Systematic Functional Interpretation of Regulatory Variants in Neuropsychiatric Disorders

摘要 尽管在神经精神疾病的全基因组关联研究（GWAS）中风险位点不断增加， 确定致病变异/基因一直是一项挑战，这阻碍了GWAS发现的转化 新的疾病生物学。一个主要的障碍是，大多数风险变异位于DNA的非编码区， 易于解释的功能。非编码调控序列通常位于开放染色质区域（OCR）。 在我们最初的生产性R 01期，我们以人诱导多能干细胞（hiPSC）神经元作为模型， 已经在影响染色质可及性的OCR中鉴定了丰富的调节变体，表现出等位基因- 特异性开放染色质（ASoC）。ASoC SNP经常影响基因表达，并强烈富集 精神分裂症风险变异然而，精神分裂症和其他神经精神疾病的大多数致病变体/基因， 疾病仍然未知。因为调控变体通常在特定的生物学背景下起作用，例如，蜂窝 刺激或干扰，我们假设许多神经精神疾病的变异可能会改变染色质 仅在激活的神经元中的可及性和基因表达。各种神经元刺激引起膜 去极化，导致小鼠神经元中的稳健的活性依赖性染色质和表达变化。 我们在KCl去极化的人类神经元中的试验数据也显示了大量的活性依赖性染色质， 值得注意的是，表达变化与数百个活性依赖性ASoC SNP有关，其中一些是 精神分裂症风险变异利用易于处理的hiPSC模型，该模型可以在以下情况下被扰动： 遗传变异，这种竞争性更新申请将解决三个具体问题：（1）在多大程度上 遗传变异影响神经活性依赖的染色质可及性和基因表达？为此， 我们将分析细胞类型特异性染色质和表达在单细胞分辨率在基线和 激活的神经元，并进行数量性状基因座（QTL）作图，以确定 活性依赖性ASoC和表达QTL（eQTL）。(2)活动依赖型的贡献是什么 神经精神疾病的调节变体为此，我们将联合分析ASoC和eQTL SNP， 神经精神病学GWAS数据集，用于精细绘制影响活性依赖性染色质的因果疾病变体 和表达，然后进行多重CRISPR碱基编辑以验证它们的功能和顺式靶基因。（三） 活性依赖性染色质变化的机制是什么？为此，我们将研究活动是否- 依赖的染色质区域富含特异性转录因子（TF），并探索 转录因子对人类神经元中染色质可及性、表达和细胞表型的CRISPR编辑这 这项研究将产生新的机制的见解神经活动依赖的染色质的贡献， 神经精神疾病的表达变化。

ABSTRACT Despite the mounting risk loci in genome-wide association studies (GWAS) of neuropsychiatric disorders, identifying the causal variants/genes has been challenging, which hinders the translation of GWAS findings into novel disease biology. A major hurdle is that most risk variants lie in noncoding regions of DNA without easily interpretable function. Noncoding regulatory sequences often reside in open chromatin regions (OCRs). With human induced pluripotent stem cell (hiPSC) neurons as a model in our initial productive R01 period, we have identified abundant regulatory variants in OCRs that affect chromatin accessibility, exhibiting allele- specific open chromatin (ASoC). ASoC SNPs frequently affect gene expression and are strongly enriched for schizophrenia risk variants. However, most causal variants/genes of schizophrenia and other neuropsychiatric disorders remain unknown. Because regulatory variants often act in specific biological context, e.g., cellular stimulation or perturbation, we hypothesize that many neuropsychiatric disease variants may alter chromatin accessibility and gene expression only in activated neurons. Various neuronal stimuli cause membrane depolarization, resulting in robust activity-dependent chromatin and expression changes in mouse neurons. Our pilot data in KCl-depolarized human neurons also showed substantial activity-dependent chromatin and expression changes, notably, with hundreds of activity-dependent ASoC SNPs some of which are schizophrenia risk variants. Leveraging the tractable hiPSC model that can be perturbed in the context of genetic variation, this competitive renewal application will address three specific questions: (1) To what extent genetic variation influences neural activity-dependent chromatin accessibility and gene expression? For this, we will assay cell type-specific chromatin and expression at single-cell resolution in both baseline and activated neurons of a well-powered hiPSC cohort, and perform quantitative trait loci (QTL) mapping to identify activity-dependent ASoC and expression QTL (eQTL). (2) What is the contribution of activity-dependent regulatory variants to neuropsychiatric disorders? For this, we will jointly analyze ASoC and eQTL SNPs with neuropsychiatric GWAS datasets to fine-map causal disease variants that affect activity-dependent chromatin and expression, followed by a multiplex CRISPR base editing to validate their function and cis-target genes. (3) What is the mechanism of activity-dependent chromatin changes? For this, we will examine whether activity- dependent chromatin regions are enriched for specific transcriptional factors (TFs), and explore the effects of CRISPR-editing of TFs on chromatin accessibility, expression, and cellular phenotypes in human neurons. This study will yield novel mechanistic insights into the contribution of neural activity-dependent chromatin and expression changes to neuropsychiatric disorders.