ARHGEF2基因缺陷引起小头畸形、中后脑发育异常和智力低下的致病机制研究

In a case with a novel form of microcephaly, midbrain-hindbrain malformation, and intellectual disability, we identified a candidate disease causal gene, namely, ARHGEF2. We confirmed the dramatically decreased expression of ARHGEF2 in the patients’peripheral blood cells in terms of both mRNA and protein. We also showed that, when injecting shRNA into the brain ventricles of mouse fetus in order to knock down the Arhgef2 expression, the proliferation and migration of the neural precursor cells were blocked; over-expression of the wild-type ARHGEF2 could rescue the affected phenotype. This gene encodes a protein that controls the Rho GTPase activation, thus affects cell cycle. Till now, there has been no report that the defect in this gene can cause disease. We intend to construct an Arhgef2-knockin mouse model by the CRISPR-Cas9 genome-editing technique, and carry out experiments, including observing the motion and cognition changes, the anatomical changes in the midbrain and hindbrain, effects on the RHOA activity, effects on cell cycle by phosphorylated histone H3, and the genome-scale assays on mRNA, lncRNA, miRNA, circRNA and DNA methylation so as to understand the perturbation in signaling pathway and cell-cycle gene network. Based on our experiments, we will manage to confirm the involvement of ARHGEF2 in the central nervous system development, and reveal the etiological mechanisms in the aspects of functional genomics.

我们前期从罕见的小头畸形、中后脑发育异常和智力低下家系中首次鉴定出致病新基因ARHGEF2。预实验证明突变患者外周血细胞ARHGEF2的mRNA和蛋白表达均显著下降；往胎鼠脑室注射shRNA敲低Arhgef2表达会干扰神经元前体细胞的增殖和迁移，而过表达野生型ARHGEF2能拯救表型。ARHGEF2控制Rho三磷酸鸟苷酸酶催化活性，影响细胞有丝分裂，以往未有报道其突变能引起疾病。本项目正在用CRISPR-Cas9技术构建小鼠Arhgef2突变敲入模型；观察突变型小鼠运动、认知能力变化和脑部结构异常；检测突变对细胞周期若干标记分子活性影响；继而检测RNA表达谱和DNA甲基化谱，结合基因网络数据库解析基因缺陷引起表型的分子调控网络。如此，我们将确证ARHGEF2突变造成中枢神经系统发育异常，并从个体、组织、细胞、分子等层面系统阐明其致病机制，为寻找有效干预分子靶点提供科学依据。

本项目针对ARHGEF2基因缺陷引起原发性小头畸形、中后脑发育异常和智力低下的临床现象展开功能研究，从个体、组织、细胞和分子等层面系统阐述其致病机制，确证ARHGEF2基因在中枢神经系统早期发育中的关键作用，并证明其缺陷会导致神经元前体细胞的增殖、迁移和分化受阻，临床表征和致病机理达成一致，为下一步寻求精准治疗靶点提供有效的科学依据。

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