Assessing genome sequence integrity in normal human cells

摘要 人体细胞每天暴露于DNA损伤剂， 和外源性来源，导致广泛的DNA损伤，从DNA断裂， 在正常体细胞中，可能多达100，000个损伤 每日几乎所有的损伤都能通过一系列复杂的基因组维护快速修复 系统，虽然有错误。这些错误导致DNA突变，这可能会因碱基而异。 取代和小的缺失或插入到较大的基因组结构变异，包括 染色体畸变突变是真正的分子终点，是细胞丧失的直接指标。 基因组序列完整性DNA突变会导致癌症，也与年龄有关- 相关疾病以及衰老本身。为了研究人类的突变，最广泛应用的 测定是选择标记测定（例如，HGPRT），但它们只能应用于可以 培养和克隆，当然只包括基因组的一小部分，排除了更多的基因。 突变负荷的全球评估。随着下一代测序技术的出现， 突变可以定量评估，但仅限于克隆谱系，如肿瘤，其中 相当一部分细胞具有相同的突变。在正常组织中，体细胞突变是 丰度非常低，不能与测序错误区分。检测基因突变 正常细胞和组织需要单细胞方法或极高的精确度， 在测序的大量DNA中区分真正的突变和伪像。我们最近开发了 经验证的下一代基于测序的检测方法，用于检测大多数（如果不是所有）类型的突变 使用大量DNA和基于单细胞的方法。在这里，我们建议进一步整合， 优化并验证这些检测试剂盒，使其成为第一个下一代基于测序的突变分析 该系统提供了对正常人类细胞中基因组序列完整性的全面了解。为 根据这项建议，该试验将专门用于测量烟草烟雾的致突变作用， 检验血液或口腔粘膜细胞突变反映基因组完整性丧失的假设 在吸烟者和非吸烟者的肺中与肺癌的关系。在目标1中，我们将开发和 验证用于测量基因组完整景观的集成和自动化测定 体外暴露于烟草烟雾冷凝物的上皮细胞的不稳定性。在目标2中，我们将验证 人支气管、口腔和血细胞基因组序列完整性的整合检测 暴露在体内，与烟草烟雾暴露和肺癌病例对照状态。的 在拟议的项目中开发的分析将有可能允许，第一次，使用全球 基因组序列完整性作为评估个体肺癌风险的终点， 暴露于烟草烟雾，反映在非侵入性标本适合流行病学 问题研究

ABSTRACT Human somatic cells are exposed to DNA damaging agents on a daily basis from both endogenous and exogenous sources, resulting in a broad range of DNA lesions, varying from DNA breaks and abasic sites to bulky adducts and crosslinks, in normal somatic cells perhaps up to 100,000 lesions per day. Virtually all of that damage is quickly repaired through a complex set of genome maintenance systems, albeit with errors. Such errors result in DNA mutations, which can vary from base substitutions and small deletions or insertions to larger genome structural variation, including chromosomal aberrations. Mutations are true molecular end points, direct indicators of loss of genome sequence integrity. DNA mutations cause cancer and have also been implicated in age- related diseases as well as aging itself. To study mutations in humans, the most widely applied assays are selectable marker assays (e.g., HGPRT), but they can only be applied on cells that can be cultured and cloned, and of course comprise only a very small part of the genome, precluding a more global assessment of mutation loads. With the advent of next-generation sequencing, somatic mutations can be quantitatively assessed, but only in clonal lineages, such as tumors, in which a substantial fraction of cells harbor the same mutations. In normal tissues, somatic mutations are of very low abundance and cannot be distinguished from sequencing errors. Detecting mutations in normal cells and tissues requires either a single cell approach or extremely high accuracy in distinguishing a true mutation from an artifact in sequenced bulk DNA. We recently developed and validated next generation sequencing-based assays for detecting most if not all types of mutations using both bulk DNA and single cell-based approaches. Here we propose to integrate, further optimize and validate these assays into the first next-generation sequencing-based mutation analysis system that provides comprehensive insight in genome sequence integrity in normal human cells. For this proposal, the assay will be tailored to measuring the mutagenic effects of tobacco smoke for testing the hypothesis that mutations in blood or buccal mucosal cells reflect loss of genome integrity in human lung in smokers and non-smokers in relation to lung cancer. In Aim 1, we will develop and validate an integrated and automated assay for measuring the complete landscape of genome instability in epithelial cells exposed in vitro to tobacco smoke condensate. In Aim 2, we will validate the integrated assay for genome sequence integrity in human bronchial, buccal and blood cells exposed in vivo, in relation to tobacco smoke exposure and lung cancer case-control status. The assay developed in the proposed project would potentially allow, for the first time, the use of global genome sequence integrity as an endpoint to assess individual risk of lung cancer in relation to exposure to tobacco smoke, as reflected in non-invasive specimens amenable to epidemiologic studies.