Genetic Perturbation of a Schizophrenia Susceptibility Locus in Isogenic Human Ne

同基因人类神经元中精神分裂症易感位点的遗传扰动

• 批准号: 8621279
• 负责人: Jubao Duan
• 金额: $ 19.5万
• 依托单位: NORTHSHORE UNIVERSITY HEALTHSYSTEM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621279
• 关键词: Address; Affect; Alleles; Ankyrins; Bipolar Disorder; Brain; Brain Diseases; Cell Line; Cell model; Cells; Characteristics; Cognitive; DNA Repository; DNA Resequencing; Development; Disease model; Drug Design; Enhancers; Etiology; Excitatory Synapse; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Risk; Genome; Glutamates; Heterogeneity; Human; Individual; Inhibitory Synapse; Knowledge; Life; Link; Luciferases; Mediating; MicroRNAs; Minor; Molecular; Molecular Profiling; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Nucleic Acid Regulatory Sequences; Pathway interactions; Patients; Phenotype; Play; Population; Predisposition; Reporter Genes; Risk; Role; Schizophrenia; Synapses; Synaptic Transmission; Therapeutic; Therapeutic Uses; Transcription Coactivator; Universities; Variant; density; effective intervention; genetic variant; genome wide association study; induced pluripotent stem cell; innovation; insight; interest; migration; molecular phenotype; neuroblastoma cell; novel; nuclease; public health relevance; rare variant; risk variant; screening; small molecule; synaptic function

ABSTRACT Schizophrenia (SZ) is a devastating brain disorder afflicting ~1% of population. Recent SZ genome-wide association studies (GWAS) have implicated dozens of loci, but causal mechanism for each locus is largely unknown - such knowledge would help to identify novel targets for more effective intervention. One of the most strongly associated SZ loci spans two brain-expressed microRNAs: MIR137 regulates neuronal differentiation, maturation and synaptic function, and about half of SZ GWAS loci contain MIR137 targets, suggesting a central hub role for MIR137 in a SZ gene network. MIR2682 has little known function, but it is predicted by TargetScan6.2 to target ankyrin 3 (ANK3), a gene associated with bipolar disorder in GWAS. To understand the causal mechanism underlying the MIR137 SZ locus, this R21 application will address these three key questions: 1) which of the many equivalently associated SNPs are functional, 2) which genes and in what types of neuron are affected by the risk variants, and 3) whether the risk variants impact SZ-relevant cellular phenotypes. We will leverage both the putatively regulatory common SNPs implicated by GWAS and a rare enhancer SNP identified by our resequencing. We expect the rare enhancer SNP to provide unparalleled insights on the causal mechanism because of its large effect; the risk allele was found to reduce reporter gene expression by >60%. Neurons differentiated from induced pluripotent stem cells (iPSCs) are emerging as a pathophysiologically relevant cellular model for studying brain disorders. We have shown robust expression of MIR137/MIR2682 in such neurons. Our hypothesis is that the risk alleles of both the rare and the common SZ- risk variants reduce expression of MIR137/MIR2682 in iPSC-derived neurons, resulting in SZ-relevant cellular phenotypic changes. We present a rigorous and innovative approach: 1) Instead of simply comparing a few iPSCs from SZ cases vs. controls, which is often underpowered to attribute phenotypic differences to a genetic variant due to variable genetic backgrounds between iPSCs, we will generate isogenic iPSC lines differing only at the SNP of interest by using an efficient transcription activator-like effector nuclease (TALEN)-mediated genome editing. 2) To overcome the heterogeneity of iPSC-derived neurons, we will simultaneously profile in single neurons the expression of different subtype-specific markers as well as of MIR137/MIR2682 and their target genes, and then examine within each subtype of neurons the effect of the SZ risk allele on gene expression. Combining the use of iPSC, genome editing, and single neuron expression profiling, we will pursue two specific aims: (1) To generate isogenic iPSC lines differing only at each of the SZ-risk variants on a unified genetic background, and (2) To determine in isogenic iPSC-derived cortical neurons the effects of SZ-risk alleles on expression of MIR137/MIR2682, and on SZ-relevant basic cellular phenotypes.

抽象的 精神分裂症（SZ）是一种毁灭性的脑部疾病，遭受了约1％的人口。近期SZ全基因组 关联研究（GWAS）与数十个基因座有关，但是每个基因座的因果机制在很大程度上是 未知 - 这种知识将有助于确定新的目标以进行更有效的干预。最大的 密切相关的SZ基因座跨越了两个脑表达的microRNA：miR137调节神经元分化， 成熟和突触功能，大约一半的SZ GWAS基因座包含miR137目标，表明一个 MiR137在SZ基因网络中的中心枢纽。 miR2682的功能鲜为人知，但由 Targetscan6.2靶向Ankyrin 3（ANK3），该基因与GWAS中的双相情感障碍有关。理解 MiR137 SZ基因座基础的因果机制，该R21应用程序将解决这三个键 问题：1）许多等效相关的SNP中的哪一个是功能性的，2）哪些基因以及什么 神经元的类型受风险变异的影响，以及3）风险变体是否影响SZ相关的细胞 表型。我们将利用GWAS涉及的推定的调节性共同SNP和罕见的 通过我们的重新陈述确定的增强剂SNP。我们希望稀有增强器SNP提供无与伦比的 洞察因果机制，因为其效果很大；发现风险等位基因可减少报告基因 表达> 60％。与诱导多能干细胞（IPSC）区分开的神经元正在作为一个 用于研究脑疾病的病理生理相关的细胞模型。我们已经显示出强大的表达 此类神经元中的miR137/miR2682。我们的假设是，罕见和常见的SZ-的风险等位基因 风险变异降低IPSC衍生神经元中miR137/miR2682的表达，导致相关的细胞 表型变化。我们提出了一种严格而创新的方法：1）而不是简单地比较一些 来自SZ病例与控件的IPSC通常不足以将表型差异归因于遗传 由于IPSC之间的遗传背景变化而引起的变体，我们将生成仅不同的IPSC线。 通过使用有效的转录激活剂样效应核酸酶（TALEN）介导的介导的SNP 基因组编辑。 2）要克服IPSC衍生神经元的异质性，我们将同时介绍 单神经元的表达不同亚型特异性标记以及mir137/mir2682及其 靶基因，然后在神经元的每个亚型中检查SZ风险等位基因对基因的影响 表达。结合IPSC，基因组编辑和单个神经元表达谱分析的使用，我们将追求 两个具体的目的：（1）生成仅在统一上的每个SZ风化变体上不同的等源IPSC线 遗传背景和（2）确定在IPENIC IPSC衍生的皮质神经元中SZ-Fisk的影响 关于miR137/miR2682的表达等位基因，以及与SZ相关的基本细胞表型。