THE HUMAN BRAINOME III: EQTL REGULATION BY NATURAL ANTISENSE RNA IN ALZHEIMER S DISEASE

项目摘要 以前，我们已经采取了一种创新的方法（人类脑组; [1-6]）来绘制风险基因座， 迟发性阿尔茨海默病（LOAD）。而不是像大多数基因组那样只看一层信息- 广泛关联（GWAS）研究，我们已经绘制了下游背景下的基因组变异， 转录组学和蛋白质组学表达。这允许映射LOAD中涉及的关键变化， 以及下游效应及其方向。此外，它还允许构建多个 对疾病进程至关重要的参与者。 目前工作的一个缺点是，我们绘制的DNA表达关系微妙地 但是我们还没有完全理解为什么这些DNA表达关系在阿尔茨海默氏症中会发生变化， 改变了我们知道，表达是由特定的等位基因改变，但必须有额外的调节，因为我们的基因， 映射输出。可以改变途径的一个靶标是天然反义转录物（NAT），其可以结合 寡核苷酸产物并改变其表达和降解。在我们的应用程序中，我们建议使用 长读测序技术（SMRT;单分子，实时）和完整的档案RNA从我们的人类 脑库样本我们将研究这些产出的位置，并开展初步工作， 确定这些新的命中是否可以作用于我们现有的结果。 我们建议通过三个目标来实现这些目标。目标1将涉及从公共数据库中跟踪命中。 目标2将涉及收集额外的RNA分析数据。最后，目标3将寻求验证和订购所有新的 目标1和2的结果。重要的是使用适合我们假设的技术来获取新数据 收藏.大多数非编码RNA属于称为长非编码RNA的转录物类别 （lncRNA），其可以跨越1000- 10，000 bp [7]。典型的短读RNA测序（SRS）技术 基于捕获~150 bp的短序列，因此，SRS难以捕获， 更长的产品。我们正在与伊坎基因组研究所的罗伯特·塞布拉合作， Multicale Biology，他是SMRT测序的专家[8]。这项技术提供了更长的读取长度， 因为大多数人类RNA分析涉及SRS。 通过这些目标的实现，我们将拥有1。人脑中新的长读段测序图谱 组织，这将是一个重要的补充领域，因为迄今为止使用的大多数技术都集中在短读 测序，并且在病理学定义的人脑组织中存在有限的谱分析，2.了解 这些新的命中如何影响感兴趣的直接有义转录本以及已知的LOAD 病理学，3.通过使用多重捕获，对靶标的精确性和再现性进行多级映射 技术和4。通过测量两种方法验证命中对已知LOAD致病性靶标的影响 在细胞培养物中的表达和蛋白水平。

PROJECT SUMMARY Previously, we have taken an innovative approach (The Human Brainome; [1-6]) to mapping risk loci for late onset Alzheimer's disease (LOAD). Rather than looking at a single layer of information as in most genome- wide association (GWAS) studies, we have mapped genomic variation in the context of downstream transcriptomic and proteomic expression. This allows for mapping both the crucial variation involved in LOAD, as well as the downstream effects and their directions. Additionally, it allows for building networks of multiple players crucial for disease processes. One shortcoming of the current work is that we have mapped DNA-expression relationships that are subtly changed in Alzheimer's, but we have yet to fully understand why those DNA-expression relationships are altered. We know that expression is altered by specific alleles, but there must be added regulation given our mapped outputs. One target that can alter pathways are natural antisense transcripts (NATs), which can bind to oligonucleotide products and alter their expression and degradation. In our application, we propose to use long read sequencing technology (SMRT; Single Molecule, Real-Time) and fully profile RNA from our human brain bank samples. We will examine where these outputs are located and perform preliminary work to determine if any of these new hits can act on our existing results. We propose to follow these targets through 3 Aims. Aim 1 will involve following hits from public databases. Aim 2 will involve collecting additional RNA profiling data. Finally, Aim 3 will seek to validate and order all novel findings from Aims 1 and 2. It is important to use technologies appropriate to our hypothesis for the new data collection. The majority of non-coding RNA belongs to the class of transcripts called long non-coding RNA (lncRNA), which can span from 1000-10,000 bp [7]. Typical short-read RNA sequencing (SRS) technologies are based on the capture of short sequences of ~150 bp, and therefore, SRS has difficulty in capture and alignment of longer products. We are working with Robert Sebra at the Icahn Institute for Genomics and Multicale Biology, who is an expert in SMRT sequencing [8]. This technology offers longer read lengths and will be unique-in-field, since most human RNA profiling involves SRS. By the completion of these Aims, we will have 1. A map of novel long read sequencing in human brain tissues, which will be a significant add-to-field, given most technologies used to date are focused on short read sequencing, and there is limited profiling in pathologically defined human brain tissues, 2. An understanding of how these novel hits are affecting both the direct sense transcript of interest as well as the known LOAD pathologies, 3. Multi-level mapping of rigor and reproducibility of targets through the use of multiple capture techniques and 4. Validation of the effect of hits on the known LOAD pathogenic targets by measuring both expression and protein levels in cell culture.