Translating preclinical studies of mitochondrial disease mice towards monitoring disease progression in patients

总体目的：利用患者特异性变异MRPL44（L156R）建立线粒体疾病的新型小鼠模型，并利用多组学技术建立线粒体疾病的综合病理生理学。线粒体疾病是一种具有异质性表现的遗传性多系统遗传疾病。线粒体有自己独立的蛋白质合成机制，线粒体核糖体是其中的一部分。新出现的一类线粒体疾病是线粒体体的突变；通常会影响线粒体DNA编码的蛋白质的产生。因此，在线粒体突变患者中发现的常见生化缺陷是氧化磷酸化复合物的缺陷。MRPL44是一种大亚基蛋白，其变体L156R最初在婴儿心肌病患者中被发现具有致病性。迄今为止，有9名MRPL44突变患者，所有患者都携带至少一个L156R等位基因，使其成为感兴趣的关键变体。此外，严重缺乏线粒体疾病的健壮动物模型，这被证明是治疗方法发展的障碍。因此，我们希望首先使用关键的患者特异性变异MRPL44（L156R）制作敲入小鼠模型，该模型准确地模拟患者表型，然后使用多组学深度表型分析观察RNA，蛋白质和代谢物的所有差异来表征小鼠。这将有助于建立线粒体疾病的发病机制，并找到候选生物标志物，这是监测疾病进展的宝贵工具，特别是与线粒体疾病相关的疾病。方法第一阶段：验证MRPL44（L156R）小鼠作为线粒体疾病的准确动物模型。由于心脏和肝脏的分子表型已经建立，我们将在骨骼肌和大脑中展示这些分子表型。该疾病的进行性将通过4个不同年龄组进行验证：1米、3米、6米和12米。第二阶段：使用组学技术对MRPL44（L156R）小鼠进行深度表型分析和表征。将对使用不同组织的更大队列进行RNA测序。此外，串联质谱法将对不同年龄组的关键组织进行蛋白质组学和代谢组学分析。这将用于寻找候选生物标志物。第三阶段：在线粒体疾病患者样本中验证从模型中鉴定的生物标志物。这将需要使用人体组织，并将按照人体组织法进行。伦理考虑：虽然本项目使用小鼠作为主要调查对象，但由于已经收集了小鼠的样本，并且没有活的小鼠，因此该项目的这一部分不需要伦理批准。这个项目将主要包括小鼠组织的分子分析。细胞系不受人体组织行为的保护。

Overall Aim: Establish a novel mice model of mitochondrial disease using patient specific variant MRPL44(L156R) and leverage multi-omics to build a comprehensive pathophysiology of mitochondrial disorders. Mitochondrial disorders are inherited multi-systemic genetic disorder with heterogeneous presentation. The mitochondria have their own separate protein synthesis mechanism which mitochondrial ribosomes (mitoribosomes) are part of. New emerging disease group of mitochondrial diseases are mutation in the mitoribosome; typically affecting production of proteins encoded by the mitochondrial DNA. Thus, a common biochemical deficiency found in patients of mitoribosome mutations are deficiency in the complexes of oxidative phosphorylation. MRPL44 is a protein of the large subunit and its variant L156R was found to be pathogenic originally in infantile cardiomyopathy patient. To date, there are 9 patients of MRPL44 mutations and all of the patients carry at least one allele of the L156R making it a key variant of interest. Additionally, there profound lack of robust animal models of mitochondrial disorders which is proving to be an hinderance in the development of therapeutics. Therefore, we want to first make a knock-in mice model using key patient specific variant MRPL44(L156R) that is accurately emulating patient phenotypes and then characterise the mice using multi-omics deep phenotyping looking at all the differences in the RNA, protein and metabolites. This will help to establish the mechanism of pathogenesis of mitochondrial disorders and find candidate biomarkers which is invaluable tool for monitoring the progression of disease which is particularly relevant in mitochondrial disorders. MethodsPhase one: validation of the MRPL44(L156R) mice as an accurate animal model of mitochondrial disorders. As the molecular phenotypes of the heart and liver were established already, we will demonstrate these molecular phenotypes in skeletal muscle and brain. The progressive nature of the disease will be validated using 4 different age groups: 1m, 3m, 6m and 12m. Phase two: deep phenotyping and characterisation of the MRPL44(L156R) mice using omics technology. RNA sequencing of a bigger cohort using different tissues will be performed. Moreover, Tandem-mass spectrometry will be performed on the key tissues in different age groups for proteomic and metabolomic analysis. This will be used to find candidate biomarkers. Phase three: the validation of biomarkers identified from the model in mitochondrial disease patient samples. This will require use of human tissues and will be done complying to the human tissue act. Ethical considerations: Although, this project uses mice as the subject of the primary investigation, this arm of the project does not require ethical approvals as the samples from the mice are already collected and there are no alive mice. This project will mainly consist of molecular analysis of mice tissues. Cell lines are not covered by human tissue act.