High throughput assessment of de novo CNV formation in eukaryotic cells

描述（由申请人提供）： 拷贝数变异（CNVs）-亚显微染色体缺失或50个碱基对的重复超过一百万碱基-是人类健康和疾病遗传变异的关键组成部分。在人类群体的所有染色体上识别出数万个CNV。人类在许多CNV位点上存在差异，通常每个个体>100个。由于它们的大小，CNV通常会影响基因内容，因此是多样性的重要贡献者。CNV具有高的从头突变率，估计1-3%的个体携带双亲中不存在的等位基因。因此，CNV是遗传性疾病中的常见突变，包括智力残疾、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。我们开发了一种人体外细胞系统，该系统允许在实验环境中通过将微阵列和/或配对测序应用于操作的细胞克隆来对全基因组的从头CNV形成进行评分。这种方法表明，两种机制不同的复制抑制剂诱导细胞培养物中的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候选环境和遗传CNV风险因素，但目前方法的缓慢、费力和昂贵的性质阻碍了快速和广泛的扩大应用。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测定法，这些测定法仅关注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样细胞的CNV热点位置及其与脆性位点和转录单位的相关性进行深入比较。这将产生一系列表征的热点，这些热点对所有人类CNV形成具有广泛的信息。在目标2中，将开发一系列用于高通量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。优化的工具将用于未来的研究，以阐明CNV的形成机制，以及这些是如何调制的环境压力。

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.