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Multiplex Ligation-dependent Probe Amplification Technology for Detection of Mitochondriopathy
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摘要:
目的 探讨多重连接探针扩增技术(multiplex ligation-dependent probe amplification, MLPA)用于常见线粒体疾病基因检测的可行性。 方法 收集2010年5月至2012年8月北京协和医院281例可疑线粒体异常病例, 包括神经科存在神经系统损伤的患者233例及眼科疑诊Leber遗传性视神经病变的患者48例, 运用MLPA法对常见线粒体疾病突变位点进行检测, 并使用线粒体基因序列测定方法进行验证。 结果 281例标本中共38例检测到线粒体基因突变, 总检出率为13.5%。眼科48例标本中, 3460G > A、11778G > A和14484T > C 3种突变共检测到19例, 检出率为39.6%;神经科233例标本中, 3243A > G、8344A > G、8993T > G和大片段缺失共检测到19例, 检出率为8.2%。除1例大片段缺失暂无PCR测序验证外, 其余MLPA结果均经序列测定验证为相符。 结论 MLPA法是一种可行的快速、准确、简便的基因诊断方法, 可作为筛查常见线粒体疾病的检测工具, 为临床诊断和治疗提供依据。 -
关键词:
- 线粒体疾病 /
- 多重连接探针扩增技术 /
- 基因突变 /
- Leber遗传性视神经病变 /
- 线粒体脑肌病伴乳酸血症和卒中样发作
Abstract:Objective To investigate the feasibility of multiplex ligation-dependent probe amplification (MLPA) for the molecular diagnosis of mitochondriopathy. Methods Totally 281 patients with suspected mitochondriopathy in Peking Union Medical College Hospital from May 2010 to August 2012 were enrolled in this study. Among them 233 with nervous system damage were from the department of neurology and 48 with suspected Leber hereditary optic neuropathy (LHON) were from the department of ophthalmology. The common mutation sites for mitochondrial disease were detected with MLPA, and the results were verified by mitochondrial DNA (mtDNA) sequencing. Results We found 38 cases carried mtDNA mutations from all 281 cases, and the total detection rate was 13.5%. Among 48 cases suspected of LHON, 19 cases (39.6%) were found containing 3460G > A, 11778G > A or 14484T > C. Among 233 cases suspected of mitochondria related neuropathies, 19 cases (8.2%) were found containing 3243A > G, 8344A > G, 8993T > G or a large deletion. Except that the large deletion could not be sequenced, the other mutations detected by MLPA were all verified as consistent with gene sequencing results. Conclusions MLPA is a rapid, accurate, and simple method for detecting mtDNA mutation for common mitochondriopathy. It is feasible to be used in clinical diagnostic laboratory. -
表 1 线粒体常见突变位点引物序列及扩增条件
表 2 MLPA检测各位点比例及序列测定验证结果
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[1] Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification[J]. Nucleic Acids Res, 2002, 30:e57. doi: 10.1093/nar/gnf056 [2] 陈竺.医学遗传学[M].北京:人民卫生出版社, 2002:158-159. [3] A human mitochondrial genome database[DB/OL].[2012-12-7]. http://www.mitomap.org/MITOMAP. [4] DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases[J]. N Engl J Med, 2003, 348:2656-2668. doi: 10.1056/NEJMra022567 [5] 陈刚, 杜卫东, 曹慧敏.线粒体DNA突变与相关人类疾病[J].遗传, 2007, 29:1299-1308. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yc200711003 [6] Chinnery PF, Howell N, Lightowlers RN, et al. MELAS and MERRF. The relationship between maternal mutation load and the frequency of clinically affected off spring[J]. Brain, 1998, 121:1889-1894. doi: 10.1093/brain/121.10.1889 [7] Howell N, Chinnery PF, Ghosh SS, et al. Transmission of the human mitochondrial genome[J]. Hum Reprod, 2000, 15:235-245. doi: 10.1093/humrep/15.suppl_2.235 [8] Cao L, Shitara H, Horii T, et al. The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells[J]. Nat Genet, 2007, 39:386-390. doi: 10.1038/ng1970 -
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