becker muscular dystrophy,DMD/BMD)患者视网膜眼电图（electroretinogram,ERG)表型与临床分型以及基因型的关系。进一步探讨不同基因型的DMD患者抗肌营养不良蛋白（dystrophin)及其同源蛋白在视网膜上的表面爱功能,揭示DMD出现ERG异常的分子机理,方法 用11对引物对22例临床确诊的DMD／BMD患者作三步多重PCR进行基因缺失分析,并行ERG检查,结果 DMD／BMD患者ERG改变与临床分型及病情严重程度无关,与DMD／BMD的基因型有关,基因中央区缺失型的ERG异常率明显高于基因非缺失型,结论 DMD／BMD的ERG改变与DMD基因突变位点有关,可能DP260转录启动子与视网膜电信号的传导关系最密切。     

一例罕见的X连锁鱼鳞病合并杜氏肌营养不良患儿家系的遗传学分析CSCD

目的对1例X连锁鱼鳞病(X-linked ichthyosis,XLI)合并杜氏肌营养不良(Duchenne muscular dystrophy,DMD)的患儿及其家系成员进行遗传学分析,明确其致病原因。方法采集先证者及其父母的外周血样,提取DNA,对先证者进行全外显子测序(whole exome sequencing,WES)。应用多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)分别检测患儿的STS与DMD基因。结果检测发现患儿携带STS基因半合子缺失及DMD基因第48~54外显子区域的半合子缺失。结论先证者同时患有XLI与DMD,非常罕见。     

假肥大型肌营养不良症的基因型与临床表型关系分析

目的 分析中国人群假肥大型肌营养不良症基因型和临床表型之间的关系.方法 临床诊断Duchenne型肌营养不良症(Duchenne muscular dystrophy,DMD)和Becker型肌营养不良症(Becketmuscular dystrophy,BMD)患者,应用多重探针连接依赖性扩增技术进行DMD基因检测,将基因检测结果与临床诊断比较进行统计分析.结果 280例DMD基因缺失或重复患者中,DMD患者238例(85.0%),BMD患者35例(12.5%),中间型患者7例(2.5%).DMD或BMD符合阅读框原则的有252例,占92.31%(252/273),不符合阅读框的有21例,占7.69%(21/273).DMD基因为整码突变而患者表现为DMD的有12例(12/273,4.40%),移码突变而患者表现为BMD的有9例(9/273,3.30%).7例中间型患者均为移码突变.结论 阅读框假说可以解释大约90%DMD或BMD基因型与表现型关系,部分移码突变患者表型为BMD可有助于了解该病的发病机制,为未来治疗提供理论依据.     

一个以肌痛为主要表现的Becker肌营养不良症家系的遗传学分析

目的:对1例以肌痛为主要临床表现的Becker肌营养不良症(Becker muscular dystrophy,BMD)的家系进行遗传学分析,并回顾分析以肌痛为主要症状的BMD患者的 DMD基因变异,以期为疾病的早期诊断提供依据。 方法:对患者进行临床资料采集并完善肌酶、肌电图等辅助检查,利用多重...     

Comparative analysis of PCR-deletion detection and immunohistochemistry in Brazilian Duchenne and Becker muscular dystrophy patients

We studied 48 patients with dystrophinopathies (29 Duchenne muscular dystrophy (DMD), 13 Becker muscular dystrophy (BMD), four possible carriers, one female with DMD, and one intermediate form, using polymerase chain reaction (PCR) analysis of muscle tissue for 20 exons and compared them with immunohistochemistry studies for dystrophin. Of these, 42 (87.5%) showed at least one intragenic deletion. Most of them (47.45%) involved exons 2 to 20. All BMD patients presented deletions on the dystrophin gene. The 29 patients with DMD showed abnormal dystrophin in immunohistochemistry studies, some with total absence (17/29), others with residual (3/29), and the remaining with scattered positive fiber (9/29). The majority of the 13 patients with BMD had abnormal immunohistochemistry studies with diffuse reduction in the majority of muscle fibers (10/13), a few with patch discontinuation in the sarcolemma (2/13), and one normal (1/13). The immunohistochemistry exam for dystrophin is still the gold-standard method for DMD/BMD diagnosis. An ethnic difference, the analysis of several exons, the sample size, and the use of muscle tissue could explain this high frequency of deletions in the dystrophin gene found in our cases.     

An isolated case of Duchenne muscular dystrophy (DMD) in a female with a deletion of DMD cDNA

An isolated case of Duchenne muscular dystrophy (DMD) in a female who has a deletion of the DMD locus is described. This patient was a 26-year-old woman born to unrelated, healthy parents. She was initially examined at age 6 because of a waddling gait. At age 15, pseudohypertrophy of calves and pes equinus were observed along with proximal muscular weakness and wasting. Her serum creatine kinase level was high and histological evidence of muscular dystrophy was apparent on muscle biopsy. The patient was ambulant at age 15 and progression of motor disability has been slow. Chromosomal studies revealed a normal karyotype, and mental retardation is moderate. DNA analysis at age 26 revealed that she has a deletion of DMD cDNA 8 mapped within Xp21 and is heterozygous for the deletion. Since diagnosis of DMD is now dependent on the evidence of mutation or deletion at Xp21, this patient is thought to have a form of DMD. Expression of the DMD gene in the heterozygous state might be due to random but unequal lyonization.     

Duchenne muscular dystrophy and myotonic dystrophy in the same patient

We report on the first patient identified with myotonic dystrophy and Duchenne muscular dystrophy (DMD). The family of the propositus had a strong history of myotonic dystrophy, and there was an intrafamilial pathological expansion of the responsible CTG repeat between the mildly affected mother (160 repeats; normal 27 repeats) and her more severely affected son (650 repeats), and his sister (650 repeats). The propositus was an isolated case of Duchenne muscular dystrophy with marked dystrophin deficiency in muscle biopsy. The patient was still ambulatory post age 16. Myotonic dystrophy could interfere to some extent with the progression of Duchenne dystrophy. However, other interpretations are possible. Twelve percent of dystrophin revertant fibers as observed by immunohistochemistry could be sufficient to ameliorate typical DMD clinical severity, or the patient may present a somatic mosaic. The pathophysiological interactions of these two unlinked disorders are discussed at the clinical and histopathological levels. © 1995 Wiley-Liss, Inc.     

Heterozygotic gene expression in endomyocardial biopsies a new diagnostic tool confirms the Duchenne carrier status

Summary Identification of the defective gene and the absent gene product dystrophin can substantiate the clinical evidence for manifesting X-linked Duchenne type muscular dystrophy (DMD). It is not always possible, however, to rule out definitely a clinically asymptomatic carrier status in question, since even in the proven carrier DNA analysis is often inconclusive, and multinucleated skeletal muscle fibers express a basically normal membrane dystrophin. To substantiate the value of endomyocardial biopsy as a new tool for detection of the DMD carrier status we examined an endomyocardial biopsy of a volunteer who met the clinical criteria of a DMD carrier. Dystrophin immunohistochemistry and western blot of her skeletal muscle biopsy were inconclusive, and polymerase chain reaction and cDNA analysis failed to locate directly the X-chromosomal defect. We observed a clearcut mosaic of dystrophin-positive and -negative mononucleated cardiac muscle cells, reflecting a heterozygote carrier status in her endomyocardial biopsy, whereas 20 controls were uniformely positive. The incidence of DMD (1:3000 males) and especially the 30% spontaneous mutation rate, still the major pitfall in DNA analysis, show the need for an additional diagnostic tool.Abbreviations DMD Duchenne type muscular dystrophy - PCR polymerase chain reaction - mAb monoclonal antibody Dedicated to Prof. Dr. N. Zöllner on the occasion of his 70th birthday     

Absence of correlation between skewed X inactivation in blood and serum creatine-kinase levels in Duchenne/Becker female carriers

The pattern of X inactivation in lymphocyte DNA was investigated in 107 Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers (102 asymptomatic and 5 manifesting carriers) and 117 normal female controls of different ages, with the aim: a) to analyze the pattern of X inactivation in blood DNA of a large number of DMD/BMD carriers as compared to normal female controls; b) to determine if there is a decrease in serum creatine kinase (CK) levels with age in obligate DMD/BMD carriers; c) to determine if there is a correlation between X-chromosome inactivation and serum CK among asymptomatic DMD/BMD carriers of different ages or with different clinical manifestations in symptomatic carriers. A high proportion of females showed extremely skewed X inactivation (>90 of one X preferentially inactivated), which was almost the same among carriers and normal controls (19 and 24, respectively). The mean serum CK was significantly greater among young (<20 years old) than adult (>20 years old) DMD/BMD carriers and it decreased significantly until age 20 with an apparent stabilization afterwards. No statistically significant correlation was found between the proportion of active X^DMD in blood and serum CK activity in DMD/BMD carriers although it was higher among those less than 20 years old. Our observations suggest that highly skewed X-chromosome pattern in blood (with preferential inactivation of the X^N chromosome) is not enough to predict that a young DMD carrier will develop muscular weakness. Am. J. Med. Genet. 80:356–361, 1998.     

