两步多重聚合酶链反应对假肥大型肌营养不良的基因诊断

应用分子生物学技术，用９组寡核苷酸引物分两步多重聚合酶链反应扩增ｄｙｓｔｒｏｐｈｉｎ基因的９段脱氧核糖核酸序列。对３６例ＤＭＤ和４例ＢＭＤ进行基因诊断。首先用缺失率较高的５对引物扩增，检出缺失者１７例，再用４对引物扩增，检出缺失者２例。这样，９对引物多重ＰＣＲ总缺失率占受检患者的４７．５％，表明此法可检测出７９．１％左右有基因缺失的患者。实验结果提示，两步多重ＰＣＲ可用于ＤＭＤ／ＢＭＤ的基因诊断。     

两步多重聚合酶链反应对假肥大型肌营养不良的基因诊断

应用分子生物学技术，用９组寡核苷酸引物分两步多重聚合酶链反应（ｍＰＣＲ）扩增ｄｙｓｔｒｏｐｈｉｎ基因的９段脱氧核糖核酸（ＤＮＡ）序列。对３６例ＤＭＤ和４例ＢＭＤ进行基因诊断。首先用缺失率较高的５对引物扩增，检出缺失者１７例，再用４对引物扩增，检出缺失者２例。这样，９对引物多重ＰＣＲ总缺失率占受检患者的４７．５％，表明此法可检测出７９．１％左右有基因缺失的患者。实验结果提示，两步多重ＰＣＲ可用于ＤＭＤ／ＢＭＤ的基因诊断。文中从ＤＭＤ／ＢＭＤ的临床表型与基因缺失的关系上进行了比较、探讨。     

X连锁肌营养不良症14例基因缺失的研究

应用９对引物的多重扩增对新疆地区的１４例Ｘ连锁肌营养不良症（ＭＤ）患者进行了基因分析。结果发现其中７例（５０％）有至少一个基因片段的缺失，且缺失热点集中于外显子４５￣５１，这与国内外报道相近。同时发现，２例Ｂｅｃｋｅｒ型肌营养不良症的基因缺失均为一段，而５例Ｄｕｃｈｅｎｎｅ型肌营养不良症中的４例缺失各为２￣３段。缺失片段多少是否与症状严重程度之间存在在某种联系尚待研究论证。     

假肥大型肌营养不良症2例

1临床资料例1：16岁,男性,因＂进行性四肢无力8a,加重半年＂于2011-06-30入院。8a前无明显诱因开始出现四肢无力,初尚能行走,端碗持筷,但步行蹒跚,逐渐加重;半年前发展至行走不能,双上肢活动费力,持物不稳,并出现四肢肌肉萎缩,于外院考虑：肌营养不良,未予以特殊处理。体格检查：     

假肥大型肌营养不良症40例临床特征与基因诊断

目的对假肥大型肌营养不良症（DMD／BMD）患者总结其临床特征并进行基因诊断,以提高对DMD／BMD疾病的认识及诊断水平。方法对40例DMD／BMD患者临床特征进行总结包括临床表现、血清肌酶、肌电图及肌肉活检等,并应用18对引物多重PCR的方法对其进行Dystrophin基因缺失诊断。结果DMD／BMD为儿童期隐匿起病、缓慢进行性加重,以肌无力和肌萎缩为特点,主要选择性侵犯四肢近端肌、盆带肌、腰带肌等,可有肌肉假性肥大,有些患者可有智能减退和心肌损害;血清肌酶水平异常增高,肌电图示肌源性损害,肌肉活检呈肌病特征。基因诊断27例存在外显子片段缺失,13例未检测到缺失。结论识别DMD／BMD的临床特征有助于提高对其的诊断水平,多重PCR作为一种简便快速的诊断方法可对DMD／BMD患者进行基因诊断。     

