Assessing genome sequence integrity in normal human cells

评估正常人类细胞的基因组序列完整性

• 批准号: 10408704
• 负责人: SIMON D SPIVACK
• 金额: $ 66.61万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408704
• 关键词: Aging; Animal Model; Biological Assay; Blood; Blood Cells; Blood specimen; Cancer Etiology; Cell Fraction; Cells; Chromosome abnormality; Complex; DNA; DNA Damage; DNA Repair; DNA Sequence Alteration; DNA lesion; Disease; Epidemiology; Epithelial Cells; Exposure to; Genes; Genome; Genomic Instability; HPRT1 gene; Human; Human Genome; Hypoxanthine Phosphoribosyltransferase; In Vitro; Individual; Lesion; Libraries; Link; Lung; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Methods; Mitosis; Molecular; Morphologic artifacts; Mus; Mutation; Mutation Analysis; Normal Cell; Normal tissue morphology; Oral mucous membrane structure; Preparation; Process; Risk; Sampling; Site; Smoker; Smoking; Somatic Cell; Somatic Mutation; Source; Specimen; Standardization; System; Systems Analysis; Testing; Time; Tobacco smoke; Transgenic Model; Variant; adduct; age related; base; bronchial epithelium; case control; cell type; computational pipelines; crosslink; data analysis pipeline; design; environmental tobacco smoke exposure; epidemiology study; former smoker; genome integrity; genome-wide; in vivo; insight; mutation assay; never smoker; next generation sequencing; non-smoker; pilot test; repaired; repository; time use; tumor; virtual; whole genome

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.

抽象的 人类体细胞细胞每天都暴露于DNA损伤剂中。 和外源性来源，导致广泛的DNA病变，与DNA断裂和 在正常的体细胞中，可笨重的加合物和交叉链接的卑鄙位点可能多达100,000个病变 每天。几乎所有这些损害通过一组复杂的基因组维护迅速修复 系统，尽管有错误。这种错误导致DNA突变，可能因碱而有所不同 替换和少量缺失或插入更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，直接指标丢失 基因组序列完整性。 DNA突变引起癌症，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，最广泛的应用 测定是可选的标记分析（例如，HGPRT），但只能应用于可以被应用的单元格 被培养和克隆，当然只包含很小的基因组，而排除了更多 突变负荷的全球评估。随着下一代测序的出现，躯体 突变可以定量评估，但仅在克隆谱系中，例如肿瘤，其中a 大量细胞具有相同的突变。在正常组织中，体细胞突变为 非常低的丰度，不能与测序误差区分开。检测突变 正常细胞和组织需要单个细胞方法或极高的精度 区分真实突变与测序的散装DNA中的伪影。我们最近开发了 经过验证的下一代测序测定法，用于检测大多数（如果不是所有类型的突变） 使用散装DNA和单细胞的方法。在这里，我们建议整合，进一步 优化并验证这些测定中的第一个基于下一代测序的突变分析 在正常人细胞中提供了基因组序列完整性的全面见解的系统。为了 该提案，该测定法将是针对测量烟草烟的诱变作用而定制的。 检验以下假设，即血液或颊粘膜细胞中反映了基因组完整性的丧失 与肺癌有关的吸烟者和非吸烟者中的人肺。在AIM 1中，我们将发展并 验证一个集成和自动化的测定，以测量基因组的完整景观 在体外暴露于烟草烟雾的上皮细胞中的不稳定性。在AIM 2中，我们将验证 基因组序列完整性的综合测定在人支气管，颊和血细胞中 与烟草烟雾暴露和肺癌病例对照状态有关。这 在拟议项目中开发的测定可能首次允许全球使用 基因组序列完整性作为评估肺癌的个体风险的终点 暴露于烟草烟雾中，反映在适合流行病学的非侵入性标本中 研究。