Mani Shalini, Rao S Narasimha, Kumar M V Kranthi
Department of Biotechnology, Centre for Emerging Diseases, Jaypee Institute of Information Technology, Noida 201301, India; Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
Government Institute of Child Health, Niloufer Hospital for Women and Children, Red Hills, Hyderabad, India.
J Neurol Sci. 2020 Aug 15;415:116870. doi: 10.1016/j.jns.2020.116870. Epub 2020 Apr 30.
Cytochrome c oxidase (COX) deficiency is known to be associated with Leigh syndrome (LS), however there are limited studies on genetic screening of mitochondrial (mt) DNA encoding COX genes as well as the functional validation of identified variants. In our previous studies, we cared for total 165 LS patients and analyzed the nucleotide variations across entire mt genome. We observed a high level of genetic heterogeneity in these patients. We identified various reported and novel variation across entire genome including COX genes. In our present study we have further studied and functionally validated the selected novel nucleotide variant of COX I and COX II gene using different in-silico tools and trans mitochondrial cybrid based assays. As a result of our study, G6036A (G45S) variant of COX I gene, reduced the COX activity in both spectrophotometric as well as In-gel BN-PAGE assays. FACS analysis also revealed this variant to affect the mitochondrial membrane potential in the respective cybrids. Interestingly most of our in-silico studies indicated that this variant might affect the secondary structure and confirmation of COX I protein. Thus we report the first missense mutation in the COX I gene of LS patients and justify its pathogenic role in these patients by different assays. Variant A7746G (N54K) in COX II gene was also predicted to affect the secondary structure as well as stability of COX II protein. Though, the effect of this variant was not significant, however it will be interesting to investigate its significance by other assays in future.
细胞色素c氧化酶（COX）缺乏症与 Leigh 综合征（LS）有关，然而，关于编码COX基因的线粒体（mt）DNA的基因筛查以及已鉴定变异的功能验证的研究有限。在我们之前的研究中，我们共诊治了165例LS患者，并分析了整个mt基因组的核苷酸变异。我们观察到这些患者存在高度的遗传异质性。我们在整个基因组中鉴定出各种已报道的和新的变异，包括COX基因。在本研究中，我们使用不同的电子计算机模拟工具和基于转线粒体杂交细胞的试验，进一步研究并在功能上验证了COX I和COX II基因选定的新核苷酸变异。作为我们研究的结果，COX I基因的G6036A（G45S）变异在分光光度法和凝胶内BN-PAGE试验中均降低了COX活性。流式细胞仪分析还显示该变异影响了相应杂交细胞中的线粒体膜电位。有趣的是，我们的大多数电子计算机模拟研究表明，该变异可能影响COX I蛋白的二级结构和构象。因此，我们报告了LS患者COX I基因中的首个错义突变，并通过不同试验证明了其在这些患者中的致病作用。COX II基因中的A7746G（N54K）变异也被预测会影响COX II蛋白的二级结构以及稳定性。虽然该变异的影响不显著，但未来通过其他试验研究其意义将很有趣。
