Measuring nucleotide excision repair in human populations

测量人群中的核苷酸切除修复

• 批准号: 9789289
• 负责人: Laura Jane Niedernhofer
• 金额: $ 43.48万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-21 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789289
• 关键词: Address; Affect; American; Amish; Autoradiography; Basal cell carcinoma; Biological Assay; Biopsy; Blood; Blood Cells; Cell Cycle; Cell Line; Cell Nucleus; Cells; Chemistry; Chemotherapy-Oncologic Procedure; Child; Clinic; Clinical; DNA; DNA Damage; DNA Repair; DNA Structure; DNA biosynthesis; DNA lesion; Defect; Deoxyuridine; Dermal; Detection; Diagnosis; Diagnostic; Disease; Dose; Enrollment; Environmental Carcinogens; Environmental Exposure; Equipment; Excision; Exposure to; Failure; Family; Fibroblasts; Flow Cytometry; General Population; Genes; Genetic Polymorphism; Genome; Genomic Instability; Goals; Grain; Head and Neck Cancer; Health; Health Care Costs; Heterozygote; Human; Individual; Inherited; Knowledge; Label; Lesion; Link; Longevity; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Maryland; Measurement; Measures; Methods; Microscopy; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Nucleosides; Nucleotide Excision Repair; Nucleotides; Oligonucleotides; Patients; Peripheral Blood Mononuclear Cell; Persons; Population; Population Study; Proteins; Radioactive; Radiolabeled; Reproducibility; Residual state; Resistance; Risk; S Phase; Sampling; Series; Severities; Site; Skin; Skin Cancer; Skin Carcinoma; Spouses; Squamous Cell; Squamous cell carcinoma; Sunburn; Sunlight; Symptoms; Testing; Thymidine; Time; Tissues; UV induced; Ultraviolet Rays; United States National Institutes of Health; Universities; Xeroderma Pigmentosum; analog; cancer therapy; clinical diagnostics; cohort; design; disorder prevention; disorder risk; early onset; environmental stressor; gene product; gene repair; genetic pedigree; high risk; inter-individual variation; lymphoblast; medical schools; melanoma; minimally invasive; nucleoside analog; oncology; photolesion; placental mammal; premature; programs; repaired; sample collection; tool; ultraviolet; ultraviolet irradiation; virtual

PROJECT SUMMARY Nucleotide excision repair (NER) is a DNA repair mechanism that recognizes and removes bulky, helix-distorting lesions from the nuclear genome. Key substrates for NER are lesions induced by ultraviolet (UV) radiation upon environmental exposure to sunlight and a subset of oxidative DNA lesions produced endogenously. This is dramatically illustrated by patients with xeroderma pigmentosum (XP), a disease caused by inherited defects in NER. XP patients have a 10,000-fold increased risk of skin cancer and early onset neurodegeneration. XP is heterogeneous, ranging from mild to profoundly debilitating. XP severity is proportional to the extent to which NER is disrupted. This suggests that subtle defects in NER, due to, for example, polymorphisms in NER genes, might modestly but significantly impact one’s risk of skin cancer. Since skin cancer affects 20% of Americans and is preventable (by avoiding environmental exposure to UV), identifying those at risk could have a tremendous impact on the health of Americans and healthcare costs. The greatest barrier to identifying those at risk is the lack of an assay to measure NER that is rapid, inexpensive and applicable to samples safely and easily collected from patients. NER occurs in a series of steps involving the recognition of a site of DNA damage, unwinding the DNA locally, excision of a single-stranded oligonucleotide containing the lesion, and templated DNA synthesis to fill the residual gap. NER is the only way that UV-induced photolesions are removed from the genome in human cells. Therefore, NER is measured by the detection and quantification of UV-induced DNA synthesis outside of the S-phase of the cell cycle, or unscheduled DNA synthesis (UDS). Historically, UDS measurement required the use of radioactively-labeled nucleosides and/or specialized equipment. We developed a method to measure NER that employs the thymidine analog 5-ethynyl-2'-deoxyuridine and Click-iT chemistry for fluorescent detection of UDS by flow cytometry. This can be applied to peripheral blood cells for rapid measurement of NER requiring minimally invasive sample collection. UDS in XP patients ranges from <10% to 50%. Nothing is known about the health implications of having a UDS between 50-100%, or how to define 100% NER capacity. This project aims to correct these gaps in knowledge through optimization of our functional assay and proof-of- concept pilot human studies. The assay will be applied to existing cohorts of patients seen at the University of Miami Skin Cancer Clinics, the NIH Undiagnosed Diseases Program or enrolled in the University of Maryland Amish Longevity Study, to interrogate associations between NER capacity and high risk of skin cancer, early onset neurodegeneration, and within family pedigrees, respectively. This project will yield an NER assay applicable to larger population studies aimed at testing associations between NER capacity, environmental exposures and disease risk, and begin to define “normal” NER capacity. The assay could have a significant impact on how risk of squamous cell or basal cell carcinoma of the skin, melanoma, lung or head and neck cancer, neurodegeneration, and resistance to cancer chemotherapy is identified and managed.

