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：
8717661
负责人：
THOMAS EDWARD WILSON
金额：
$ 19.24万
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 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8717661
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 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 Structural Congenital Anomalies 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）：项目摘要拷贝数变体（CNV） - 超显微染色体缺失或50个碱基对的重复与超级巨头比更多 - 是健康和疾病中人类遗传变异的关键组成部分。在所有染色体的人口中，数以万计的CNV被认可。人类在许多CNV站点上有所不同，通常每个人> 100。由于其大小，CNV通常会影响基因含量，因此是多样性的重要贡献。 CNV具有较高的从头突变率，估计有1-3％的个人在任何父母中都不存在等位基因。一致地，CNV是遗传疾病中的常见突变，包括智力障碍，神经精神疾病，结构性先天缺陷等，以及以体细胞突变形式的癌症。确定确定个人形成新CNV风险的遗传和环境因素，要求在前瞻性，假设驱动和受控的实验中评估候选因素。我们开发了人类的体外细胞系统，该系统允许通过在操纵细胞克隆中应用微阵列和/或伴侣测序在实验环境中对新的CNV形成基因组进行评分。这种方法表明，两个机械上不同的复制抑制剂在细胞培养中诱导CNV，这表明任何导致复制应激的条件都可能引起有害的CNV。在这种假设测试方法中，必须研究许多进一步的候选环境和遗传CNV风险因素，但是当前方法的缓慢，费力和昂贵的性质阻碍了快速而广泛的扩展应用。该项目的目的是开发改进和快速的实验CNV检测方法，这将通过利用从先前的努力中获得的知识来实现。将创建和比较理性设计的测定法，这些测定最有限地关注增加CNV形成的热点。在AIM 1中，将对CNV热点的位置以及它们与脆弱的位点和转录单元的关联进行深入比较。这将为所有人类CNV形成提供广泛信息的特征热点。在AIM 2中，将开发一系列用于高通量CNV检测的创新方法，并将其用于实验和筛选应用的效用。将来的研究将使用优化的工具来阐明CNV形成的机制以及如何通过环境应力调节这些机制。