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

高通量鉴定神经精神疾病相关 GWAS 命中的因果变异

基本信息

批准号：
10569114
负责人：
JOSEPH CORBO
金额：
$ 68.34万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-10 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10569114
关键词：
Acceleration Address Affect Bar Codes Behavioral Binding Binding Sites Biological Assay Bipolar Disorder Brain Catalogs Cerebral cortex Cerebrum Chromatin Complement DNA Data Dependovirus Development Disease Electroporation Enhancers Evaluation Gene Expression General Population Genes Genomic Segment Genotype-Tissue Expression Project Goals Human Human Genetics Human Genome Individual Libraries Linkage Disequilibrium Location Maps Measures Mediating Mus Mutation Neuroglia Neurons Nucleotides Organoids Patients Persons Process Prosencephalon Protein Microchips Proteins Regulatory Element Reporter Reporter Genes Rodent Schizophrenia Techniques Technology Time Tissues Transcript Untranslated RNA Variant autism spectrum disorder candidate identification causal variant cognitive performance disorder risk falls genetic disorder diagnosis genetic variant genome wide association study genome-wide genome-wide analysis human data in utero in vivo induced pluripotent stem cell neuropsychiatric disorder neuropsychiatry promoter rare variant trait transcription factor transcriptome sequencing

项目摘要

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.

项目摘要神经精神疾病会影响全球数百万的人。全基因组关联研究（GWAS）已经确定了越来越多的与神经精神疾病相关的序列变体相关性状，但这些GWA的大多数命中均属于非编码区域，其功能效应是难以破译。我们假设大多数功能性非编码变体与神经精神疾病属于大脑顺式调节元件（CRE；即增强子/启动子），并发挥作用它们通过破坏转录因子（TF）结合位点的影响，从而改变基因的表达水平编码在大脑中表达的蛋白质，尤其是大脑皮层。识别基本的因果变体与神经精神病相关的GWAS命中并绘制与神经精神病相关的CRE，我们建议实施一种称为CRE-SEQ的技术（通过测序进行顺式调节元件分析）。在Cre-Seq中，个人 CRE与报告基因融合，每个基因都包含独特的DNA条形码。由此产生的Cre-Reporter库，由数千种结构组成，被引入活组织，并定量报告基因表达通过用RNA-Seq计数条形码转录本。 Cre-Seq有望极大地加速我们的测量能力顺式调节变体对神经精神疾病的影响。为了实现这一目标，我们提出了两个具体的目标。在AIM 1中，我们将使用CRE-SEQ来识别所有已知GWAS Loci相关的因果关系调节变体神经精神疾病和相关特征。我们将测量数千个的顺式调节活性小鼠脑脑皮质中的野生型和变体CRE在体内和人IPSC衍生的前脑器官中通过腺相关病毒（AAV）介导的Cre-Seq库的传递。然后，我们将评估使用包含所有已知人类TF的蛋白质微阵列在TF结合上的选定变体。最后，我们会的将我们的Cre-Seq分析结果与脑EQTL数据相关联。在AIM 2中，我们将建立一个模板通过系统地绘制人脑的位置来解释罕见的神经精神疾病相关的变体 CRES。我们将利用“捕获和克隆”策略进行Cre-Seq库的构建，该策略允许分析每个位置的长（即约500 bp）瓷砖记者。这样，我们将确定必需的TF绑定位点（TFBS）这是稀有功能变体的可能目标。接下来，我们将使用Cre-Seq分析将所有可能的单核苷酸取代引入已识别的TFBS。就像在AIM 1中一样，我们将执行CRE- 小鼠脑和人IPSC衍生的大脑器官的SEQ。两者一起，这两个目标将在人类基因组中启用对常见变体和稀有变异的功能解释，从而促进患者神经精神疾病风险的评估。