Somatic Mutations in Human Aging

描述（由申请人提供）：体细胞突变的积累已被提出在人类衰老过程中发挥重要作用。然而，由于突变的基线率极低，精确地量化这种基因损伤的增加一直是一项重大的技术挑战。迄今为止，未修复的复制错误随着年龄的增长而积累的最佳证据来自培养或转基因小鼠模型中的细胞类型特异性选择系统。由于缺乏足够敏感和通用的生化工具，在大多数人体组织中无法直接测量突变频率。我们的团队最近开发了一种基于限制性内切酶的突变检测方法，称为随机突变捕获（RMC），该方法能够在超过108个野生型序列中识别单个碱基变化。我的初步数据建立了使用这种方法测定多拷贝人类核糖体DNA （rDNA）位点突变频率的可行性。我建议使用这种分析来确定在人类的一生中，不同组织类型中单碱基替换和较大缺失的流行程度是如何变化的。

DESCRIPTION (provided by applicant): The accumulation of somatic mutations has been proposed to play an important role in the human aging process. Precisely quantifying this increase in genetic damage, however, has been a major technical challenge because of the extremely low baseline rate of mutation. To date, the best evidence that unrepaired replication errors accumulate with age comes from cell type-specific selection systems in culture or from transgenic mouse models. Direct measurement of mutation frequency in most human tissues has been precluded by a lack of sufficiently sensitive and generalizable biochemical tools. Our group has recently developed a restriction endonuclease-based method for mutation detection known as Random Mutation Capture (RMC), which has the ability to identify a single base change among more than 108 wild type sequences. My preliminary data establish the feasibility of using this approach to assay mutation frequency at the multi-copy human ribosomal DNA (rDNA) locus. I propose to use this assay to determine how the prevalence of single base substitutions and larger deletions changes in different tissue types over the human lifespan. In Specific Aim 1, I will adapt the RMC method to interrogate a site in the multi-copy 18s ribosomal RNA gene to overcome sample-size limitations inherent to the very low mutation frequency of normal human tissues. In Specific Aim 2, I will use this improved assay to compare the prevalence and, spectrum of single base substitutions in different tissues obtained from 24 individuals ranging eighty years in age. In Specific Aim 3, I will use these tissue samples and a form of the assay that is specific for detection of deletions and rearrangements, to determine how the load of such genetic alterations develops with time. The aging phenotype is highly diverse in how it manifests across different individuals with respect physiological rate of progression and mode of disability and disease induced. It is possible that a similar diversity in the quantity of genetic damage that accrues over the course of a lifetime exists to explain this variation and might ultimately have utility as a life-year-independent marker of molecular aging to predict clinically relevant outcomes such as disease risk or lifespan.