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实验室进行。在2018财年，我调查了9例患者中4个基因/基因簇的候选突变的致病性。进行生物信息学、分子遗传学和生化分析，包括RNA测序、Sanger测序、DNA克隆、qPCR、SDS-PAGE和BN-PAGE western blotting、呼吸速率分析和OXPHOS酶检测。我们在几个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区域重排。我的研究强调了阐明基因簇区域中确切的dna突变的技术挑战。5例患者中2例确诊为分子诊断。这是与澳大利亚默多克儿童研究所的David Thorburn教授合作进行的一项正在进行的研究。其中一种新型致病基因是与澳大利亚墨尔本大学的戴安娜·斯托亚诺夫斯基博士合作研究的。结果，由于缺乏致病性的支持性证据，该变异被排除在两例患者的进一步分析之外。编码OXPHOS复合物III亚基的核基因中的两个新的DNA变体在Leigh综合征患者中被鉴定出来。对患者进行基因组DNA、RNA和蛋白质分析，以确定这两种复合杂合变异体引起的致病性证据。这项研究导致了与David Thorburn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.