Genetic analysis and investigation of molecular pathogenesis for mitochondrial diseases

线粒体疾病患者的全外显子组测序在我们的实验室进行。在2018财年，我研究了9例患者中4个基因/基因簇的候选突变的致病性。通过生物信息学、分子遗传学和生物化学分析，包括RNA测序、桑格测序、DNA克隆、qPCR、SDS-PAGE和BN-PAGE蛋白质印迹、呼吸速率分析和OXPHOS酶分析，我们在几例确诊或疑似线粒体疾病的患者中检测到大的染色体缺失和基因重排。我的研究强调了阐明基因簇区域中确切gDNA突变的技术挑战。在所调查的5例患者中，2例证实了分子诊断。这是一项与澳大利亚默多克儿童研究所的大卫·索伯恩教授合作进行的研究，其中一个新的疾病基因是与澳大利亚墨尔本大学的戴安娜·斯托贾诺夫斯基博士合作研究的。结果，该变异被排除在进一步的分析中，在两名患者由于缺乏支持性证据致病性。两个新的DNA变异的核基因编码的OXPHOS复合物III亚基被确定在Leigh综合征患者。进行基因组DNA、RNA和蛋白质分析，以确定患者中这2种复合杂合变体引起的致病性证据。这项研究导致了与大卫索伯恩教授的持续合作。正在编写一份联合出版物。

Whole exome sequencing of mitochondrial disease patients was performed in our laboratory. In fiscal year 2018, I investigated candidate mutations in 4 genes/gene cluster for their pathogenicity in 9 patients. Bioinformatic, molecular genetic and biochemical analyses including RNA sequencing, Sanger sequencing, DNA cloning, qPCR, SDS-PAGE and BN-PAGE western blotting, respiration rate analysis and OXPHOS enzyme assays were performed.We detected large chromosomal deletions and rearrangements of the gene region in several patients with confirmed or suspected mitochondrial disease. My research highlighted the technical challenge in elucidating the exact gDNA mutations in the gene cluster region. Molecular diagnosis was confirmed in 2 of the 5 patients investigated. This is an on-going research in collaboration with Prof. David Thorburn from Murdoch Children’s Research Institute in Australia.One of the novel disease genes was investigated in collaboration with Dr Diana Stojanovski from the University of Melbourne in Australia. As a result, the variant was excluded from further analysis in two patients due to the lack of supportive evidence for pathogenicity.Two novel DNA variants in a nuclear gene encoding an OXPHOS complex III subunit were identified in a patient with Leigh Syndrome. Genomic DNA, RNA and protein analyses were performed to identify evidence for pathogenicity caused by those 2 compound heterozygous variants in the patient. This study led to an on-going collaboration with Prof. David Thorburn. A joint-publication is in preparation.