维甲酸降解关键酶CYP26B1基因新发突变致神经管畸形的作用机制

Retinoic acid (RA) is a known teratogen of Neural tube defects (NTDs). Its distribution and levels in developing central nervous system are precisely controlled by the degradative enzymes of the cytochrome P450 subfamily (CYP 26). It has been reported that CYP26B1 gene haploinsufficiency or homozygous mutation in human cause lethality, craniofacial abnormalities. Our preliminary finding from high-though target sequencing indicates that single nuclide variant of CYP26B1 significantly attacked in NTDs compared with the matched controls (case vs. control: 9:0). To study whether NTDs pathogenesis induced by CYP26B1 mutations through RA dysregualtion, we will detect the expression level of CYP26B1, RA and its receptors by human brain tissues and mouse neural stem cells carrying missense mutation found in human cases. Furthermore, using ChIP-Seq, RNA-Seq et al. methods, the changes in RA receptors regulated candidate genes which are crucial for neural tube closing will be explored. Subsequently, the screening profile of changes will be validated in human samples. It is respected that the study will explain the pathogenic mechanism of rare genetic variant of CYP26B1 in the aspect of dysregualtion of RA pathways, further display the experimental evidence in heritable etiology of NTDs.

维甲酸（RA）信号异常通路可导致神经管畸形（NTDs）发生，胚胎中枢神经系统RA水平和分布受其降解酶CYP26的精确控制。人CYP26B1基因的纯合点突变和杂合缺失可引起颅面部畸形，我们前期大样本高通量测序发现CYP26B1基因变异富集于NTDs样本（病例 vs. 对照 9∶0），其中一个变异位于剪接区, 这些变异是否引起NTDs，机制如何？为探索该基因变异是否通过RA信号通路异常导致NTDs发生，本课题拟采用人NTDs脑组织样本和CYP26B1错义突变转染的小鼠神经干细胞，检测分析CYP26B1基因表达、RA代谢水平及RA受体表达；通过ChIP-Seq、RNA-Seq方法，在构建的突变体中筛选被RA异常调控的与神经管发育直接相关的靶基因，并在突变携带者中验证，以揭示CYP26B1基因变异导致NTDs的分子机制，为进一步阐明罕见变异导致NTDs发生提供实验依据。

罕见变异被认为是复杂疾病的原因。维甲酸（RA）信号异常通路可导致神经管畸形（NTDs）发生，胚胎中枢神经系统RA水平和分布受其降解酶CYP26的精确控制。课题组前通过对355例NTD病例和225例对照的基因测序，筛查到编码RA降解酶的CYP26B1基因的5个新发突变位点，这些变异是否引起NTDs，机制如何？本项目对5个新发突变位点进行功能分析。. 为了检测CYP26B1的功能，我们将CYP26B1表达质粒瞬时转染到NE-4C细胞中，然后用RA（1μM）处理细胞24小时。利用实时定量PCR，评估RA激活和/或与神经管闭合或神经分化有关基因的mRNA水平。当引入WT CYP26B1时，与空白对照组相比，所有基因的表达显著降低，WT CYP26B1降低了细胞中RA的水平，导致RA介导的转录反应降低。而所有的CYP26B1突变体显示与空白对照相似的表达谱，CYP26B1突变体不能调节RA信号传导，基本上是功能性等位基因的丧失。Rara、Rarb和Rarg相对于WT的westernblot分析显示，RA介导的转录活性的这些变化对RA受体蛋白表达没有明显影响。. 为了测试CYP26B1影响神经元分化的能力，我们用1μM RA处理瞬时转染CYP26B1的NE-4C细胞以诱导分化。然后，我们通过量化神经元标记物Tuj1、Map2和Tbr2阳性染色的细胞数量，评估RA治疗后7-8天的神经元分化。WT CYP26B1抑制所有三种神经元标记物的神经元分化。相比之下，所有CYP26B1突变体显示出抑制分化的能力减弱，并且更类似于空白或空载体对照。. 实验结果表明CYP26B1突变体在RA诱导的转录和神经元分化实验中对RA的降解降低。