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，由于基线突变率极低，准确量化这种遗传损害的增加一直是一项重大的技术挑战。到目前为止，未修复的复制错误随年龄积累的最好证据来自培养中的细胞类型特定选择系统或转基因小鼠模型。由于缺乏足够灵敏和可推广的生化工具，直接测量大多数人类组织中的突变频率是不可能的。本课题组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)，它能够从108多个野生型序列中识别单个碱基变化。我的初步数据证实了使用这种方法检测多拷贝人类核糖体DNA(RDNA)基因座突变频率的可行性。我建议使用这项测试来确定单个碱基替换和更大的缺失在不同组织类型中的流行程度如何在人类寿命中发生变化。 在具体目标1中，我将采用RMC方法来询问多拷贝18S核糖体RNA基因中的一个位点，以克服正常人类组织非常低的突变频率所固有的样本量限制。在具体目标2中，我将使用这种改进的方法来比较不同组织中单碱基替换的发生率和谱，这些组织来自24个年龄在80岁之间的人。在具体目标3中，我将使用这些组织样本和一种专门用于检测缺失和重排的分析形式，以确定此类基因变化的负荷如何随时间发展。 衰老表型在生理进展速度、残疾和诱发疾病的方式方面表现在不同的个体之间有很大的差异。在一生中累积的基因损伤量有可能存在类似的多样性，以解释这种变异，并最终可能作为分子衰老的独立于寿命年的标记来预测临床相关结果，如疾病风险或寿命。

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.