项目概要 核苷酸切除修复 (NER) 是一种 DNA 修复机制，可识别并去除庞大的螺旋扭曲片段 来自核基因组的损伤。 NER 的关键底物是紫外线 (UV) 辐射引起的损伤 环境暴露于阳光下以及内源性产生的氧化 DNA 损伤的子集。这是 着色性干皮病 (XP) 患者就是一个戏剧性的例子，这是一种由遗传性缺陷引起的疾病 NER。 XP 患者患皮肤癌和早发性神经退行性疾病的风险增加 10,000 倍。经验值是 异质性，从轻度到严重衰弱。 XP 严重性与以下程度成正比： NER 被破坏。这表明 NER 中的细微缺陷，例如由于 NER 基因的多态性， 可能会适度但显着地影响一个人患皮肤癌的风险。由于 20% 的美国人患有皮肤癌 并且是可以预防的（通过避免环境暴露于紫外线），识别处于危险中的人可能会产生巨大的影响 对美国人的健康和医疗费用的影响。识别高危人群的最大障碍是 缺乏快速、廉价且适用于安全、轻松收集样本的 NER 测定方法 来自患者。 NER 发生在一系列步骤中，涉及识别 DNA 损伤位点、解开 DNA 损伤位点。 局部 DNA，切除含有病变的单链寡核苷酸，以及模板化 DNA 合成 以填补剩余的空白。 NER 是从基因组中去除紫外线诱导的光损伤的唯一方法 人体细胞。因此，NER是通过检测和定量UV诱导的DNA合成来测量的 细胞周期 S 期之外，或非计划 DNA 合成 (UDS)。从历史上看，UDS 测量 需要使用放射性标记的核苷和/或专用设备。我们开发了一种方法 使用胸苷类似物 5-乙炔基-2'-脱氧尿苷和 Click-iT 化学进行荧光测量 NER 通过流式细胞术检测UDS。这可以应用于外周血细胞以快速测量 NER 需要微创样本采集。 XP 患者的 UDS 范围为 <10% 至 50%。什么都不知道 有关 UDS 介于 50-100% 之间对健康的影响，或如何定义 100% NER 容量。这 该项目旨在通过优化我们的功能分析和证明来纠正这些知识差距 概念试点人类研究。该检测将应用于伦敦大学现有的患者队列 迈阿密皮肤癌诊所、NIH 未确诊疾病计划或就读于马里兰大学 阿米什长寿研究，旨在探讨 NER 能力与早期皮肤癌高风险之间的关系 分别是发病神经退行性变和家族谱系内。该项目将产生 NER 检测 适用于更大的人口研究，旨在测试 NER 能力、环境之间的关联 暴露和疾病风险，并开始定义“正常”NER 能力。该测定可能具有显着的 对皮肤鳞状细胞癌或基底细胞癌、黑色素瘤、肺癌或头颈癌风险的影响 癌症、神经退行性变和癌症化疗耐药性得到识别和管理。