Ultrasensitive measure of human mutagenesis: Connecting the exposome to disease

项目总结 体细胞突变是癌症和其他与年龄相关的疾病背后的驱动力，但 它们在人类身上的起源机制是模棱两可的。这项提议寻求从根本上解决 核诱变过程和确定体细胞突变作为生物标记物的有效性 在具有良好特征的人群中的个人环境暴露。这些目标现在是可行的。 由于一种名为CypherSeq的新突变检测技术的开发，该技术利用循环 条形码模板、滚动圆圈放大和大规模并行测序可准确量化 整个基因组的体细胞突变具有前所未有的敏感性。首先，CypherSeq方法将是 用于建立无疾病人类体细胞突变的全基因组图谱，并测试 在整个基因组中，突变的频率、分布和谱随年龄变化。第二, 增殖和静止的人类细胞将用已知的诱变剂处理，诱变将被 由CypherSeq追踪。这些实验将探索细胞增殖和细胞生长之间的关系， 转录、DNA修复和体细胞突变，同时也发现了基因组的高度突变区域， 可作为监测人群中急性和慢性诱变剂暴露的场所。此外，这一点 AIM还将测试这一假设，即修复和突变固定需要增殖 基因组的转录区域。最后，在第三个目标中，将描述强大的突变靶点 并用来监测长期忍受癌症的人的血液中的突变频率， 暴露在烟熏煤中会致癌。这项横断面研究将检验以下假设： 烟煤的致癌性是其可能增加接触者体细胞突变频率的原因 个人。因此，预计终生接触烟雾煤的增加将与 突变诱变。成功完成拟议的目标将为人类提供新的见解 突变，并强调了监测体内突变诱导作为一种生物标志物的潜在用途 诱变性环境暴露；从而识别出患癌症风险增加的个人。 最终，基于突变的生物标记物有可能对癌症风险进行分层，为指导 医疗干预、生活方式改变(即限制诱变剂暴露)、早期诊断和/或应用 化学预防措施，从而拯救生命。

PROJECT SUMMARY Somatic mutations are the driving force behind carcinogenesis and other age-related diseases, yet the mechanisms underlying their genesis in humans are ambiguous. This proposal seeks to resolve fundamental processes of nuclear mutagenesis and ascertain the utility of monitoring somatic mutation as a biomarker of individual environmental exposure within a well-characterized population. These objectives are now feasible thanks to the development of a novel mutation detection technology termed CypherSeq, which utilizes circular barcoded templates, rolling circle amplification, and massively parallel sequencing to accurately quantify somatic mutation throughout the genome with unprecedented sensitivity. First, the CypherSeq method will be used to establish a genome-wide profile of somatic mutation in disease-free humans, and test how the frequency, distribution, and spectrum of mutation change with age throughout the genome. Second, proliferating and quiescent human cells will be treated with known mutagens, and induced mutagenesis will be tracked by CypherSeq. These experiments will explore the relationships between cell proliferation, transcription, DNA repair, and somatic mutation, while also uncovering highly mutable regions of the genome, which might serve as sites to monitor acute and chronic mutagen exposure in human populations. Further, this Aim will also test the hypothesis that proliferation is required for repair and mutation fixation within non- transcribed regions of the genome. Lastly, in the third Aim, robust mutational target sites will be characterized and used to monitor mutation frequency in blood draws from individuals who have endured a long-term, carcinogenic exposure to smoky coal. This cross-sectional study will test the hypothesis that underlying the carcinogenicity of smoky coal is its potential to increase the frequency of somatic mutation in exposed individuals. As such, it is expected that increased lifetime exposure to smoky coal will positively correlate with mutation induction. Successful completion of the proposed Aims would provide new insights into human mutagenesis, and highlight the potential utility of monitoring in vivo mutation induction as a biomarker of mutagenic environmental exposure; and thus identify individuals with an elevated risk of developing cancer. Ultimately, mutation-based biomarkers have the potential to stratify cancer risk, providing a basis to direct medical intervention, lifestyle changes (i.e. limiting mutagen exposure), early diagnosis, and/or the application of chemopreventive measures, and thus save lives.