CNTNAP2基因在新生儿惊厥中的作用机制

Neonatal seizures are common diseases, which have complex causes. The causes in some newborns are unknown. Genetic factors may be important in neonatal seizures. In our previous study, we found a neonate with seizures carrying CNTNAP2 gene mutation, suggesting that CNTNAP2 gene is a candidate gene that responsible for neonatal seizures. Previous studies on the CNTNAP2 gene, confirmed that the genes involved in a variety of developmental disorders of the nervous system, but its role in neonatal seizures is unclear. Therefore, we will do the following things in this project: 1. Analyze the genotype of CNTNAP2 in unexplained seizures in newborns; 2. Get the fibroblasts from the patient with CNTNAP2 gene mutations, and induced the fibroblasts into pluripotent stem cells, and then further induced into neurons. Neuronal morphology and electrophysiological characteristics, and the role of CNTNAP2 gene in neuronal differentiation and maturation will be investigated; 3. Reverse the mutation into wild type by CRISPR/Cas9 method, and analyze whether the function of the nervous system is reversed or not; 4. Build zebrafish model with CNTNAP2 gene mutation, and observe the behavioral changes and investigate the associated mechanisms. Through these studies, we want to clarify the role and mechanism of CNTNAP2 gene in neonatal seizures.

新生儿惊厥是常见疾病，部分患儿原因不明，遗传因素可能是其重要致病机制。本课题组通过全外显子组测序，在一家系中发现携带CNTNAP2基因突变的惊厥新生儿，提示CNTNAP2基因是导致新生儿惊厥的候选基因。CNTNAP2基因涉及多种神经系统发育障碍，但在新生儿惊厥发生中的作用尚不明确。因此，本课题拟：1.在不明原因惊厥新生儿队列中，进行CNTNAP2基因型分析；2.将CNTNAP2基因突变患儿皮肤成纤维细胞诱导分化为多能干细胞后进一步分化为神经元，观察CNTNAP2基因突变神经细胞的形态及电生理特征，并观察CNTNAP2基因对神经细胞分化成熟及功能的影响及其调控机制；3.利用CRISPR/Cas9的方法，将突变回复为野生型，分析是否对神经系统功能产生回复作用；4.构建CNTNAP2基因突变斑马鱼模型，进行行为学观察和惊厥关关联机制研究，以期阐明CNTNAP2基因在新生儿惊厥中的作用及其机制。

CNTNAP1基因编码蛋白CASPR是一种参与髓鞘形成的粘附分子，然而到目前为止报道的CNTNAP1相关神经发育异常病例均表现出胚胎晚期及新生儿期的神经发育异常表型，这与典型的CHN存在不同。典型的CHN如CMT综合征、异染性脑白质营养不良MLD、肾上腺脑白质营养不良ALD，主要表现为初生时和婴儿早期小儿发育正常，以后逐渐出现运动少，肌肉张力低，逐渐失去维持姿势的能力，而癫痫发作较少。提示CNTNAP1基因在神经元发育中的重要作用，但其机制尚不清楚。本课题组发现两例患儿携带CNTNAP1复合杂合突变，均表现出胚胎期的发育异常。通过体外培养小鼠原代皮层神经元及小鼠胚胎晚期体内皮层神经元的迁移实验，发现当CNTNAP1基因被敲减后，小鼠原代皮层神经元细胞发育异常包括数突长度及分支明显减少，且小鼠皮层神经元胚胎晚期迁移障碍。这些研究结果提示，CNTNAP1基因在神经元的形态发育及胚胎晚期皮层发育过程中具有重要的作用，解释了CNTNAP1基因相关神经发育异常疾病的胚胎晚期及新生儿期严重的疾病表型。