Molecular mechanisms underlying secondary mitochondrial dysfunction in patients diagnosed in the 100,000 Genomes Project

原发性线粒体疾病（PMDs）是一种复杂的神经系统和/或多系统疾病，其特征是巨大的临床、生化和遗传异质性。这些疾病造成严重的残疾，常常限制幼儿期的生命，目前没有治愈疾病的治疗方法。近400种疾病基因已被证明可导致pmd。已知的疾病机制包括编码线粒体氧化磷酸化酶亚基和组装因子的基因突变，线粒体DNA维持、蛋白质合成、辅因子生物合成和脂质代谢紊乱。最近，在英国的100,000基因组计划中，我们在最初被怀疑患有PMD的儿童中发现了先前与PMD无关的基因缺陷。这些儿童表现出提示线粒体疾病的临床特征（复杂的神经系统和/或多系统表现），骨骼肌活检证实线粒体呼吸链缺陷。在这些儿童中发现的遗传缺陷包括涉及RNA代谢和转录、DNA修复和钙信号传导的基因的致病变异，以及涉及细胞对缺氧反应的脯氨酸羟化酶。这些基因缺陷和线粒体功能障碍之间的联系尚不清楚。我们假设，了解这些罕见的遗传性小儿神经退行性疾病继发性呼吸链缺陷的分子机制将有助于阐明更常见的神经退行性疾病的致病基础，这些疾病涉及继发性线粒体功能障碍，包括帕金森病、阿尔茨海默病和亨廷顿病。这些神经退行性疾病也缺乏治疗方法，针对导致继发性呼吸链功能障碍的机制将是一种新的治疗途径。由于可能涉及常见的下游通路，因此本研究可能为罕见的原发性儿科线粒体疾病和更常见的成人神经退行性疾病确定新的治疗靶点。拟议的项目将包括使用RNAseq进行全基因组转录组分析，以确定原代患者细胞中共有的转录差异，以及从患者来源的诱导多能干细胞重编程的神经细胞系的详细生化功能表征。Nanna Therapeutics开发了一个小分子化合物和技术库，使用独特的高通量平台分析这些化合物对线粒体功能的影响，需要微小的样品。我们将确定已确定的基因缺陷对细胞生物化学和生理学的影响，我们将使用Nanna Therapeutics分析来筛选他们的化合物库，以确定潜在的治疗效果。

Primary mitochondrial diseases (PMDs) are complex neurological and/or multisystem disorders characterised by enormous clinical, biochemical and genetic heterogeneity. These disorders cause severe disability, are frequently life-limiting in early childhood and currently have no curative disease-modifying therapies. Approaching 400 disease genes have been shown to cause PMDs. Known disease mechanisms include mutations in genes encoding subunits and assembly factors of the mitochondrial oxidative phosphorylation enzymes, and disorders of mitochondrial DNA maintenance, protein synthesis, cofactor biosynthesis and lipid metabolism. Recently, in the UK 100,000 genomes project we have identified defects in genes not previously linked to PMDs in children initially suspected to have a PMD. These children presented with clinical features suggestive of mitochondrial disease (complex neurological and/or multisystem manifestations) and had confirmed mitochondrial respiratory chain deficiencies in skeletal muscle biopsies. The genetic defects identified in these children include pathogenic variants in genes involved in RNA metabolism and transcription, DNA repair and calcium signalling, and a proline hydroxylase implicated in the cellular response to hypoxia. The links between these gene defects and mitochondrial dysfunction are not known. We hypothesise that understanding the molecular mechanisms underlying secondary respiratory chain deficiencies in these rare genetic paediatric neurodegenerative disorders will help to elucidate the pathogenic basis of more common neurodegenerative diseases where secondary mitochondrial dysfunction has been implicated, including Parkinson, Alzheimer and Huntington diseases. These neurodegenerative disorders also lack curative therapies, and targeting the mechanisms leading to secondary respiratory chain dysfunction would be a new therapeutic avenue. Since common downstream pathways may be involved, this research is thus likely to identify novel therapeutic targets for both rare primary paediatric mitochondrial disorders and more common adult neurodegenerative diseases. The proposed project will involve genome-wide transcriptome analysis using RNAseq to identify shared transcriptional differences in primary patient cells, and detailed biochemical functional characterization in neuronal cell lines reprogrammed from patient-derived induced pluripotent stem cells. Nanna Therapeutics have developed a library of small molecule compounds and technologies to assay the effects of these compounds on mitochondrial function using a unique high throughput platform requiring minute samples. We will establish the impact of the identified gene defects on cell biochemistry and physiology and we will use the Nanna Therapeutics assays to screen their library of compounds to identify potential therapeutic hits.