The Roles of Polymerase Gamma Accessory Subunit Gene Mutations in Human Disease.

描述（由申请人提供）：人类线粒体DNA （mtDNA）聚合酶（Polg）由两个核基因编码的两个亚基组成：1。编码催化亚基p140和2的POLG基因。POLG2基因编码同二聚体副亚基p55。Polg是唯一用于mtDNA复制和修复的细胞mtDNA聚合酶。线粒体疾病，包括与POLG2突变相关的疾病，是一种毁灭性疾病，包括连续的重叠表型，这些疾病的发病年龄从幼儿期到成年后期不等。目前还没有治愈这些疾病的方法。迄今为止，所有报道的与人类线粒体疾病相关的POLG2突变都以杂合突变的形式发生。与POLG2突变相关的体内功能障碍可能是由于p55的显性负性变异对野生型基因产物，即p140和p55的功能产生负面影响或阻断。这一研究领域的主要障碍包括缺乏对异二聚体p55变异的表征，以及排除致病等位基因分析的生化方法的局限性。该领域的另一个障碍是缺乏含有POLG2突变的细胞模型来确定和表征呼吸功能障碍的细胞机制。在K99/指导期，Young博士的目标是确定POLG2突变的显性负性机制。Young博士已经开发并将利用p55异二聚体变异来确定体外显性负性机制。在异二聚体研究的联合研究中，Young博士开发了具有POLG2突变的稳定人类细胞系，以确定体内的显性负性机制。Young博士将接受mtDNA缺失定性分析和mtDNA拷贝数定量测量的结构化培训，以研究与POLG2突变相关的mtDNA维持的细胞功能障碍。在该奖项的R00/Independent阶段，Young博士将前瞻性地研究人类稳定细胞系模型，以解决确定与线粒体疾病突变相关的呼吸缺陷机制的第二个目标。他将利用已经开发出的含有POLG2突变的人类细胞系，并开发新的细胞模型来确定呼吸缺陷的机制。这些研究对于基础研究和确定线粒体响应疾病突变的生物学机制至关重要。申请人的长期目标是进行暴露研究，并确定药物和毒素如何影响野生型和疾病状态下mtDNA的维持和呼吸，即所谓的线粒体-环境相互作用。首席研究员Young博士基于他之前在线粒体研究领域的训练，非常适合执行拟议的研究计划。他之前的培训包括酵母线粒体遗传学，人类稳定细胞系的发展，包含POLG2突变，以及Polg变异的生化表征。拟议的培训和职业发展将使Young博士成为一名独立的生物医学研究科学家，并为管理线粒体研究实验室的领导角色做好准备。

DESCRIPTION (provided by applicant): The human mitochondrial DNA (mtDNA) polymerase, (Polg) is composed of two subunits encoded by two nuclear genes: 1. the POLG gene encoding the catalytic subunit, p140 and 2. The POLG2 gene encoding the homodimeric accessory subunit, p55. Polg is the sole cellular mtDNA polymerase for mtDNA replication and repair. Mitochondrial diseases, including those linked to POLG2 mutations, are devastating disorders that comprise a continuum of overlapping phenotypes and the age of onset of these diseases range from early childhood to late adulthood. Currently there are no cures for these diseases. To date, al reported POLG2 mutations associated with human mitochondrial disease occur as heterozygous mutations. In vivo dysfunction associated with POLG2 mutations likely results from dominant negative p55 variants that negatively affect or block the function of wild-type gene products, namely p140 and p55. Major barriers to this field of research include the lack of characterization of heterodimeric p55 variants and the limitations of biochemical approaches that exclude analysis of pathogenic alleles. Another barrier in the field is the absence of cell models harboring POLG2 mutations to determine and characterize cellular mechanisms of respiratory dysfunctions. During the K99/Mentored phase Dr. Young's aim is to determine dominant negative mechanisms of POLG2 mutations. Dr. Young has developed and will exploit p55 heterodimeric variants to determine dominant negative mechanisms in vitro. In alliance with heterodimeric studies Dr. Young has developed stable human cell lines with POLG2 mutations to determine dominant negative mechanisms in vivo. Dr. Young will receive structured training in qualitative analysis of mtDNA deletions and quantitative measurements of mtDNA copy number to investigate cellular dysfunctions of mtDNA maintenance linked to POLG2 mutations. In the R00/Independent phase of this award Dr. Young will study prospectively human stable cell line models to address the second aim of determining mechanisms of respiratory defects associated with mitochondrial disease mutations. He will exploit already developed human cell lines harboring POLG2 mutations and develop new cell models to determine mechanisms of respiration deficiency. These studies are essential to fundamental research and determining the biological mechanisms of the mitochondrion that respond to disease mutations. The applicant's long-term objective is targeted at exposure research and determining how drugs and toxins affect mtDNA maintenance and respiration in wild-type and disease states, so called mitochondrion-environment interactions. The principle investigator, Dr. Young, is well suited to carry out the proposed research plans based on his prior training in the mitochondrial research field. His prior training includes yeast mitochondrial genetics, development of human stable cell lines harboring POLG2 mutations, and biochemical characterization of Polg variants. The proposed training and career development will enable Dr. Young to become an independent biomedical research scientist and prepare him for a leadership role managing a mitochondrial research laboratory.