建立基于神经发育数据的孤独症表观调控网络解析孤独症发生机制

Autism is a complex disorder of neural development, and associates with abnormal development of embryo. Genetics has a key role in the aetiology of autism, in conjunction with developmentally early environmental factors. There are a number of factors that seriously hinder our understanding of the pathogenesis of the disease, such as difficulty in drawing materials and ethical constraints. To construct epigenetic regulatory network of autism based on data of neural development, we integrated and analyzed the microarray, RNA-seq and ChIP-seq that associated with autism, human fetal brain and mouse embryo neurodevelopment by using bioinformatics analysis methods. Then we detected gene expression level of our pre-existing autism sample with RNA-seq, and compared with the previous results to obtain the key genes. Meanwhile, we inserted the four stem cell transcription factor genes OCT4, SOX2, C-MYC and KLF4 into foreskin fibroblasts of autism, and obtained induced pluripotent stem cells (iPSCs). RT-PCR was used to detect the expression level of the key genes in iPSC-derived neural stem cells. ChIP-PCR was used to verify the surface modification. Finally, we obtained the potential biological marker and exploring the pathogenesis of autism. Our studies would provide a new clue to early intervention in autism, a new idea on clinical molecular diagnosis and target therapy, and had the double significance in basic research and clinical application.

孤独症是神经发育相关的一种复杂性疾病，与胚胎大脑发育异常有关，受遗传因素和发育早期环境因素共同调控。取材困难制约以及伦理约束等诸多因素，严重阻碍理解疾病发生机制。本研究利用GEO数据库收录的孤独症、人胎儿大脑和小鼠胚胎神经发育相关的microarray、RNA-seq和ChIP-seq实验数据，采用生物信息学分析方法，构建基于神经发育的孤独症表观调控网络。并结合RNA-seq技术检测现有样本的基因表达水平，比对前期获得结果，筛选出关键基因。与此同时，采用经典4因子法诱导患者包皮成纤维获得多潜能干细胞（iPSCs），并定向分化神经干细胞（NSCs），检测关键基因在NSCs中的表达。最后，采用ChIP-PCR进行表观修饰验证，确定孤独症潜在的生物学标记，解析孤独症的发生机制。本研究为早期干预孤独症发生提供新的线索，为临床分子诊断和靶点治疗提供新的思路，具有基础研究和临床应用双重意义。

孤独症是一种受遗传和环境因素影响的高度异质性神经发育疾病。缺乏疾病胚胎样本和表型明确动物模型，导致发生机制尚不明确。本研究中我们利用公共数据库的转录和表观遗传组学数据，采用生物信息、统计遗传学分析和实验技术相结合，构建基于神经发育的孤独症表观调控网络，确定了孤独症潜在生物学标记，解析孤独症发生机制。.1、重要结果.（1）开展孤独症和人脑发育相关通路挖掘工作，识别了与神经系统、代谢和免疫相关10条共同关联通路。通过比较人和鼠脑发育过程中基因时序动态变化和组蛋白修饰调控，构建了基于神经发育的孤独症表观调控网络，确定了小鼠疾病模型研究的脑发育时间契合点。论文撰写完毕，投稿中。.（2）已构建Cfp1模型小鼠，鉴定了皮质区和神经干细胞的增殖能力。基于自测RNA-seq数据获得了鼠脑发育基因转录的时序动态变化。已整理完结果，论文撰写中。.（3）获得孤独症来源的iPSCs并定向分化NPCs。.（4）开展了鉴定多种神经障碍性疾病（阿尔兹海默症、帕金森、孤独症、亨廷顿氏舞蹈症、多发性硬化症、痴呆、抑郁症和精神分裂症等）共甲基化区域的工作，找寻疾病之间的关联性。已整理完结果，论文撰写中。.（5）开发了适用神经精神疾病破译EWAS分析的模块，集成到EWAS2.0转件中，并完成了发表工作。.（6）完成了孤独症等多种神经精神疾病共67个EWAS分析，并录入EWASdb数据库中及发表工作。.（7）本项目研究期间共开发数据库1个，软件升级1个，投稿2个，满足原计划SCI收录论文1-3篇的任务，均已按规定标注基金资助。主要结果发表在杂志：《Bioinformatics》和《Nucleic Acids Research》。.2、科学意义.本项目的完成将有助于确定神经发育相关孤独症潜在生物学标记，对从胚胎神经发育角度深入理解孤独症发生机制做出重要贡献。利用交叉学科研究方式，揭示神经发育过程中母体代谢和免疫失调影响孤独症发病。