假肥大型肌营养不良症基因治疗新进展

假肥大型肌营养不良症(DMD)的基因治疗涉及有关基因缺陷、启动基因、病毒载体、免疫系统的监控和载体介入骨骼肌的方法等。近来把研究焦点集中在辨别截短的、保留正常功能的抗肌萎缩蛋白(Dys)的翻译,设计一些微小型Dys cDNA以利于基因携带,并通过改进病毒载体携带基因功能,从而提高基因治疗DMD的效果并减轻免疫反应。基因修复、直接介入外源或上调utrophin也能改善DMD肌肉收缩功能。     

假肥大型肌营养不良患者dystrophin基因缺失断裂点的分子结构特点

目的分析假肥大型肌营养不良患者dystrophin基因缺失断裂点的分子结构特点，探讨dystrophin基因缺失的发生机制。方法以PCR步移法定位2例DMD患者基因断裂位点，克隆其缺失连接片段并测序，通过Pubmed文献检索获取既往55例缺失连接片段序列资料，对以上57例缺失连接片段5′端和3′端断裂点两侧的序列进行重复序列、基质附着区、TTTAAA序列以及基因缺失后修复方式的分析。结果57例缺失连接片段中40．4％断裂点位于重复序列，其中主要是L1元件和Alu元件；36．3％断裂点在邻近基质附着区5kb之内的区域；15．0％断裂点两侧50bp的范围内发现有，TTTAAA序列；基因缺失后修复的方式仅1例通过Alu元件同源连接，其余56例通过非同源末端连接进行修复，非同源末端连接以形成1～4bp的微小同源序列为主。结论重复序列、基质附着区、TTTAAA序列以及非同源末端连接修复机制均在一定程度上参与dystrophin基因的断裂重组。dystrophin基因缺失可能是由以上多种因素的综合作用所导致，染色体的物理结构可能在基因缺失中起主要作用。     

假肥大型肌营养不良症与氧自由基

Duchenne肌营养不良症(DMD)是一种X连锁隐性遗传性疾病,但对其发病机理仍不清楚.本文综述了近年来国内外有关DMD发病中氧自由基损害的研究进展.对自由基的特性,肌细胞破坏的机制及DMD抗氧化系统等方面作了阐述.     

迪谢内/贝克肌营养不良症基因缺失的分布

目的分析迪谢内／贝克肌营养不良症（ＤＭＤ／ＢＤＭ）基因缺失类型及其分布规律。方法采用全长ｃＤＮＡ探针和１８对引物多重聚合酶链反应（ｍＰＣＲ）检测１３８例ＤＭＤ／ＢＭＤ。结果用全长ｃＤＮＡ探针，ＤＮＡ印迹法检测出８６例患者基因缺失和４例重复，缺失率为６２．３％。用１８对引物ｍＰＣＲ检测出８２例缺失，占全长ｃＤＮＡ探针检出缺失的９５．４％。结论基因缺失的分布具有一定的规律性。８６例缺失主要集中在两个缺失热区内，其中５９例（６８．６％）分布于外显子４４～５２，相当于ｃＤＮＡ８和７的３′端４个外显子覆盖的区域内。２３例缺失（２６．７％）分布于５′端外显子１～１９，相当于探针１～２ａ和２ｂ～３的检测区域内。仅４例（４．７％）缺失分布于基因的中心区。基因缺失的类型及分布与表型有一定关系。     

假肥大型肌营养不良基因突变检测方法的应用比较

目的比较假肥大型肌营养不良基因突变各种检测方法的优缺点,为不同条件下选择最佳的检测方案提供借鉴。方法分别应用18对引物多重PCR和5×4DNA微阵列对30例假肥大型肌营养不良患者进行dystrophin基因(缺失)检测分析,并结合文献中的方法进行应用比较。结果 30例假肥大型肌营养不良患者中有21例至少存在一个外显子片段缺失,占总例数的70%;9例未被检测到缺失,点30%。末检测到全部缺失的患者。PCR、DNA微阵列检测结果一致。结论对于假肥大型肌营养不良患者及携带者可选择MLPA检测缺失和重复突变,对于阴性者再利用PCR联合DHPLC结合测序检测点突变。经济方案是先选择18对引物定量PCR或DNA微阵列检测常见外显子缺失和重复突变,对于阴性者再用MLPA检测其它外显子缺失和重复突变。对于可疑胎儿产前诊断行MLPA或PCR-STR连锁分析。     

