线粒体DNA低频点突变在衰老中的影响及机制

High frequent mutations of mitochondrial DNA (mtDNA) were reported associated with many human diseases. Recent studies demonstrated that low frequent mtDNA mutations (<2%) also play important roles in cancer and aging-associated diseases, which was neglected before. The applicant have developed a reliable method for detecting low frequent mtDNA mutations (as low as 0.3%), and found out that the increased mtDNA mutations in the mouse with deficient mtDNA polymerase (Polg) were mainly contributed by increased number of low frequent mutations, not the increased frequency of existing mutation sites. Further study showed that Polg deficient mice tend to have more C to T transitions than wild-type control mice, the result of which is the corresponding hydrophilic amino acid changed to hydrophobic ones, indicating low frequent mtDNA mutations might be an important cause leading to premature aging. In the following study, we are planing to identify low frequent point mutations in multiple aging stages, figure out their features, and to find out if there were low frequent mtDNA mutations can have higher frequency of expression, and probe into the mechanism causing aging. Moreover, by creating Polg deficient cell lines derived from different tissues and detecting the low frequent mtDNA mutations in different passages, we aim to unclose why different tissues can have different mtDNA mutation load. Overcoming the technical limits, the studies in our present proposal provides the promise to the understand the connection between low frequent mtDNA mutations and aging from new aspect.

线粒体DNA(mtDNA)高频点突变可导致众多人类疾病。最新的研究发现线粒体DNA的低频点突变(<2%)在癌症及衰老相关疾病中也发挥了之前所忽视的重要作用。申请人前期发展了mtDNA低频点突变深度测序方法，并发现mtDNA聚合酶(Plog)基因突变导致早衰的小鼠中mtDNA突变水平的升高是由于低频点突变的位点数目增加而非已有突变位点的频率增加。进而发现Polg基因突变小鼠中有更多的C变成T，使得更多亲水氨基酸变成疏水性，提示低频点突变的累积可能是导致早衰表型的原因。后续拟系统鉴定Polg突变早衰和自然衰老小鼠不同衰老阶段的mtDNA低频点突变，通过分析其突变特征、变化规律及验证低频点突变的高表达存在与否来揭示其致衰机制。通过细胞系中定位点突变Polg基因阐明不同组织mtDNA低频点突变种类和数目不同的机制。本研究突破技术限制，针对新角度，有望加深mtDNA低频点突变与衰老关系的理解。

本项目旨在理解线粒体低频点突变在衰老鼠中的突变规律以及不同组织的特征，为理解由线粒体DNA改变所导致的衰老及相关疾病提供新线索。在4年时间里，我们通过干湿结合的技术手段，建立了线粒体低频点突变精确检测的新方法，可检测低至1ng水平的总DNA中线粒体DNA低至0.3%的点突变。利用该方法发现不同组织的低频点突变谱的差异，进而发现线粒体DNA的低频点突变先于早衰表型的出现。随后发现自然衰老鼠线粒体突变变化规律与Polg早衰鼠的差别，并且探索了单细胞水平线粒体DNA点突变的检测新方法，为后续从新角度研究单细胞中线粒体DNA点突变的异质性及其生物学意义奠定了基础。项目发表了6篇标注项目号的相关研究论文，均为最后通讯/共同通讯论文，其中三篇论文为1区（Genome Research, Nucleic Acids Research, BMC Biology）。申请并获批了一项中国发明专利，并且技术转让正在进行中。因此，本项目总体实施情况良好。