基因组微缺失病人特异性iPSCs基因修饰及其表型效应研究

Microdeletion/microduplication syndromes are genomic disorders that result from the loss or gain of DNA material, which is caused by genomic recombination and refers to copy number variations (CNVs). Because CNVs usually involve more than one gene and/or regulatory sequence and are responsible for complex phenotypes, the underlying pathogenesis are mostly unknown. This proposal focuses on a rare case with pachygria and a de novo heterozygous microdeletion harboring only gene A and B. Having prepared the patient-specific iPSCs (induced pluripotent stem cells) for a disease model, we try to add gene A and B in situ via MMEJ (microhomology-mediated end-joining) using TALENs (Transcription activator-like effector nucleases). Meanwhile, we try to knock out gene A and/or B in normal control iPSCs by using CRISPR/Cas9 and ssODNs. Then the patient’s iPSCs and normal iPSCs with or without gene editing will be respectively differentiated to neurons in vitro. The differential transcriptome will be analyzed at specific differentiation periods to test the expression of genes correlated with neuronal migration and development, characterizing the dynamic gene regulation network and the effects of gene A and B on the pathogenesis of pachygria, which would provide a hopeful tool to explore the potential mechanisms underlying the genotype-phenotype correlation of genomic disorders..

微缺失/微重复综合征是由于基因组重排导致DNA片段的缺失/重复，即拷贝数变异，而引起临床表型的基因组疾病，因涉及多个基因和调控序列，其发病机制复杂，迄今缺乏可行的研究手段。本研究以一个携带新发微缺失的特殊病例——存在A和B基因单拷贝缺失巨脑回畸形患者为研究对象，利用病人特异性诱导多潜能干细胞（iPSCs）为疾病细胞模型，通过联合TALENs，以微同源介导的末端连接(MMEJ)方式进行A、B基因的原位添加，实现目的基因修复；同时联合CRISPR/Cas9与ssODN对正常人iPSCs行A或B基因敲除，获得目的基因缺失的iPSCs。对基因编辑前后的iPSCs进行体外神经分化，在各特殊分化时期进行转录组分析，检测神经移行和神经发育相关基因的表达情况，构建动态基因调控网络，以研究A、B基因在巨脑回畸形发生过程中的作用，并发现其可能潜在的调控机制，为基因组疾病的“基因型-表型关系”研究提供新的途径。

巨脑回畸形是由于神经元迁移异常引起的大脑皮质发育畸形，本室收集了一例典型巨脑回畸形病人，该病人携带STXBP6和NOVA1的杂合缺失突变。已有研究发现表明敲低Nova1，不影响小鼠的神经元迁移。因而在本研究中，我们成功构建了Stxbp6敲除的小鼠，对模型小鼠的大脑皮层厚度和大脑皮层分层结构进行分析，未发现Stxbp6敲除小鼠大脑皮层异常，RNA-Seq分析未发现影响神经元增殖分化和神经元迁移相关的基因和信号通路。随着干细胞技术和3D培养技术的发展，类脑器官的出现为神经系统疾病的研究提供了一个新的模型，因此我们利用CRISPR/Cas9技术在H9细胞中同时敲除STXBP6和NOVA1，并将其分化为类脑器官，重现人大脑皮层发育过程中STXBP6和NOVA1的影响。