利用CRISPR/Cas9技术研究先天性眼球震颤相关FRMD7基因突变的致病机制

Idiopathic congenital nystagmus (ICN) is a genetic heterogenous disorder that characterized by involuntary horizontal oscillations of both eyes, and there is no effective treatment available. Mutations of the FERM domain containing protein 7 (FRMD7) gene are involved in the pathogenesis of X-linked ICN, which is related to neuronal outgrowth. In the previous study, we have showed that FRMD7 interacts with one of the Rho GTPase regulators RhoGDIα, then specifically activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth, and mutant FRMD7 influences Rac1 signaling activation; however, so far, the exact process of how FRMD7 regulates neuronal outgrowth and its pathogenesis of ICN are remain unclear. Recently, a new technique based on the introduction of DNA double-strand breaks by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to facilitate homologous recombination has been demonstrated to target endogenous genes in human cells effectively, which brings a new direction for the research of gene function and disease pathogenesis. On the basis of preliminary studies, this project aims to establish cell models of FRMD7 gene knock-out and ICN-associated mutations by CRISPR/Cas9 technology, furthermore to explore the role of FRMD7 in neuronal growth and the pathogenesis of ICN, providing new ideas of the pathphysiology and treatment of ICN.

先天性特发性眼球震颤（congenital idiopathic nystagmus，ICN）是一种以发作性双侧眼球摆动为典型症状的遗传异质性疾病，尚无有效治疗方法。FRMD7是X连锁ICN的致病基因，被认为与神经元的生长发育相关；前期我们研究发现FRMD7蛋白能与神经元细胞骨架的主要调节者RhoGDIα相互作用，特异性激活Rac1信号通路，突变FRMD7明显影响该功能；但FRMD7蛋白调节神经元生长的具体过程及突变的致病机制仍不清楚。近年来新的基因靶向修饰工具--规律成簇的间隔短回文重复序列（CRISPR）/Cas9 的出现为疾病机制和基因功能的研究开辟了新的方向。因此，本课题拟在前期研究基础上采用CRISPR/Cas9基因编辑技术建立FRMD7基因knock-out及ICN相关突变的细胞模型，进而深入研究其对神经元生长的调节过程及突变的致病机制，以期为ICN的发病和治疗提供新思路。

FRMD7基因是X连锁先天性特异性眼球震颤（idiopathic congenital nystagmus，ICN）的致病基因，其突变致ICN的机制尚不清楚。本项目在原有突变检测及相关信号通路研究的基础上，采用近年来较新的基因靶向修饰工具--规律成簇的间隔短回文重复序列（CRISPR）/Cas9，建立了眼球震颤相关FRMD7基因敲除和突变的细胞模型。在本研究中，我们在建立人源性神经母细胞瘤细胞系SHSY-5Y 的基础上进一步拓展，结合细胞重编程技术（直接诱导神经元技术及人诱导多潜能干细胞技术），先后在人成纤维细胞系MRC-5及AG22056 中成功敲除了FRMD7 基因，并通过直接诱导神经元技术，将相应的基因修饰细胞系诱导为神经元，发现在直接诱导神经元中，FRMD7 突变不影响成纤维细胞重编程为神经元的效率，但影响了神经元的生长，诱导后神经元突起的总长度和节点数较正常对照明显减少；同时FRMD7基因突变影响Rho GTPases的表达。另一方面，我们亦成功建立FRMD7 基因敲除的人类诱导多潜能干细胞iPSCs 模型，并进一步分化为神经元，探索突变FRMD7 蛋白对神经元生长的影响。同时我们成功建立了ICN相关FRMD7突变的SHSY-5Y细胞模型，发现FRMD7突变对神经元相关细胞骨架蛋白及Rho GTPases的影响，为ICN的发病机制提供新的理论依据。

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