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.

项目摘要 核苷酸惊喜修复（NER）是一种DNA修复机制，可识别和去除庞大的螺旋延伸 核基因组的病变。 NER的关键底物是紫外线（UV）辐射引起的病变 内源性产生的环境暴露于阳光和氧化DNA病变的一部分。这是 由心脏病患者（XP）动态说明，这种疾病是由遗传的缺陷引起的 ner。 XP患者患皮肤癌的风险增加了10,000倍，并提早发作神经退行性变化。 XP是 异质性，从轻度到深度衰弱。 XP严重程度与 ner被破坏了。这表明，由于NER基因中的多态性，NER中的细微缺陷， 可能适度，但会显着影响一个人的皮肤癌风险。由于皮肤癌影响20％的美国人 并且是可以预防的（避免环境暴露于紫外线），确定有风险的人可能会有很大的 影响美国人和医疗保健费用的健康。确定有危险者的最大障碍是 缺乏评估来测量快速，廉价且适用于安全易于收集的样品的NER 来自患者。 NER发生在一系列步骤中，涉及对DNA损伤部位的识别，并放松 DNA局部，切除含有病变的单链寡核苷酸和模板DNA合成 填补剩余差距。 NER是从基因组中删除紫外线诱导的照片的唯一途径 人类细胞。因此，通过检测和定量UV诱导的DNA合成来测量NER 在细胞周期的S期之外，或者外的DNA合成（UDS）。从历史上看，UDS测量 需要使用放射标记的核苷和/或专用设备。我们开发了一种方法 测量采用胸苷类似物5-乙基尼尔2'-脱氧尿苷的NER和Click-It化学性质 通过流式细胞仪检测UDS。这可以应用于外围血细胞，以快速测量NER 需要微创样本收集。 XP患者的UDS范围从<10％至5​​0％不等。什么都不知道 关于使UD在50-100％之间或如何定义100％NER容量之间的健康含义。这 项目旨在通过优化我们的功能测定和证明证明，旨在纠正知识中的这些差距 概念飞行员人类研究。该测定法将应用于大学的现有患者队列 迈阿密皮肤癌诊所，NIH未诊断的疾病计划或马里兰州大学 阿米什人的寿命研究，以询问NER容量与皮肤癌高风险之间的关联，早期 发作神经变性，分别在家庭血统中。该项目将产生NER分析 适用于较大的人群研究，旨在测试NER容量，环境之间的关联 暴露和疾病风险，并开始定义“正常”的能力。该测定可能具有重要的 影响皮肤，黑色素瘤，肺或头颈的鳞状细胞或基本细胞癌的风险 鉴定和管理癌症，神经退行性和对癌症化学疗法的抗性。