Surveyor™ nuclease:: A new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects
Surveyor™ nuclease:: A new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects
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DOI：
10.1002/humu.20177
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发表时间：
2005-06-01
期刊：
影响因子：
3.9
通讯作者：
Paquis-Flucklinger, V
中科院分区：
文献类型：
--
作者：
Bannwarth, S;Procaccio, V;Paquis-Flucklinger, V
线粒体DNA（mtDNA）的分子分析是呼吸链缺陷诊断和遗传咨询的关键步骤。目前还没有快速的方法来鉴定未知的mtDNA点突变。我们已经开发了一种新的策略，基于完整的mtDNA PCR扩增，然后用错配特异性DNA内切酶，Surveyor”核酸酶消化。这种酶是植物DNA内切酶的CEL核酸酶家族的成员，在任何错配位点切割双链DNA，包括碱基取代和小的插入/缺失。消化后，分离裂解产物并通过琼脂糖凝胶电泳进行分析。消化产物的大小指示突变的位置，然后通过测序确认和表征。虽然这种方法可以分析2kb mtDNA扩增子并检测同一片段内的多个突变，但它不能识别同质碱基替换。同质致病突变已被描述。然而，大多数同质碱基取代是中性多态性，而有害突变通常是异质的。在这里，我们报告说，这种方法可用于检测mtDNA突变，如m.3243A > G tRNA（Leu）和m.14709T > C tRNA（Glu），即使它们存在于来自呼吸链缺陷患者的DNA样本中，其水平低至3%。然后，我们测试了五名患有线粒体呼吸链缺陷的患者，并在其中两名患者中发现了一种变体（m16189 T> C），该变体之前与糖尿病和心肌病的易感性有关。总之，这种方法可以有效地用于快速和完整地筛选整个人类线粒体基因组的异质性突变，在这种情况下，代表了线粒体疾病诊断的重要进展。(c)2005 Wiley-Liss，Inc.
Molecular analysis of mitochondrial DNA (mtDNA) is a critical step in diagnosis and genetic counseling of respiratory chain defects. No fast method is currently available for the identification of unknown mtDNA point mutations. We have developed a new strategy based on complete mtDNA PCR amplification followed by digestion with a mismatch-specific DNA endonuclease, Surveyor" Nuclease. This enzyme, a member of the CEL nuclease family of plant DNA endonucleases, cleaves double-strand DNA at any mismatch site including base substitutions and small insertions/deletions. After digestion, cleavage products are separated and analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. Although this method allows the analysis of 2 kb mtDNA amplicons and the detection of multiple mutations within the same fragment, it does not lead to the identification of homoplasmic base substitutions. Homoplasmic pathogenic mutations have been described. Nevertheless, most homoplasmic base substitutions are neutral polymorphisms while deleterious mutations are typically heteroplasmic. Here, we report that this method can be used to detect mtDNA mutations such as m.3243A > G tRNA(Leu) and m.14709T > C tRNA(Glu) even when they are present at levels as low as 3% in DNA samples derived from patients with respiratory chain defects. Then, we tested five patients suffering from a mitochondrial respiratory chain defect and we identified a variant (m16189T > C) in two of them, which was previously associated with susceptibility to diabetes and cardiomyopathy. In conclusion, this method can be effectively used to rapidly and completely screen the entire human mitochondrial genome for heteroplasmic mutations and in this context represents an important advance for the diagnosis of mitochondrial diseases. (c) 2005 Wiley-Liss, Inc.