Three cases of manifesting female carriers in patients with Duchenne muscular dystrophy

Duchenne muscular dystrophy usually affects males. However, females are also affected in rare instances. Approximately 8% of female Duchenne muscular dystrophy (DMD) carriers are manifesting carriers and have muscle weakness to some extent. We investigated the clinical features of 3 female patients with dystrophinopathy diagnosed by clinical, pathological, and genetic studies at our neuromuscular disease clinic. The onset age of manifesting symptoms varied (8-28 years). Muscle weakness grade varied as follows: patient 1 showed asymmetrical bilateral proximal upper and lower extremities weakness, patient 2 showed asymmetrical bilateral upper extremities weakness similar to scapulohumoral muscular dystrophy, and patient 3 had only bilateral asymmetric proximal lower extremities weakness. Two patients had familial histories of DMD (their sons were diagnosed with DMD), but the 1 remaining patient had no familial history of DMD. The serum creatine kinase level was elevated in all patients, but it was not correlated with muscular weakness. An electromyography study showed findings of myopathy in all patients. One patient was diagnosed with a DMD carrier by a muscle biopsy with an immunohistochemical stain (dystrophin). The remaining 2 patients with familial history of DMD were diagnosed by multiplex ligation-dependent probe amplification (MLPA). There were inconsistent clinical features in the female carriers. An immunohistochemical analysis of dystrophin could be useful for female carrier patients. Also, multiplex ligation-dependent probe amplification is essential for the diagnosis of a manifesting female carrier DMD in female myopathic patients because conventional multiplex PCR could not detect the duplication and is less accurate compared to MLPA.     

Duchenne/Becker muscular dystrophy in the family: have potential carriers been tested at a molecular level?

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease. After identification of the mutation in the index patient, family members can be reliably investigated. Carriers should be informed about their risk of having offspring with the disease and about their own risk for cardiomyopathy for which regular cardiac surveillance is recommended. In a small country like the Netherlands with well-organized genetic services, one would expect that most DMD families are adequately informed about the above mentioned risks for carriers. We have investigated whether women at risk had been tested at a molecular level. In the national Duchenne/Becker database 311 DMD and 99 Becker muscular dystrophy (BMD) patients had been registered up to 1 July 2009. These patients were asked to give information about the number of sisters and maternal aunts of the DMD/BMD patient and anything that was known about their genetic status and that of the mother. This information was compared with the information known at the genetic laboratory. Thirty-five of 104 adult sisters/maternal aunts of DMD patients with a 50% risk of being a carrier and 45 of 148 adult women with a 4.3% risk because of germ line mosaicism for DMD had not been tested by DNA analysis. Our study indicates that about one third of the potential carriers have not been tested. Given the possible far-reaching clinical consequences of being a carrier, further studies are needed to investigate the reasons why potential female carriers have not been tested.     

Utility of dystrophin and utrophin staining in childhood muscular dystrophy

To determine the utility of dystrophin and utrophin staining in the differential diagnosis of childhood muscular dystrophy. Fifty muscle biopsies of histologically confirmed cases of childhood muscular dystrophy, below 16 years of age, were stained immunohistochemically for dystrophin and utrophin. All the 30 muscle biopsies of patients with Duchenne muscular dystrophy (DMD) showed all or majority of muscle fibers deficient for dystrophin and positive for utrophin. In the 4 female DMD carriers there was mosaic pattern of staining for dystrophin and reciprocal positivity for utrophin. All the muscle biopsies of patients with other childhood onset muscular dystrophies were positive for dystrophin and negative for utrophin. This study shows that dystrophin staining differentiates DMD and DMD carriers from other childhood muscular dystrophies and utrophin staining is of no added value. Utrophin up-regulation may compensate for structural deficiency in dystrophic muscle.     

In utero fetal muscle biopsy for the diagnosis of Duchenne muscular dystrophy in a female fetus “suddenly at risk”

DNA methods to diagnose Duchenne muscular dystrophy (DMD) are not always informative, and we have published previously the first instance of in utero muscle biopsy to assess dystrophin in a male fetus having the same “X” as an affected sib. We present here a female fetus with a de novo X,1 translocation with breakpoint at Xp21, detected on amniocentesis for advanced maternal age. The translocation breakpoint placed her at high risk for DMD. In utero muscle biopsy at 20 weeks of gestation produced a specimen positive for dystrophin immunofluorescence indcating a likely normal fetus. The pregnancy was continued, and at term the baby girl was found to have normal serum creatine kinase levels, and was therefore unaffected with DMD. Our experiences add de novo Xp21 translocation to the indications for in uteromuscle biopsy for diagnosis of DMD. © 1993 Wiley-Liss, Inc.