The role of blood cell dynamics and intracellular selection in the decline of the heteroplasmic mitochondrial DNA mutation m.3243A>G in blood

线粒体疾病是由核和线粒体（mt）基因组突变引起的。携带最普遍的异质性mtDNA突变m.3243A>G的个体显示出从临床沉默到严重神经变性的广泛表型。疾病的严重程度部分由突变的mtDNA分子（异质性）的比例解释，但这种关系是复杂的，使患者咨询具有挑战性。在有丝分裂组织（例如血液）中，异质性随时间而下降。模拟表明，这与细胞分裂的数量有关。细胞内负选择也可能有贡献，但其机制尚不清楚。线粒体疾病无法治愈;了解这一过程可能会导致新的治疗靶点的确定。目标1.确定外周血白细胞组成的变化与异质性水平和下降的相关程度，以及特定血细胞类型中的异质性与疾病负荷的相关性是否优于全血。2.确定记忆T细胞克隆的有丝分裂史是否与异质性有关。3.研究细胞内选择在降低异质性中的作用。新奇不同类型白细胞的异质性似乎存在自然变异。个体白细胞群体的群体动力学可能阐明异质性随时间下降的机制，这是一个知之甚少的过程。访问一个独特的，deepenotyped患者队列将使大量的高质量的数据在博士学位的时间范围内生产。已获得招募100例患者的及时伦理批准（REC：19/LO/0117）。已经收集了25人的血液样本;剩下的75人将在2020年底招募，让学生立即开始实验。实验方法学生将设计一组荧光激活细胞分选的抗体，将血液分离成代表主要白细胞发育阶段的亚群。将通过焦磷酸测序和实时PCR测量m.3243A>G异质性和mtDNA拷贝数。在T细胞中，有丝分裂历史最长，异质性最低，单个细胞之间可能存在异质性。单个T细胞可以被跟踪并扩增成克隆，使学生能够将异质性与有丝分裂历史相关联，并通过刺激体外增殖来操纵细胞分裂的数量。使用MitoCohort的数据，学生将模拟细胞亚群异质性和疾病负担之间的关系。为了研究活性细胞内选择在降低水平中的作用，将在设计用于操纵对氧化磷酸化的依赖的条件下培养细胞。

Mitochondrial diseases are caused by mutations in nuclear and mitochondrial (mt) genomes. Individuals carrying the most prevalent heteroplasmic mtDNA mutation, m.3243A>G, display a wide range of phenotypes from clinically silent to severe neurodegeneration. Disease severity is partly explained by the proportion of mutated mtDNA molecules (heteroplasmy) but the relationship is complicated, making patient counselling challenging. In mitotic tissues (e.g. blood), heteroplasmy declines over time. Simulations suggest this is linked to the number of cell divisions. Intracellular negative selection may also contribute but the mechanism for this is unknown. There is no cure for mitochondrial disease; understanding this process could lead to the identification of new therapeutic targets. Objectives 1. Determine the extent to which variation in the leukocyte composition of peripheral blood is associated with heteroplasmy level and decline and whether heteroplasmy in a specific blood cell type is better correlated with disease burden than that of whole blood. 2. Determine whether the mitotic history of memory T cell clones associates with heteroplasmy. 3. Investigate the role of intracellular selection in declining heteroplasmy. Novelty There appears to be natural variation in heteroplasmy in different types of leukocytes. Population dynamics of individual leukocyte populations may illuminate the mechanisms of heteroplasmy decline over time, a poorly understood process. Access to a unique, deeply-phenotyped patient cohort will enable the production of a large quantity of high quality data within the time-frame of a PhD. Timeliness Ethical approval for recruiting 100 patients has been granted (REC:19/LO/0117). Blood samples have been collected for 25; the remaining 75 will be recruited by late 2020, allowing the student to begin experiments immediately. Experimental Approach The student will design a panel of antibodies for fluorescence activated cell sorting, separating blood into subpopulations representing the major leukocyte developmental stages. m.3243A>G heteroplasmy and mtDNA copy number will be measured by pyrosequencing and real-time PCR. Within T cells, which have the longest mitotic history and lowest heteroplasmy, there is likely to be heterogeneity among individual cells. Single T cells can be tracked and expanded into clones, allowing the student to correlate heteroplasmy with mitotic history and manipulate the number of cell divisions by stimulating proliferation in vitro. Using data from the MitoCohort, the student will model the relationship between cellular sub-population heteroplasmy and disease burden. To investigate the role of active intracellular selection in declining levels, cells will be cultured in conditions designed to manipulate reliance on oxidative phosphorylation.