Martins Fábio Tadeu Arrojo, Miranda Paulo Maurício do Amor Divino, Fernandes Marcela Scabello Amaral, Maciel-Guerra Andréa Trevas, Sartorato Edi Lúcia
Human Molecular Genetics Laboratory - Center for Molecular and Genetic Engineering (CBMEG) - University of Campinas (UNICAMP) - Campinas/Brazil.
Department of Medical Genetics - Faculty of Medical Sciences- University of Campinas (UNICAMP) - Campinas/Brazil.
Mol Vis. 2017 Jul 21;23:495-503. eCollection 2017.
Leber hereditary optic neuropathy (LHON) is a mitochondrial inherited disease characterized by bilateral vision problems, such as reduced visual acuity, dyschromatopsia, and central or centrocecal scotoma. Of these cases, 95% are caused by three mutations in mitochondrial DNA (mtDNA): m.G11778A, followed by m.T14484C and m.G3460A. The remaining 5% of cases of LHON are caused by rare mutations also present in mtDNA. Although conventional molecular tools for molecular screening of LHON are becoming popular, in most cases these tools are still expensive and time-consuming and are difficult to reproduce. Therefore, to meet the need for more accurate, faster, and cheaper techniques for molecular screening, as well as make it more accessible, we used the high-throughput method TaqMan OpenArray Genotyping platform for developing a customized high-throughput assay for the three main mutations related to LHON.
The assay was performed for 87 individuals diagnosed with LHON or acquired optic neuropathy of unknown origin. The three main mutations were screened using the customized assay with the TaqMan OpenArray Genotyping platform, and all reactions were performed in triplicate. The positive and negative results were revalidated with restriction fragment length polymorphism PCR (RFLP-PCR) and Sanger sequencing.
The main mutations related to LHON were detected in 34 patients with genotyping reactions, of which 27 cases had the m.G11778A mutation, and seven had the m.T14484C mutation.
The TaqMan OpenArray Genotyping platform was shown to be an effective tool for molecular screening of the most common mutations related to LHON without presenting false positive or negative results for the analyzed mutations. In addition, this tool can be considered a cheaper, faster, and more accurate alternative for molecular screening of LHON mutations than PCR and Sanger sequencing, as 94 genotyping reactions can be performed within 6 h and specific TaqMan probes are used.
Leber遗传性视神经病变（LHON）是一种线粒体遗传性疾病，其特征为双侧视力问题，如视力下降、色觉障碍以及中心或中心暗点。在这些病例中，95% 由线粒体DNA（mtDNA）中的三种突变引起：m.G11778A，其次是m.T14484C和m.G3460A。其余5%的LHON病例由mtDNA中也存在的罕见突变引起。尽管用于LHON分子筛查的传统分子工具越来越普及，但在大多数情况下，这些工具仍然昂贵、耗时且难以重复。因此，为了满足对更准确、更快且更便宜的分子筛查技术的需求，并使其更易于获得，我们使用高通量方法TaqMan OpenArray基因分型平台开发了一种针对与LHON相关的三种主要突变的定制高通量检测方法。
对87例被诊断为LHON或不明原因获得性视神经病变的个体进行了该检测。使用TaqMan OpenArray基因分型平台的定制检测方法对三种主要突变进行筛查，所有反应均重复进行三次。阳性和阴性结果通过限制性片段长度多态性PCR（RFLP-PCR）和桑格测序进行重新验证。
在34例进行基因分型反应的患者中检测到与LHON相关的主要突变，其中27例有m.G11778A突变，7例有m.T14484C突变。
TaqMan OpenArray基因分型平台被证明是一种有效的分子筛查工具，可用于筛查与LHON相关的最常见突变，且对分析的突变不会出现假阳性或假阴性结果。此外，与PCR和桑格测序相比，该工具可被视为一种更便宜、更快且更准确的LHON突变分子筛查替代方法，因为94次基因分型反应可在6小时内完成，且使用了特异性TaqMan探针。
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