Genetic Perturbation of a Schizophrenia Susceptibility Locus in Isogenic Human Ne

摘要 精神分裂症（SZ）是一种毁灭性的大脑疾病，约占人口的1%。最近SZ全基因组 关联研究（GWAS）涉及几十个基因座，但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，以便进行更有效的干预。一个最 强相关的SZ基因座跨越两个脑表达的microRNA：MIR 137调节神经元分化， 成熟和突触功能，大约一半的SZ GWAS基因座含有MIR 137靶点，这表明 MIR 137在SZ基因网络中的中心枢纽作用。MIR 2682的功能知之甚少，但它被预测为 TargetScan6.2靶向锚蛋白3（ANK 3），一种与GWAS中双相情感障碍相关的基因。了解 MIR 137 SZ基因座的因果机制，本R21申请将解决这三个关键问题 问题：1）在许多同等相关的SNP中，哪些是功能性的，2）哪些基因和哪些基因 神经元的类型受风险变体的影响，以及3）风险变体是否影响SZ相关细胞 表型。我们将利用GWAS所涉及的常见的监管常见SNP和罕见的 增强子SNP通过我们的重测序鉴定。我们希望这种罕见的增强子SNP能提供无与伦比的 对因果机制的见解，因为它的影响大;发现风险等位基因减少报告基因 表达> 60%。从诱导多能干细胞（iPSC）分化的神经元正在成为一种新的神经元。 病理生理学相关的细胞模型，用于研究大脑疾病。我们已经展示了 MIR 137/MIR 2682在这些神经元中的表达。我们的假设是，罕见和常见的SZ- 风险变体降低了iPSC衍生神经元中MIR 137/MIR 2682的表达，导致SZ相关细胞凋亡。 表型变化我们提出了一个严谨和创新的方法：1）而不是简单地比较几个 来自SZ病例与对照的iPSC，这通常不足以将表型差异归因于遗传因素。 由于iPSC之间可变的遗传背景，我们将产生仅不同的同基因iPSC系， 通过使用有效的转录激活因子样效应物核酸酶（TALEN）介导的 基因组编辑2)为了克服iPSC衍生的神经元的异质性，我们将同时分析iPSC衍生的神经元。 单个神经元表达不同亚型特异性标志物以及MIR 137/MIR 2682及其受体。 靶基因，然后在每种亚型的神经元内检查SZ风险等位基因对基因表达的影响。 表情结合使用iPSC，基因组编辑和单神经元表达谱，我们将继续研究 两个具体的目的：（1）在统一的基因库上产生仅在每个SZ风险变体上不同的同基因iPSC系， 遗传背景，和（2）确定在同基因iPSC衍生的皮层神经元中SZ风险的影响 等位基因对MIR 137/MIR 2682表达的影响，以及对SZ相关基本细胞表型的影响。

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.