Systematic Functional Interpretation of Regulatory Variants in Neuropsychiatric Disorders

摘要 尽管神经精神疾病的全基因组关联研究(GWAS)中的风险基因不断增加， 确定因果变异/基因一直是具有挑战性的，这阻碍了GWAS调查结果的翻译 变成了新的疾病生物学。一个主要的障碍是大多数风险变异存在于DNA的非编码区，而不是 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)。 以人类诱导多能干细胞(HiPSC)神经元为模型，在我们最初的R01生产性时期，我们 已经在OCR中发现了大量影响染色质可及性的调控变异体，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，并强烈富含 精神分裂症的风险变种。然而，精神分裂症和其他神经精神疾病的大多数因果变异/基因 疾病仍然是未知的。因为调节变异体通常在特定的生物环境中起作用，例如细胞 刺激或干扰，我们假设许多神经精神疾病的变种可能改变染色质 仅在激活的神经元中可及性和基因表达。各种神经元刺激引起膜 去极化，导致强健的活性依赖的染色质和小鼠神经元的表达变化。 我们在氯化钾去极化的人神经元上的实验数据也显示了大量的活性依赖染色质和 值得注意的是，表达发生了变化，数百个依赖于活动的ASOC SNP，其中一些是 精神分裂症的风险变种。利用易于处理的HiPSC模型，该模型可以在 关于遗传变异，这一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？为了这个， 我们将在基线和单细胞分辨率下检测特定细胞类型的染色质和表达 激活功能强大的HiPSC队列中的神经元，并执行数量性状基因座(QTL)定位以识别 活性依赖的ASOC和表达QTL(EQTL)。(2)依赖活动的贡献是什么？ 神经精神障碍的调控变种？为此，我们将联合分析asoc和eQTL SNPs 神经精神病学GWAS数据集精细定位影响活动依赖染色质的因果疾病变异 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。(3) 依赖活性的染色质改变的机制是什么？为此，我们将审查活动是否- 依赖的染色质区域富含特定的转录因子(TF)，并探索 CRISPR-编辑转录因子对人类神经元染色质可及性、表达和细胞表型的影响。这 这项研究将对神经活动依赖的染色质和 神经精神障碍的表情变化。

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.