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.

项目概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）已经鉴定出越来越多与神经精神疾病相关的序列变异相关性状，但这些 GWAS 命中中的大多数属于非编码区域，其功能影响是难以破译。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) 结合位点从而改变基因的表达水平来发挥作用编码在大脑，特别是大脑皮层中表达的蛋白质。识别潜在的因果变异神经精神疾病相关的 GWAS 命中并绘制神经精神疾病相关的 CRE，我们建议实施一种称为 CRE-seq（顺式调控元件测序分析）的技术。在 CRE-seq 中，个体 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 报告库，由数千个构建体组成，被引入活体组织，并对报告基因表达进行定量通过 RNA-seq 计数带条形码的转录本。 CRE-seq 有望大大提高我们的测量能力顺式调节变异对神经精神疾病的影响。为实现这一目标，我们提出两个具体方案目标。在目标 1 中，我们将使用 CRE-seq 来识别所有已知 GWAS 相关基因座的因果顺式调控变异。患有神经精神疾病和相关特征。我们将衡量数千个的顺式监管活动小鼠大脑皮层体内和人类 iPSC 衍生的前脑类器官中的野生型和变异型 CRE 腺相关病毒（AAV）介导的CRE-seq文库交付。然后我们将评估功能效果使用包含所有已知人类 TF 的蛋白质微阵列选择 TF 结合变体。最后，我们将将我们的 CRE-seq 分析结果与大脑 eQTL 数据关联起来。在目标 2 中，我们将建立一个模板通过系统地绘制人脑位置来解释罕见的神经精神疾病相关变异商业地产 (CRE)。我们将利用“捕获和克隆”策略来构建 CRE-seq 文库，从而可以分析每个位点都有长（即~500 bp）平铺报告基因。通过这种方式，我们将查明重要的 TF 结合位点 (TFBS) 这些是罕见功能变体的可能目标。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到已识别的 TFBS 中。与目标 1 一样，我们将执行 CRE- 小鼠大脑和人类 iPSC 衍生的大脑类器官中的 seq。综合起来，这两个目标将能够对人类个体基因组中常见和罕见的变异进行功能解释，从而促进患者神经精神疾病风险的评估。