Burden and signatures of somatic mutations in genomes of healthy individuals.

由于遗传和环境因素，体细胞突变会在个体的一生中积累。 越来越明显的是，体细胞基因组的变化与一系列病理有关，包括 癌症。对不同癌症类型的基因组测序表明，不同细胞之间的突变负荷有所不同 体型和整个身体。这些变异与不同的dna损伤暴露有关。 药物和细胞的复制潜力。此外，DNA损伤导致的突变负载 还取决于细胞以无错误的方式修复损伤的能力。然而， 在健康个体中，可归因于环境和内在因素的跨细胞类型的突变负荷不是 为人所知。此外，目前尚不清楚DNA修复基因中的多态如何影响修复效率和 改变暴露在环境DNA损伤中的细胞以及未暴露的细胞中的突变格局。 这项建议的目标是确定体细胞基因组在体内和不同物种中的变化程度 并审查造成这种差异的机制。为了实现这一目标，我将 探索以下目标。在Aim1中，我将直接分析与DNA修复相关的多态的影响 用正交法研究癌症对DNA修复能力的影响。利用基于质粒的宿主细胞再激活 在检测中，我将测试带有纯合子次要(突变)或主要(野生型)等位基因的淋巴细胞的修复效率。 我还将确定这些突变的人类基因是否会增加酵母和 人类细胞在暴露于外源DNA损伤时和在无挑战的生长期间。在AIM2中，我将 确定有害单核苷酸多态(SNPs)在DNA修复基因中的作用。突变 导致功能性MBD4糖基酶的丢失，已被证明增加了C--T的变化 癌症基因组中的二核苷酸。我将测试被预测对MBD4蛋白有害的SNPs 还会增加健康个体的体细胞中的突变负载和改变特征。在Aim3，我会 估计从来自不同身体部位的相同个体分离的不同细胞类型中的突变负荷。这就做 评估与已知环境和内源相关的突变特征的贡献 诱变源对样本中的突变负荷。这项建议的研究工作完成后， 为我提供细胞培养，人类细胞系的基因操作和生物信息学方面的专业知识，铺平 成为一名独立研究人员的成功之路。 意义：这些研究将增加我们对环境和遗传相互作用的理解 决定体细胞诱变的因素。这些结果对于理解易感性是很重要的。 个体对癌症和其他与体细胞突变相关的疾病的风险，以及在设计个体- 具体的疾病预防策略。

Somatic mutations accumulate over the lifetime of an individual due to both genetic and environmental factors. It is becoming evident that somatic genome changes are associated with a host of pathologies including cancers. Sequencing genomes of different cancer types suggested that mutation loads vary between cell types and across the body. The variations have been associated with differential exposure to DNA damaging agents and the replicative potential of the cells. In addition, mutation loads due to DNA damaging lesions would also be dependent on the ability of the cells to repair damage in an error-free manner. However, the mutation loads attributable to environmental and intrinsic factors across cell types in healthy individuals are not known. Also, it is not known how polymorphisms within DNA repair genes compromise repair efficiency and alter the mutation landscape in cells exposed to environmental DNA damage as well as in unexposed cells. The goal of this proposal is to determine the extent of somatic genome changes within the body and in different individuals and to examine the mechanisms that contribute to this variability. To address this goal, I will explore the following aims. In Aim1, I will directly analyze the impact of DNA repair polymorphisms associated with cancers on DNA repair capacity using orthogonal systems. Using plasmid-based host cell reactivation assays, I will test repair efficiency in lymphocytes with homozygous minor (mutant) or major (wild-type) alleles. I will also determine if these mutant human genes increase mutation and recombination rates in yeast and human cells upon exposure to exogenous DNA damage and during unchallenged growth. In Aim2, I will determine the role of deleterious single nucleotide polymorphisms (SNPs) in DNA repair genes. Mutations leading to loss of a functional MBD4 glycosylase, have been shown to increase CT changes in CpG dinucleotides in cancer genomes. I will test if SNPs that are predicted to be deleterious to the MBD4 protein also increase mutation loads and altering signatures in somatic cells from healthy individuals. In Aim3, I will estimate mutation loads in different cell-types isolated from the same individuals from different body sites. I will assess the contributions of mutation signatures associated with known environmental and endogenous mutagenic sources to mutation loads in the samples. The completion of the studies in this proposal will provide me with expertise in cell culture, genetic manipulation of human cell lines and bioinformatics, paving the way for a successful career as an independent researcher. Significance: These studies will increase our understanding of the interplay of environmental and genetic factors that determine somatic mutagenesis. These results are important for understanding the susceptibility of individuals to cancers and other diseases associated with somatic mutagenesis, and in designing individual- specific disease prevention strategies.