High throughput assessment of de novo CNV formation in eukaryotic cells

描述(由申请人提供)： 项目摘要拷贝数变异(CNV)--50个碱基对的亚显微染色体缺失或复制，超过一个百万碱基--是人类遗传变异在健康和疾病中的关键组成部分。在人类群体中，所有染色体上都能识别出数以万计的CNV。人类在许多CNV地点都有不同，通常每个人有100英镑。由于其大小，CNV经常影响基因含量，因此是多样性的重要贡献者。CNV有很高的从头突变率，估计有1-3%的个体携带父母双方都不存在的等位基因。一直以来，CNV是遗传疾病中的一种常见突变，包括智力残疾、神经精神障碍、结构性出生缺陷和其他疾病，以及体细胞突变形式的癌症。确定决定个人形成新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对候选因素进行评估。我们开发了一种人类体外细胞系统，通过将微阵列和/或配对测序应用于操纵的细胞克隆，允许在实验环境中对全基因组新生CNV的形成进行评分。这一方法证明了两种不同的复制抑制物在细胞培养中诱导CNV，这表明任何导致复制应激的条件都可能诱导有害的CNV。在这种假设检验方法中，必须研究许多进一步的环境和遗传CNV风险因素，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推广应用。这个项目的目的是开发改进和快速的实验性CNV检测方法，这将通过利用从先前工作中获得的知识来实现。将创建合理设计的检测方法并进行比较，这些检测方法仅限于CNV形成增加的热点。在目标1中，将对人成纤维细胞和淋巴母细胞进行深入的比较，以确定CNV热点的位置及其与脆性部位和转录单位的关联。这将产生一系列特征性的热点，对所有人的CNV形成具有广泛的信息。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查应用中的应用。在未来的研究中将使用优化的工具来阐明CNV的形成机制以及这些机制是如何受到环境压力的调节的。

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.