High throughput assessment of de novo CNV formation in eukaryotic cells

真核细胞中从头 CNV 形成的高通量评估

基本信息

批准号：
8582129
负责人：
THOMAS EDWARD WILSON
金额：
$ 22.49万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-06 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8582129
关键词：
Address Affect Alleles Aphidicolin Base Pairing Biological Assay Cell Culture Techniques Cell Line Cells Chromosomal Duplication Chromosome Deletion Chromosome Fragile Sites Chromosome abnormality Chromosomes Chromosomes, Human, Pair 6 Clone Cells Computational algorithm Congenital Abnormality DNA Polymerase Inhibitor Data Detection Development Disease Dose Ensure Environmental Risk Factor Eukaryotic Cell Event Fibroblasts Frequencies Future Genes Genetic Genetic Transcription Genetic Variation Genome Genomics Hand Health Hereditary Disease Human Human Genetics In Vitro Individual Intellectual functioning disability Knowledge Libraries Location Loss of Heterozygosity Malignant Neoplasms Maps Methods Mutation Nature Parents Partner in relationship Pathway interactions Population Positioning Attribute RNA Replication Origin Ribonucleotide Reductase Inhibitor Risk Risk Factors Series Site Somatic Cell Somatic Mutation Specificity Stress Structure System Techniques Testing Variant cell type density design digital genome-wide hydroxyurea improved inhibitor/antagonist innovation insight neuropsychiatry novel prospective public health relevance rapid technique repaired research study screening skills tool

项目摘要

DESCRIPTION (provided by applicant): PROJECT SUMMARY Copy number variants (CNVs) - submicroscopic chromosomal deletions or duplications of 50 base pairs to more than a megabase - are a critical component of human genetic variation in both health and disease. Tens of thousands of CNVs are recognized in the human population across all chromosomes. Humans differ at many CNV sites, typically >100 per individual. Because of their size, CNVs often affect gene content and are thus an important contributor to diversity. CNVs have a high de novo mutation rate, with an estimated 1-3% of individuals bearing an allele not present in either parent. Consistently, CNVs are a common mutation in genetic disorders, including intellectual disability, neuropsychiatric disorders, structural birth defects and others, as well as cancer in the form of somatic mutations. Identifying the genetic and environmental factors that determine an individual's risk of forming new CNVs requires that candidate factors be assessed in prospective, hypothesis- driven and controlled experiments. We developed a human in vitro cell system that allows scoring of de novo CNV formation genome-wide in an experimental setting through application of microarrays and/or mate-pair sequencing to manipulated cell clones. This approach demonstrated that two mechanistically distinct replication inhibitors induce CNVs in cell culture, suggesting that any condition that leads to replication stress might induce deleterious CNVs. Many further candidate environmental and genetic CNV risk factors must be studied in this hypothesis-testing approach, but the slow, laborious and expensive nature of current methods has impeded rapid and extensive expanded application. The purpose of this project is to develop improved and rapid methods for experimental CNV detection, which will be achieved by exploiting knowledge gained from prior efforts. Rationally designed assays will be created and compared that have a restricted focus on hotspots of increased CNV formation. In Aim 1, an in-depth comparison of human fibroblasts and lymphoblastoid cells will be performed with respect to the location of CNV hotspots and their association with fragile sites and transcription units. This will yield a se of characterized hotspots broadly informative to all human CNV formation. In Aim 2, a series of innovative approaches for high throughput CNV detection will be developed and their utility compared for experimental and screening applications. Optimized tools will be used in future studies to elucidate the mechanisms of CNV formation and how these are modulated by environmental stresses.

描述（由申请人提供）：项目摘要拷贝数变异 (CNV)——50 个碱基对至超过 1 个兆碱基的亚显微染色体缺失或重复——是人类健康和疾病遗传变异的重要组成部分。人类所有染色体上都有数以万计的 CNV。人类在许多 CNV 位点上存在差异，通常每个人 >100 个。由于其大小，CNV 通常会影响基因内容，因此是多样性的重要贡献者。 CNV 具有很高的从头突变率，估计有 1-3% 的个体携带亲本中不存在的等位基因。一致地，CNV 是遗传性疾病的常见突变，包括智力障碍、神经精神疾病、结构性出生缺陷等，以及体细胞突变形式的癌症。确定决定个体形成新 CNV 风险的遗传和环境因素需要在前瞻性、假设驱动和对照实验中评估候选因素。我们开发了一种人类体外细胞系统，通过将微阵列和/或配偶对测序应用于操纵的细胞克隆，可以在实验环境中对全基因组范围内的从头 CNV 形成进行评分。这种方法证明了两种机制不同的复制抑制剂在细胞培养物中诱导 CNV，这表明任何导致复制应激的条件都可能诱导有害的 CNV。许多进一步的候选环境和遗传 CNV 风险因素必须在这种假设检验方法中进行研究，但当前方法的缓慢、费力和昂贵的性质阻碍了快速和广泛的扩展应用。该项目的目的是开发改进且快速的实验性 CNV 检测方法，这将通过利用先前工作中获得的知识来实现。将创建并比较合理设计的检测方法，这些检测方法将重点放在 CNV 形成增加的热点上。在目标 1 中，将对人成纤维细胞和类淋巴母细胞的 CNV 热点位置及其与脆弱位点和转录单位的关联进行深入比较。这将产生一系列对所有人类 CNV 形成具有广泛信息的特征热点。在目标 2 中，将开发一系列高通量 CNV 检测的创新方法，并比较它们在实验和筛选应用中的效用。未来的研究将使用优化的工具来阐明 CNV 形成的机制以及环境压力如何调节这些机制。