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。     

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。     

中国人抗肌营养不良蛋白基因缺失的分布特点

目的了解国内Ｄｕｃｈｅｎｎｅ／Ｂｅｃｋｅｒ型肌营养不良症（ＤＭＤ／ＢＭＤ）患者基因缺失的分布特点。方法用１２对引物以多重ＰＣＲ检测与５６例ＤＭＤ／ＢＭＤ患者,分析缺失型患者抗肌营养不良蛋白（ｄｙｓｔｒｏｐｈｉｎ）基因缺失的断裂点分布,并结合国内文献报道的２２１例病例资料,分析９对引物的总检出率及各引物的最佳组合。结果国内ｄｙｓｔｒｏｐｈｉｎ基因缺失断裂点７２％位于４４～５１号内含子内,以４４号内含子最多（２２％）,５０号内含子次之（１７％）,位于４５～５１号内含子内的断裂点是４４号内含子的２．３倍。７号内含子断裂较少,仅４％的断裂点位于该内含子。９对引物的总检出率为４８％,其中５对引物的总检出率为４６％。两对引物的最佳组合为４８号和１７号,可检出３５％的患者。结论国内ｄｙｓｔｒｏｐｈｉｎ基因４４～５１号内含子高度不稳定,容易发生断裂。７号内含子可能不是国内ｄｙｓｔｒｏｐｈｉｎ基因缺失断裂的高发区域。在国内５对引物的总检出率接近９对引物,４８号和１７号外显子的二重ＰＣＲ扩增对于全国范围内ＤＭＤ／ＢＭＤ的筛选,尤其是结合其他方法进行大范围的产前筛选,不失为一种可取的选择。     

迪谢内肌营养不良症基因治疗的评价和问题

迪谢内肌营养不良症 (Ｄｕｃｈｅｎｎｅｍｕｓｃｕｌａｒｄｙｓｔｒｏｐｈｙ,ＤＭＤ)是一种以骨骼肌进行性变性、坏死为主要病理特征的致死性的Ｘ 性连锁隐性遗传性肌病。其发病率为男性活婴的 1/ 35 0 0 ,患者通常 3～ 5岁起病 ,主要表现为骨骼肌进行性近端无力、萎缩、腓肠肌假性肥大 ,12岁前丧失行走能力 ,通常于 2 0岁左右因呼吸衰竭或心力衰竭死亡[1] 。分子病理学研究证实 ,由于ＤＭＤ基因部分区域的缺失、重复或点突变 ,导致肌细胞膜上该基因编码的抗肌萎缩蛋白[2 ] (ｄｙｓｔｒｏｐｈｉｎ ,Ｄｙｓ)的缺乏或结构变异 ,引起肌细胞膜的…     

迪谢内/贝克肌营养不良患者视网膜眼电图改变特征

目的 研究迪谢内／贝克肌营养不良（ＤＭＤ／ＢＭＤ）患者视网膜眼电图（ＥＲＧ）的改变特征,探讨ＥＲＧ对ＤＭＤ／ＢＭＤ的诊断价值及ｄｙｓｔｒｏｐｈｉｎ在视网膜信号传导上的作用。方法 对２２例临床确诊的ＤＭＤ／ＢＭＤ患者进行详细的眼科检查和ＥＲＧ检测,采用ＥＲＧ国际测量标准记录结果。结果 全部患者眼科检查无明显异常。２２例患者中１６例（７２．７％）出现异常ＥＲＧ改变;其中１５例为暗适应蓝光ｂ波波幅下降或（和）消失（Ｐ〈０．００１）。１３例暗适应白光ｂ波波幅下降或（和）替伏期延长（Ｐ〈０．００１）。１２例ｂ／ａ波波幅比≤（Ｐ〈０．００１）。１６例振荡电位ＯＰｓ１～４波波幅下降。结论 ＥＲＧ可作为ＤＭＤ／ＢＭＤ诊断和症状的一项有价值的检查。ｄｙｓｔｒｏｐｈｉｎ及同源蛋白在视网膜电信号的传导上起着重要作用。     

