Leber遗传性视神经病相关的三种原发性线粒体突变的无创检测方法建立

目的：建立1种Leber遗传性视神经病（LHON）相关的3种原发性线粒体突变11778G＞A、14484T＞C和3460G＞A的无创检测方法。方法：研究对象为在温州医科大学附属眼视光医院就诊的3例（WZ1481、WZ1478、WZ1435）基因检测确定为m.11778G＞A、m.14484T＞C和m.3460G＞A突变的LHON患者。采集3例LHON患者和2例野生型健康对照的静脉血样及口腔黏膜细胞样本，然后分别用手工法和试剂盒法提取全基因组DNA，分析比较2种样本、2种方法提取的DNA的纯度及浓度有无差别。最后以携带m.11778G＞A、m.14484T＞C和m.3460G＞A突变的LHON患者和野生型的口腔黏膜细胞和外周血提取的全基因组DNA作为模板，通过PCR扩增分别包含以上3种突变的DNA片段，然后进行焦磷酸测序、斑点杂交和Southern blot分析PCR产物。结果：口腔黏膜细胞和血液提取的DNA浓度的差异无统计学意义（P＞0.05）。手工法提取的口腔黏膜细胞DNA纯度低于试剂盒法提取的口腔黏膜细胞和血液DNA的纯度，差异有统计学意义（P＜0.05）。口腔黏膜细胞提取的DNA产量与血液相似。DNA测序结果显示血液样本和口腔黏膜细胞样本具有很好的一致性，对照组均检测不到突变，而突变组可以检测到相应的突变。斑点杂交和Southern blot的结果也一致，对照组均为阳性而突变组均为阴性。结论：口腔黏膜细胞DNA可用于筛查和鉴定LHON的3个主要线粒体突变。本研究建立了一种方便、无创的方法来检测LHON相关的m.11778G＞A、m.14484T＞C和m.3460G＞A突变。

Establishment of a non-invasive detection assay for three primary mitochondrial mutations associated with Leber’s hereditary optic neuropathy

Objective: To establish a non-invasive detection assay for three Leber’s hereditary optic neuropathy (LHON) families with m.11778G>A, m.14484T>C and m.3460G>A mutations. Methods: Three patients (WZ1481, WZ1478, WZ1435) with m.11778G>A, m.14484T>C and m.3460G>A mutations who visited School of Ophthalmology and Optometry, Wenzhou Medical University were included in this study. Purity and concentration of genes from buccal cell samples via manual sampling and reagent kits were determined. The whole genomic DNA of samples carrying m.11778G>A, m.14484T>C and m.3460G>A mutation with LHON as well as wild type were used as templates, amplification of DNA fragments each containing the above three mutations by PCR. The PCR products were analyzed by DNA sequencing, Dot blot, and Southern blot. Results: There were no significant differences in DNA concentration between buccal cells and blood (P>0.05). However, the purity of buccal cell DNA extracted by manual sampling was lower than the buccal cell DNA and blood DNA extracted by reagent kits (P<0.05). The DNA yielding of buccal cell DNA was similar to that of blood DNA. The results of DNA sequencing, Dot blot and Southern blot were also highly consistent. Both groups were positive in the control group and negative in the mutant group. Conclusion: The results of DNA sequencing, Dot blot and Southernblot demonstrate that buccal cell DNA can be used for the screening and identification of three primary mitochondrial mutations of LHON. This study has established a convenient and non-invasive detection assay suitable for clinical determination in three primary mitochondrial mutations associated with LHON.