High-throughput identification of causal variants underlying neuropsychiatric disease-related GWAS hits

项目摘要 神经精神疾病影响着全世界数百万人。全基因组关联研究 已经确定了越来越多的与神经精神疾病相关的序列变异和 相关特征，但这些GWAS命中的大多数属于非编码区，它们的功能效应是 很难破译。我们假设大多数功能非编码变体与 神经精神疾病属于大脑顺式调节元件(CRE；即增强剂/启动子)，并 它们通过破坏转录因子(TF)结合位点从而改变基因的表达水平来发挥作用 编码在大脑中表达的蛋白质，尤其是大脑皮层。找出潜在的因果变异 为了绘制与神经精神疾病相关的CRE图，我们建议 实施一种称为CRE-SEQ(顺式排序的调控元素分析)的技术。在CRE-SEQ中，个人 CRE与报告基因融合，每个基因包含一个唯一的DNA条形码。得到的Cre-Report文库， 由数千个构建物组成，被引入活组织，并对报告基因的表达进行量化 用RNA-seq对条码转录本进行计数。CRE-SEQ承诺极大地提高我们的测量能力 顺式调节变异体在神经精神疾病中的作用。为了实现这一目标，我们提出了两个具体的建议 目标。在目标1中，我们将使用cre-seq在所有已知的与Gwas相关的基因座上识别因果顺式调控变异 有神经精神疾病和相关特征。我们将衡量数以千计的顺式监管活动 野生型和变异型Cres在活体小鼠大脑皮层和人IPSC来源的前脑器官中的表达 腺相关病毒(AAV)介导的Cre-seq文库传递。然后，我们将评估其功能效果 使用包含所有已知人类TF的蛋白质微阵列对TF结合的选定变体。最后，我们将 将我们的CRE-SEQ分析结果与大脑eQTL数据相关联。在目标2中，我们将为 通过系统定位人脑来解释罕见的神经精神疾病相关变异 克雷斯。我们将利用“捕获和克隆”策略构建CRE-seq文库，这将允许分析 每个地点的记者都长(即~500个BP)。通过这种方式，我们将精确定位必需的转铁蛋白结合位点(TFBS) 它们可能是稀有功能变体的目标。接下来，我们将使用CRE-seq来分析 将所有可能的单核苷酸替换引入已识别的TFBS。与目标1相同，我们将执行CRE- 在小鼠大脑和人类IPSC衍生的脑有机体中都存在SEQ。这两个目标加在一起，将 能够对单个人类基因组中常见和罕见的变异进行功能解释，从而 促进对患者神经精神疾病风险的评估。

Project Summary Neuropsychiatric diseases affect millions of people world-wide. Genome-wide association studies (GWAS) have identified a growing number of sequence variants associated with neuropsychiatric diseases and related traits, but the majority of these GWAS hits fall within non-coding regions and their functional effects are difficult to decipher. We hypothesize that the majority of functional non-coding variants related to neuropsychiatric disease fall within brain cis-regulatory elements (CREs; i.e., enhancers/promoters), and exert their effects by disrupting transcription factor (TF) binding sites and thereby altering the expression level of genes encoding proteins expressed in the brain, particularly the cerebral cortex. To identify causal variants underlying neuropsychiatric disease-related GWAS hits and to map neuropsychiatric disease-related CREs, we propose to implement a technique called CRE-seq (Cis-Regulatory Element analysis by sequencing). In CRE-seq, individual CREs are fused to reporter genes, each containing a unique DNA barcode. The resultant CRE-reporter library, consisting of thousands of constructs, is introduced into living tissue, and reporter gene expression is quantified by counting barcoded transcripts with RNA-seq. CRE-seq promises to greatly accelerate our ability to measure the effects of cis-regulatory variants in neuropsychiatric disease. To achieve this goal, we propose two Specific Aims. In Aim 1, we will use CRE-seq to identify causal cis-regulatory variants at all known GWAS loci associated with neuropsychiatric diseases and related traits. We will measure the cis-regulatory activity of thousands of wild-type and variant CREs in mouse cerebral cortex in vivo and in human iPSC-derived forebrain organoids via adeno-associated virus (AAV)-mediated CRE-seq library delivery. We will then evaluate the functional effects of selected variants on TF binding using protein-microarrays containing all known human TFs. Lastly, we will correlate the results of our CRE-seq analyses with brain eQTL data. In Aim 2, we will establish a template for interpreting rare neuropsychiatric disease-related variants by systematically mapping the location of human brain CREs. We will utilize a 'capture and clone' strategy for CRE-seq library construction, which permits analysis of long (i.e., ~500 bp) tiled reporters at each locus. In this way, we will pinpoint essential TF binding sites (TFBSs) which are the likely targets of rare functional variants. Next, we will use CRE-seq to analyze the effects of introducing all possible single-nucleotide substitutions into identified TFBSs. As in Aim 1, we will perform CRE- seq in both mouse brain and human iPSC-derived cerebral organoids. Taken together, these two Aims will enable functional interpretation of both common and rare variants in individual human genomes and thereby facilitate assessment of neuropsychiatric disease risk in patients.