Screening of gene deletions by polymerase chain reaction in Japanese patients with Duchenne muscular dystrophy

Summary Gene deletions were screened in 49 Japanese Duchenne muscular dystrophy patients from 43 families, using the polymerase chain reaction. Enzymatic amplification was carried out on six regions prone to deletion. Fifteen of 43 families (33%) had gene deletions in at least one of the six regions. This frequency was almost the same as that previously reported in Caucasians. The mid-part of the dystrophin gene was the location most frequently deleted. The frequency of deletion of the region encompassing exon 45 was higher in Japanese families (18.4%) than in Caucasians.     

实时荧光定量聚合酶链反应诊断面肩肱型肌营养不良

目的 建立面肩肱型肌营养不良(FSHD)的实时荧光定量PCR(FQ-PCR)检测方法.方法 常规酚-氯仿法抽提基囡组DNA,通过EcoR Ⅰ酶切及琼脂糖凝胶电泳,回收38 kb以下DNA作为模板,根据4号染色体上D424序列设计特异的引物和探针,对115例研究对象进行FQ-PCR检测,根据荧光曲线与阳性对照的比较判断结果.结果 16例已知EcoR Ⅰ片段大小的FSHD患者FQ-PCR检测结果为13例阳性,78名健康人检测结果除3例阳性外其余均为阴性,16例经临床诊断的新FSHD患者和5名高危者中分别有15例和3例检测结果为阳性.统计学分析FQ-PCR方法与传统印迹杂交方法(κ=0.765,P=0.002)及临床诊断(κ=0.844,P=0.000)之间的一致性,结果有统计学意义.结论 我们创建了以FQ-PCR技术对FSHD进行基因诊断的新方法.该方法能克服印迹杂交方法费时费力、有放射性污染的缺点,且能较好解决由于4q-10q易位及p13E-11探针结合部位缺失造成传统杂交基因诊断方法欠准确的问题,具有较好的临床应用价值.     

重复顺序多态性及基因剂量分析诊断迪谢内及贝克肌营养不良基因携带者

目的：在18个缺失型迪谢内及贝克肌营养不良家系中,比较有效检测基因携带者的方法。方法：用多重聚合酶链反应方法扩增dystrophin基因9个外显子,检测先证者有无外显子缺失;用聚合酶链反应方法扩增dystrophin基因内含子及5’和3’端的短串联重复顺序,对先证者及家族成员进行扩增片段长度多态性分析;用基因剂理分析方法判断女性亲属相关外显子的基因组靶序列的原始拷贝数。结果：在18个肌营养不良家系中检测到外显子或重复顺序片段缺失,在17个家系中得到女性亲属重复顺序片段,通过分析识别了2组纯合,6组杂合和11例半合状态的重复顺序片段,对11个缺失型家系的女性亲属进行基因剂量分析,确定了9例女性为缺失基因携带者。结论：重复顺序多态性与基因剂量分析结合可有效地检测缺失型迪谢内和贝克肌营养不良的女性携带者。     

Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis.