Duchenne/Becker肌营养不良症家系基因分析

目的　对 1 8个DMD BMD基因 1 31例成员进行基因分析。　　方法　采用多重聚合酶链反应(mPCR)和短串联重复序列 (STR)多态单体连锁分析。　　结果　 1 0例男性胎儿中 4例存在基因缺失 ,4例非缺失型男性胎儿单体型与其携带者母亲相同 ,确定为DMD胎儿。 8例女性胎儿中 4例继承了母亲的单体型 ,为致病基因携带者。 46例先证者母亲及其他女性血亲为杂合型。 5′CA ,MP1P和内含子 44,49这 4个位点的杂合率分别为 65 2 % ,47 8% ,80 4%和 93 5%。　　结论　同时选择DMD基因两端和基因内 4个CA或TTGA位点进行联合分析 ,可大大提高诊断的准确率。     

两步—多重PCR诊断134例假肥大型进行性肌营养不良症基因缺失

研究临床检测假肥大型进行性肌营养不良症（ＤＭＤ／ＢＭＤ）基因缺失的有效手段。方法运用两步－多重聚合酶链反应（ＰＣＲ）技术诊断ＤＭＤ／ＢＭＤ基因缺失。结果１３４例病人中，检测阳性率为４９．３％（６６／１３４）。结论多重ＰＣＲ诊断ＤＭＤ／ＢＭＤ基因缺失敏感、快速、准确，可在临床推广应用。     

我国迪谢内肌营养不良分子遗传学研究概况

迪谢内肌营养不良（ＤＭＤ）是一种常见的致死性神经肌肉系统Ｘ连锁隐性遗传病,发病率为活产男婴的１／３５００。患者一般３～５岁发病,主要表现为全身骨骼肌进行性无力、萎缩和小腿腓肠肌假性肥大,随着病情加重,２０岁左右因呼吸衰竭死亡,迄今无有效的治疗方法。１...     

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。     

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某种联系尚待研究论证。     

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.     

用短串联重复序列单体连锁分析进行肌营养不良症产前基因诊断

目的对２０个迪谢内／贝克肌营养不良症（ＤＭＤ／ＢＭＤ）家系进行产前基因诊断。方法应用多重聚合酶链反应（ｍＰＣＲ）和短串联重复序列（ＳＴＲ）多态单体连锁分析对２０个ＤＭＤ／ＢＭＤ家系的成员进行产前基因分析。结果１６个家系可提供充足的多态性信息，４个散发型家系不能提供多态性信息。１１例男性胎儿中，３例存在基因缺失，４例非缺失型男性胎儿的单体型与其携带者母亲相同，确定为ＤＭＤ胎儿。９例女性胎儿中５例继承了母亲的单体型，为致病基因携带者。５１例先证者母亲及其他女性血亲为杂合型。结论ＳＴＲ单体连锁分析快速、准确，基因组ＤＮＡ需要量小，可提供的信息量高，适合非缺失型ＤＭＤ／ＢＭＤ家系产前诊断的需要。同时选择ＤＭＤ基因两端和基因内４个位点进行连锁分析，可大大提高诊断的准确率     

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.     

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.     

应用多重PCR检测DMD/BMD患者的基因缺失

目的:了解Duchenne型肌营养不良(DMD)/Becker型肌营养不良(BMD)患者基因缺失的分布规律,并探讨检测技术。方法:应用18对引物多重聚合酶链反应(mPCR)分两组对40例DMD/BMD患者和5例健康者进行Dys-trophin基因缺失检测分析。结果:27例(67.5%)存在外显子缺失,13例(32.5%)未检测到缺失;16例缺失片段集中于44～52号外显子,6例集中于2～20号外显子,5例在以上两个区域均有缺失;45、48、51号外显子缺失频率最高。结论:基因缺失为主要突变类型,缺失片段主要分布于44～52、2～20号外显子两个缺失热区;mPCR具有临床应用价值。     

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。     

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.     

MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy

Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60–65% of mutations, duplications for 5–10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009–2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45–54 and 3–21, whereas most duplications involved exons 3–18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots – different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers.