抗肌萎缩蛋白基因非缺失/重复突变引起的Becker型肌营养不良症的研究

目的 探讨Becker型肌营养不良症(Becker muscular dystrophy,BMD)的基因突变类型,增加对抗肌萎缩蛋白基因非缺失/重复突变引起BMD的认识.方法 收集2例BMD患者的临床资料,应用多重连接依赖式探针扩增(multiplex ligation-dependent probe amplification assay,MLPA)方法对抗肌萎缩蛋白基因进行分析,并对其肌肉进行苏木素-伊红(hematoxylin-eosin,HE)染色、抗肌萎缩蛋白(dystrophin)染色及电镜检测.结果 2例患者经MLPA方法检测抗肌萎缩蛋白基因均呈非缺失/重复突变类型,肌肉活检光镜和电镜均呈肌营养不良改变.例1患者染色示肌膜dystrophin大部分呈不连续弱阳性,部分为阴性.例2患者染色示肌膜dystrophin-C为阴性,dystrophin-N为阳性.结论 对于抗肌萎缩蛋白基因非缺失/重复突变的临床症状较轻的患者,进行肌肉活检和抗肌萎缩蛋白免疫组化将有助于明确诊断BMD及判断其预后.

Abstract：

Objective To identify potential mutations in patients featuring Becker muscular dystrophy (BMD) and to enhance the understanding of non-deletion/duplication mutations of the dystrophin gene causing BMD. Methods linical data of two patients affected with BMD were collected. Potential mutations in the dystrophin gene were screened with multiplex ligation-dependent probe amplification assay (MLPA). Biopsied muscle samples were examined with HE staining, immnostaining with anti-dystrophin antibody, and electronic microscopy. Results MLPA assay suggested that both cases were probably due to non-deletion/duplication mutations of the dystrophin gene. Light and electronic microcopy of skeletal muscle biopsies confirmed dystrophic changes in both patients. For patient A, immunostaining showed non-contiguous weak staining for most parts of sarcolemma. For patient B, immunostaining showed positive result with N-terminal anti-dystrophin antibody and negative result with C-terminal anti-dystrophin antibody. Conclusion For patients with mild phenotypes but without dystrophin gene deletion/duplication, muscle biopsy and immunochemistry are helpful for diagnosis and prognosis.     

DMD患者骨骼肌抗肌萎缩蛋白表达与临床病理改变

目的探讨抗肌萎缩蛋白（dystrophin）免疫组织化学检查的临床价值及与Duchenne型肌营养不良（DMD）临床病理改变之问的相关性。方法通过组织学观察和免疫组织化学方法，对36例DMD患者骨骼肌dystrophin的表达情况、临床表现和肌肉病理改变进行观察分析。结果发现25例年龄在4岁以上的患儿多有比较典型的DMD临床表现，而11例4岁以下患儿症状比较轻。肌肉病理显示15例早期改变，17例中期改变，4例晚期改变，病理改变的严重程度与年龄相关。免疫组化染色显示36例患者的肌肉标本均有严重的dystrophin缺失，其中9例完全缺失，10例部分肌纤维膜有微弱着色，17例极少数肌纤维膜清楚着色，dystrophin的表达分级与病理改变分期及年龄无明显相关。结论检查dystrophin在肌纤维膜上的表达对DMD具有特异性诊断价值，但临床病理改变的严重程度主要与年龄和病程有关。     

抗肌萎缩蛋白的疏水结构与DMD／BMD的关系

用Ｇｅｎｅｐｒｏ程序在计算机上分析抗肌萎缩蛋白的疏水性和亲水性，首次发现该蛋白存在４个疏水肽段，分别位于第９５～１２０，１９９０～２０１０，２４３８～２４９３和３１５０～３３００位氨基酸。其中第３疏水肽段（２４３８～２４９３位氨基酸）的疏水性最强，由抗肌萎缩蛋白基因第５１号外显子碥码，称之为缺失热区疏水肽段。通过对６５例整码缺失的ＤＭＤ和１３６例整码缺失的ＢＭＤ病例分析，７５％（４９例）的ＤＭＤ患者缺失热区疏水肽段消失，９８％（１３３例）的ＢＭＤ患者缺失热区疏水肽段存在。提示缺失热区疏水肽段是抗肌萎缩蛋白的一个重要结构功能区。     

脐血干细胞移植治疗假肥大型肌营养不良症

目的比较假肥大型肌营养不良症(Duchennemusculardystrophy,DMD)患者经脐血干细胞移植治疗前后其肌肉再生、抗肌萎缩蛋白表达和运动功能的改变;以及评价治疗的安全性。方法对1例经基因分析和肌肉活检及抗肌萎缩蛋白检测确诊的、已丧失行走能力的DMD患儿,经HLA配型,在脐血库中寻找到一个全相合的脐血供体。采用白消安+环磷酰胺+兔抗胸腺淋巴细胞球蛋白预处理后进行异基因脐血干细胞移植;术后采用环孢素A和骁悉方案预防移植物抗宿主反应(graftversushostreaction,GVHD)。同时定期检测原发病的生化指标如血清肌酸激酶(creatinekinase,CK)、造血重建的植入证据(血型转变、肌肉和血液系统的聚合酶链反应短串联重复序列分析)、缺陷基因是否纠正、新生肌肉是否出现、肌肉中抗肌萎缩蛋白是否表达和运动功能是否改善。结果(1)中性粒细胞在脐血干细胞移植后第15天(+15天)达到0.5×109/L,白细胞在+25天达正常水平;血小板于+22天达到20×109/L;血红蛋白维持于85～100g/L。术后140天骨髓穿刺提示三系生长旺盛;(2)移植后140天血型转为供体AB型。至今没有出现移植物抗宿主反应。(3)术后18天、30天、43天、55天、74天、233天患者外周血DNA和术后140天、183天、235天骨髓细胞DNA经PCRSTR检测为供者独立植入;(4)患儿术后60天取外周血做基因分析,显示19号缺失的外显子得到完全纠正,患儿转变为正常基因型;(5)患儿在移植后75天的肌肉活检可见新生肌管形成,抗肌萎缩蛋白免疫组化呈弱阳性,少数为强阳性反应,DNA分析:供者基因DNA占1%～13%;移植后126天抗肌萎缩蛋白免疫组化检测显示阳性的肌纤维明显增多,供者基因DNA上升至2.5%～25%;(6)患儿血清CK从移植治疗前的5735U/L降至274U/L;(7)术后100天体检发现患儿肌力略有改善,肢端温暖。结论异基因脐血干细胞移植治疗DMD,可在移植后短期内重建造血功能、血清CK显著下降、肌肉抗肌萎缩蛋白表达,患儿运动有所改善,提示造血干细胞移植将有益于DMD的治疗。     

假肥大型进行性肌营养不良的诊断与遗传分析

目的总结假肥大型进行性肌营养不良患者的临床特征、病因及病理变化,提高其诊治水平。方法对患者进行肌电图、磷酸肌酸激酶浓度、肌肉活体组织、智力、神经反射等项检查,收集完整的家系资料进行遗传分析,判断致病基因携带者并评估再发风险。结果得到一假肥大型进行性肌营养不良的家系,并进一步确定其X连锁隐性遗传的遗传方式,明确了患者的表型特征,总结了目前的诊治方法,给与家系中相关人员以必要的遗传指导。结论假肥大型进行性肌营养不良是抗肌萎缩蛋白基因发生突变所致,目前应用PCR的相关技术、变性高效液相色谱结合测序技术可对突变基因进行筛查,从而预防患者的出生,对该病的治疗已取得了一定的进展。     

Duchenne肌营养不良基因缺陷及基因治疗

Duchenne肌营养不良(DMD)是常见的神经肌肉遗传病之一,由于骨胳肌肌膜上的抗肌萎缩蛋白(dystrophin)完全或部分缺失引起.本文介绍了dystrophin的结构和功能,对DMD基因治疗的目的基因,基因治疗方式(包括病毒载体和非病毒载体),基因转染途径作了较为全面的介绍,指出腺相关病毒载体介导的基因治疗及干细胞移植是有希望的治疗方向,经全身途径使目的基因广泛转染骨胳肌并实现心肌和膈肌的转染,是基因治疗研究的难点.     

进行性肌营养不良症116例临床与家系分析

进行性肌营养不良症是一组原发于肌肉组织的遗传性疾病，临床上主要表现为进行性加重的肌肉萎缩和无力。但其病因和发病机制目前还未十分清楚。我们对１９８３１９９８年间，证实为肌营养不良症的１１６例进临床分型，家族遗传史及治疗探讨如下。病例与家系一般资料：本组１１６例，男１０２例，女９例，男：女为１２－８９：１。年龄：６５８岁，６岁，４５例；１０岁，３９例；２０岁，１４例；３０岁，１０例；４０岁，６例；５０岁，２例。共中６２０岁８４例，占７２－４％。家族史：１１６例中有阳性家族史１８个例…     

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.     

Mutation analysis in Duchenne and Becker muscular dystrophy patients from Bulgaria shows a peculiar distribution of breakpoints by intron

For the first time in Bulgaria, a deletion/duplication screening was performed on a group of 84 unrelated Duchenne/Becker muscular dystrophy patients, and the breakpoint distribution in the dystrophin gene was analyzed. Intragenic deletions were detected in 67.8% of patients, and intragenic duplications in 2.4%. A peculiar distribution of deletion breakpoints was found. Only 13.2% of the deletion breakpoints fell in the “classical” hot spot in intron 44, whereas the majority (>54%) were located within the segment encompassing introns 45–51, which includes intron 50, the richest in breakpoints (16%) in the Bulgarian sample. Comparison with data from Greece and Turkey points at the probable existence of a deletion hot spot within intron 50, which might be a characteristic of populations of the Balkan region. © 1996 Wiley-Liss, Inc.     

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.     

肌特异性miRNA在肌细胞分化过程及肌营养不良中的表达

目的:观察肌特异性miRNA(miR-1,-133a和-206)在肌细胞增殖和分化过程中的变化,探讨肌特异性miRNA与肌营养不良的关系。方法:培养小鼠成肌细胞C_2C_(12)并诱导分化成熟,应用实时荧光定量PCR(q-PCR)分别检测C_2C_(12)在增殖期和分化期(1、3、5 d)miR-1、-133a、-206的变化;免疫组织化学法筛选dystrophin缺失型营养不良病例(DMD),q-PCR检测miR-1、-133a、-206在DMD肌组织中的表达情况。结果:增殖期C_2C_(12)细胞的miR-1、-133a、-206表达量较低,分化期3者表达均升高,并随着成肌细胞分化时间的延长,miRNAs表达显著升高,其中miR-1表达升高最明显;筛选的10例DMD标本均存在不同程度的dystrophin蛋白表达减弱或缺失,与非特异性改变肌组织对比,miR-1、-133a、-206表达均升高,其中miR-206在DMD)患者肌组织的表达水平显著升高。结论:miR-1、-133a、-206均能促进成肌细胞的分化,其中miR-206在肌营养不良的病变过程中可能发挥重要作用。     

Duchenne muscular dystrophy and spinal muscular atrophy type I segregating in the same family

We report on a family with two severe neuromuscular diseases: Duchenne muscular dystrophy (DMD) and acute infantile spinal muscular atrophy (SMA I). One boy has DMD, and his brother died of SMA I at 11 months of age. Both boys had received the same DMD allele from their mother. Analysis of dystrophin by immunohistochemistry and Western blot showed complete lack of dystrophin in both brothers. The mother had a partial deficiency of dystrophin. The boy with SMA I had increased levels of creatine kinase in serum, compatible with DMD, but the muscle biopsy and post-mortem examination of the spinal cord showed the typical changes of SMA I. There were no cytogenetic abnormalities explaining the occurrence of both DMD and SMA I in this family. Molecular genetic prenatal diagnosis of DMD and SMA I, using analysis of RFLPs and dinucleotide repeats, has been performed in one foetus in the family. The results showed that the foetus had a high risk of developing SMA I. An abortion was planned but the pregnancy was terminated by miscarriage.     

Variable dystrophin expression in different muscles of a Duchenne muscular dystrophy carrier

The majority of Duchenne muscular dystrophy (DMD) female carriers show dystrophin immunostaining abnormalities, although a significant proportion of clinically non-manifesting carriers are normal following this analysis. We had the opportunity to study dystrophin immunostaining in two different muscles, the vastus lateralis and the rectus abdominis of a possible DMD carrier. While the vastus showed normal dystrophin immunostaining, pathological staining was detected in her rectus abdominis. These findings seem to indicate that dystrophin expression can vary in different muscle groups of a DMD carrier. The implications of these findings in DMD carrier detection and possible dystrophin function are discussed.     

Relatively low proportion of dystrophin gene deletions in Israeli Duchenne and Becker muscular dystrophy patients

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55–65%). Seventy-eight percent of the deletions were confined to exons 44–52, half of these to exons 44–45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. © 1994 Wiley-Liss, Inc.     

ERG phenotype of a dystrophin mutation in heterozygous female carriers of Duchenne muscular dystrophy

PURPOSE: Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD). DMD is associated with an abnormal electroretinogram (ERG) if the mutation disrupts the translation of retinal dystrophin (Dp260). Our aim was to determine if incomplete ERG abnormalities would be associated with heterozygous carriers of dystrophin gene mutations. METHODS: Ganzfeld ERGs were obtained under scotopic and photopic testing conditions from a family which includes the heterozygous maternal grandmother, the heterozygous mother, and her children, two affected boys and dizygotic twin sibs, an unaffected male and heterozygous female. Southern blot analyses were done to characterise the dystrophin mutation. RESULTS: The dystrophin gene was found to contain a deletion encompassing exon 50. The ERGs in the two affected boys were abnormal, consistent with the DMD ERG phenotype. Serial ERGs of the heterozygous females were abnormal; however, they were less severely affected than the DMD boys. The ERG of the female sib showed a greater abnormality than her mother and maternal grandmother. The unaffected twin had a normal ERG. CONCLUSIONS: The ERG shows abnormalities associated with carrier status in this family with a single exon deletion. A large study of confirmed obligate carriers is planned to clarify further the value of the ERG in detecting female heterozygous carriers of dystrophin gene mutations.     

Additional dystrophin fragment in Becker muscular dystrophy may result from proteolytic cleavage at deletion junctions

Becker muscular dystrophy is usually caused by intragenic dystrophin gene deletions that result in production of an internally deleted protein. Previous studies have detected what appears to be a unique dystrophin degradation product that appears only in muscle biopsies from patients with Becker muscular dystrophy. This dystrophin fragment is always seen in addition to the “full-size” dystrophin of the expected size for a given gene deletion. It is only found in biopsies from patients with mutations in the deletion-prone region encompassing exons 45–53, but it does not appear to correlate with any observable phenotype at the clinical level. By correlating the size and locations of dystrophin gene deletions with the size of this degradation product, together with use of region-specific dystrophin antisera, we find that proteolytic cleavage may occur at the deletion breakpoints, perhaps due to alterations of the secondary and/or tertiary structures of the protein. This cleavage results in loss of the carboxy-terminal domains that are thought to be important for interactions between dystrophin and other membrane-bound proteins. © Wiley-Liss, Inc.     

Intragenic deletions in 164 boys with Duchenne muscular dystrophy (DMD) studied with dystrophin cDNA

DNA from 164 unrelated Duchenne muscular dystrophy patients was screened with cDNA probes from the dystrophin gene. Molecular deletions were demonstrated in 82 (50%) subjects. Sixty-two deletions (76%) were detected using cDNA probes Cf56a (cDNA 8) and Cf56b (cDNA 6-7) which map to the centre of the gene, while 22 deletions (27%) mapped to the 5' end of the gene. In three subjects, the deletion extended from the 5' end to the centre of the gene. One deletion was identified by probe 47-4 (cDNA 5b-7) alone. In six of the deletions, junction fragments of altered size were observed. Using the three cDNA probes, RW2kb, Cf56a (cDNA 8) and Cf56b (cDNA 6-7), 99% of the deletions were detected. This will have implications for prenatal diagnosis in deletion families. Unlike Becker muscular dystrophy, where the deletions are more homogeneous, the deletions in the present study were heterogeneous both in size and position. No correlation between intelligence and either site or extent of deletion was found.