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 （GWAS）已经鉴定了越来越多的与神经精神疾病相关的序列变异， 相关性状，但这些GWAS命中的大多数属于非编码区，其功能效应是 很难破译我们假设，大多数功能性非编码变异与 神经精神疾病属于脑顺式调节元件（克雷斯;即，增强子/启动子），并发挥 通过破坏转录因子（TF）结合位点，从而改变基因的表达水平， 编码在大脑，特别是大脑皮层中表达的蛋白质。为了识别潜在的因果变异， 神经精神疾病相关的GWAS命中和映射神经精神疾病相关的克雷斯，我们建议 CRE-seq（顺式调控元件测序分析）。在CRE-seq中，个体 克雷斯与报告基因融合，每个基因含有独特的DNA条形码。由此产生的CRE记者库， 由数千个构建体组成，被引入活组织中， 通过RNA-seq计数条形码转录本。CRE-seq有望大大加快我们测量 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来鉴定所有已知的GWAS基因座的因果顺式调节变体， 患有神经精神疾病和相关特征。我们将测量数千个 在体内小鼠大脑皮层和人iPSC衍生的前脑类器官中的野生型和变体克雷斯， 腺相关病毒（AAV）介导的CRE-seq文库递送。然后，我们将评估 使用含有所有已知人类TF的蛋白质微阵列对TF结合的选择变体。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，我们将建立一个模板 通过系统地绘制人类大脑的位置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”策略进行CRE-seq文库构建，其允许分析 长（即，~500 bp）在每个基因座上平铺报告基因。通过这种方式，我们将精确定位必需的TF结合位点（TFBS）。 这是罕见的功能性变体的可能目标。接下来，我们将使用CRE-seq来分析 将所有可能的单核苷酸取代引入鉴定的TFBS中。如目标1所述，我们将进行综合招聘考试- seq在小鼠脑和人iPSC衍生的脑类器官中的表达。这两个目标合在一起， 能够对个体人类基因组中的常见和罕见变异进行功能解释， 有助于评估患者的神经精神疾病风险。

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.