Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing
Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing
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DOI：
10.1089/gte.2005.9.190
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发表时间：
2005-09-01
期刊：
影响因子：
--
通讯作者：
Cross, NCP
中科院分区：
文献类型：
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作者：
White, HE;Durston, VJ;Cross, NCP
线粒体DNA（mtDNA）中的致病突变通常是异质性的，因此对线粒体疾病的基因检测的解释可能是有问题的。低水平异质性的检测在技术上要求很高，并且通常难以区分特定组织中不存在突变或检测突变的技术失败。不同组织异质性的可靠测量可能有助于识别有发生特定并发症风险的个体，并为患者和家庭成员提供更好的预后建议。我们已经评估了焦磷酸测序技术用于检测和估计与以下疾病相关的六种线粒体点突变的异质性：Leber遗传性视神经病变（LHON），G3460 A，G11778 A和T14484 C;线粒体脑病伴乳酸酸中毒和中风样发作（MELAS），A3243 G;肌阵挛癫痫伴不规则红纤维（MERRF），A8344 G，神经源性肌无力、共济失调和视网膜色素变性（NARP）/Leighs：T8993 G/C。从焦磷酸测序检测50例推定线粒体疾病的患者获得的结果进行了比较，使用常用的诊断技术的聚合酶链反应（PCR）和限制性内切酶消化。焦磷酸测序分析提供了准确的基因分型和突变负荷的定量测定，灵敏度和特异性均为100%。MELAS A3243 G突变在1%异质性水平下被可靠地检测到。我们的结论是焦磷酸测序是一种快速和强大的方法检测异质性线粒体点突变。
Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.