We developed a method for the detection of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers. The method is based on the quantitative analysis of the products of standard multiplex polymerase chain reaction (PCR) from 18 different exons of the dystrophin gene, and is designated "QM-PCR." We detected deletions of one or more exons by standard multiplex PCR in DMD/BMD patients in 14 of 18 families examined (77.7%). The same deletions were readily demonstrated by QM-PCR in nine of 14 mothers (64.3%) and in another six of 22 possible carriers in these families. In five families where deletions were detectable in DMD/BMD patients, the mothers did not exhibit any deletions in their peripheral blood (35.7%). We obtained evidence for germinal mosaicism in at least two of these families and confirmed carrier identification by haplotype analysis using CA repeat polymorphisms at the 5' and 3' ends of the dystrophin gene. Furthermore, analysis of 17 coded DNA samples from normal females and obligatory carriers by QM-PCR showed that this technique could directly identify carriers of deletions in any of 18 different exons of the dystrophin gene. Its application in combination with existing techniques is expected to significantly improve the accuracy of carrier diagnosis in many families, and it may also be applicable to families in which pedigree and polymorphism information is insufficient for carrier diagnosis.     

Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal

There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most frequently affected by a deletion includes exons 47-51. The distal region of the dystrophin gene was most frequently affected by the deletion in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.     

A screening for dystrophin gene deletions in Japanese patients with Duchenne/Becker muscular dystrophy by the multiplex polymerase chain reaction.

A new screening method involving the multiplex polymerase chain reaction was developed to detect dystrophin gene deletions in Japanese patients with Duchenne and Becker muscular dystrophy (DMD/BMD). Eleven exonic regions including deletion "hot spots" were analyzed. Gene deletions were found in 33% of 92 unrelated Japanese patients, mainly in the central portion (exons 43-52) and at the 5' end (exons 1-17). This is a useful laboratory test for the rapid genetic diagnosis of DMD/BMD.     

Dystrophin在不同类型肌营养不良症中的变化及诊断价值

目的研究dystrophin在不同类型肌营养不良症中的变化及分型诊断价值.方法用抗dystrophin抗体对107例肌营养不良症患者肌组织标本行免疫组织化学分析.结果Duchenne型肌营养不良(DMD)患者肌细胞膜上无显色,Becker型肌营养不良(BMD)患者肌细胞膜上显色浅淡、不连续或呈斑片状.肢带型肌营养不良(LGMD)患者肌细胞膜上染色正常.结论dystrophin免疫组化染色对于年龄较小临床不易区分的DMD/BMD患者,可区分开来,以早期预测功能影响程度.该方法也有助于区分临床表现相似的成年散发BMD和LGMD患者,对于正确地进行遗传咨询具有重要意义.     

Detection of deletions within the dystrophin gene in Polish families affected with Duchenne/Becker muscular dystrophy

DNA analysis was performed in 190 cases of Duchenne and Becker muscular dystrophies (DMD/BMD), including 150 cases with DMD and 40 cases with BMD, using Southern blotting and PCR multiplex techniques with application of 25 pairs of primers. Deletions in the overall material were found in 109 cases: 81 (54%) in patients with DMD and 28 (70%) in patients with BMD. All the deletions in DMD were out of frame with the exception of two cases, whereas in BMD all the deletions but two were in frame. Junction fragments were detected in 12 cases of DMD. In five cases duplications were found: four in patients with DMD and one in a patient with BMD.     

Polymerase chain reaction fiber analysis and somatic mosaicism in autopsied tissue from a man with Duchenne muscular dystrophy

Single muscle fibers, obtained at autopsy from a 22-year-old man with Duchenne muscular dystrophy were examined immunocytochemically and also using polymerase chain reaction (PCR). Dystrophin-positive cells were widespread in skeletal, cardiac, smooth muscle, and in brain cells. PCR and Southern blot analyses of DNA from peripheral blood lymphocytes revealed a deletion of exon 45 in the dystrophin gene. With PCR of single fibers, three bands corresponding to exons 44, 45, and 47 were present in the normal control muscle fibers and dystrophin-positive fibers from the patient, while only two bands, exons 44 and 47, were observed in dystrophin-negative fibers. Therefore, in this patient, the genotype of dystrophin-positive fibers differed from that of the dystrophin-negative fibers, possibly because of a somatic mosaicism for deletion in the dystrophin gene. A mutation of the dystrophin gene may have occurred in one cell at an early stage of ontogenesis