Genomic and epigenomic effects of large CNV in neurons from iPSC

描述(由申请人提供) 摘要：我建议研究大基因组拷贝数变异(CNV)通过哪些分子机制控制基因，动态地影响细胞分化和细胞功能。我们的目标是在我们的实验室建立一种新的研究计划和范例，将诱导多潜能干细胞(IPSC)的使用与对基因组和表观基因组层控制和活性的多层次、全面和集成的分析相结合。在这个过程中，我们将测试几个关于CNV影响基因调控的分子机制的新假说，从而影响细胞和最终的生物表型。这也将为更小的CNV及其对健康和疾病表型的潜在微妙和累积或组合但潜在的实质性影响提供一个模型。拷贝数变异(CNV)在健康人的基因组中很常见，并与表型变异有关。CNV还经常与主要疾病表型密切相关，特别是在涉及大脑异常发育的神经精神疾病中，如精神分裂症和自闭症。现在有几个与精神分裂症、自闭症、智力低下和癫痫等神经精神疾病密切相关的CNV的突出例子(22q11.2缺失、1q21.1缺失和复制、15q13.3缺失、16p11.2缺失和复制、3q29微复制)。CNV(有时是如上所述的相同CNV)也与心脏畸形疾病以及涉及免疫系统功能的表型密切相关。因此，CNV本身是一个值得研究的重要现象，既是疾病的强烈易感因素，也是更好地了解具有强烈和复杂的遗传和基因组成分的复杂疾病的分子病因学的诱人切入点。更好地理解CNV如何影响表型的分子机制的一个主要障碍是无法接触到携带与CNV相关的特定疾病的相关人体组织，例如，具有与精神分裂症或自闭症密切相关的大量基因组缺失的神经组织培养。使用来自具有给定CNV的先证者的IPSC系有望克服这一障碍，并将使我们能够在分子水平上观察CNV在相关组织中的影响，随着CNV沿着发育轨迹发展，然后稳定到神经元表型。基于大规模并行DNA下一代测序的完整和多水平高分辨率基因组学和表观基因组学分析将包括通过RNA-Seq、meDNA-Seq和ChIP-Seq进行全面的转录组分析，以研究DNA甲基化模式和绘制调节组蛋白修饰和转录因子网络。 公共卫生相关性：神经发育和神经精神障碍，如精神分裂症和自闭症谱系障碍，是一个主要的公共卫生问题。拷贝数变异是与这类疾病有最强关联的候选基因座之一，但它们通常影响多个基因，而且对其分子作用机制只有很少的基本知识。我们的项目将通过将IPSC技术与全面的下一代基因组分析相结合，创建一种新的研究范式，研究CNV在神经元发育过程中的作用。

DESCRIPTION (Provided by the applicant) Abstract: I propose to study through which molecular mechanisms of genetic control it is that large genomic Copy Number Variants (CNVs) exert their effects on gene regulation, dynamically over cellular differentiation and cellular functioning. We aim to establish a novel research program and paradigm in our laboratory that combines the use of induced Pluripotent Stem Cells (iPSC) and multi-level, comprehensive and integrated analysis of genomic and epigenomic layers of control and activity. In the process we will be testing several novel hypotheses on the molecular mechanisms through which CNVs affect gene regulation and thus cellular and eventually organismal phenotypes. This will also serve as a model for smaller CNVs and their potentially subtle and cumulative or combinatorial yet potentially substantive effects on phenotype in health and disease. Copy Number Variation (CNV) is common in the genome of healthy humans and is associated with phenotypic variation. CNV is also frequently and strongly associated with major disease phenotypes, especially in neuropsychiatric diseases that involve an aberrant development of the brain such as schizophrenia and autism. There are now several prominent examples for CNVs that are strongly associated with neuropsychiatric disorders such as schizophrenia, autism, mental retardation and epilepsy (22q11.2 deletions, 1q21.1 deletions and duplications, 15q13.3 deletions, 16p11.2 deletions and duplications, 3q29 microduplications). CNVs (and sometimes the same CNVs as mentioned above) are also strongly associated with malformation diseases of the heart as well as in phenotypes that involve the functioning of the immune system. CNVs are therefore an important phenomenon to study both in its own right as a strong predisposing factor for disease as well as an enticing point of entry for the better understanding of the molecular etiology of complex diseases with a strong and complex genetic and genomic component. A major barrier to a better understanding of how the molecular mechanisms through which CNV affect phenotype is the lack of access to relevant human tissues that carry a given disease associated CNV, for example neuronal tissue cultures with a large genomic deletion that is strongly associated with schizophrenia or autism. The use of iPSC lines from probands with a given CNV promises to overcome this barrier and will allow us to observe the effects of CNVs on a molecular level in the relevant tissue as it progresses along a developmental trajectory and then settles into the neuronal phenotype. The complete and multilevel high-resolution genomics and epigenomics analyses based on massively parallel DNA next-generation-sequencing will include comprehensive transcriptome analyses by RNA-Seq, meDNA- Seq to study DNA methylation patterns and ChIP-Seq to map regulatory histone modifications and transcription factor networks. Public Health Relevance: Neurodevelopmental, neuropsychiatric disorders such as schizophrenia and autism spectrum disorders are a major public health concern. Copy Number Variations are amongst the candidate loci with the strongest association with such diseases but they typically affect multiple genes and only very little basic knowledge about their molecular mechanisms of action exists. Our project will create a new research paradigm into the effects of CNV during neuronal development by combining iPSC techniques with comprehensive nextgeneration genomics analyses.