Functional genomic consequences of MBD5 knockdown in mouse brain and CRISPR-derived neurons [mouse]

Background: MBD5, encoding the methyl-CpG-binding domain 5 protein, has been proposed as a necessary and sufficient driver of the 2q23.1 microdeletion syndrome. De novo missense and protein-truncating variants from exome sequencing studies have directly implicated MBD5 in the etiology of autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs). However, little is known concerning the specific function(s) of MBD5. Methods: In an effort to gain insight into the complex interactions associated with genetic alteration of MBD5 in individuals with ASD and related NDDs, we explored the transcriptional landscape of MBD5 haploinsufficiency across multiple mouse brain regions of the heterozygous hypomorphic Mbd5+/GT mouse model and compared these results to CRISPR-mediated mutations of MBD5 in human iPSC-derived neuronal models. Results: Gene expression analyses of three brain regions from Mbd5+/GT mice showed cortex as the region most affected by the knockdown, indicating context-dependent effects. Gene co-expression network analyses revealed gene clusters that are associated with MBD5 knockdown and enriched for GPCR signaling and terms related to ciliary function. We also identified genes that were downregulated in both models such as EPHA3, OLIG1, related to neuronal development and glial function. Limitations: These analyses were performed in a limited number of brain regions and neuronal models, and the effects of the gene knockdown are subtle. As such, these results will not reflect the full extent of MBD5 disruption across brain tissues during early brain developmentneurodevelopment in ASD. Conclusions: Our study points to transcriptional consequences of Mbd5 disruption in the brain and suggests a link between MBD5 and pathways such as ciliary function that are associated with established developmental disorders and syndromes. 10 mice with Mbd5 heterozygous deletion, 8 mice with WT genotype from same litters, for each mice three tissues are sequenced: cerebellum(Cb), cortex (Cx) and striatum(St).

背景：MBD5基因编码甲基CpG结合域5蛋白，已有研究提出其为2q23.1微缺失综合征的必要且充分驱动因子。过往外显子组测序研究中鉴定出的新生错义变异与蛋白截短变异，已直接将MBD5与自闭症谱系障碍（autism spectrum disorder, ASD）及相关神经发育障碍（neurodevelopmental disorders, NDDs）的病因学关联起来。然而，目前学界对MBD5的具体功能仍知之甚少。
方法：为深入解析ASD及相关NDDs患者体内MBD5遗传改变所涉及的复杂相互作用机制，本研究针对携带低功能型Mbd5杂合等位基因的Mbd5+/GT小鼠模型的多个脑区，探究了MBD5单倍体不足状态下的转录组特征，并将该结果与人类诱导多能干细胞（induced pluripotent stem cell, iPSC）衍生神经元模型中CRISPR介导的MBD5突变结果进行了对比。
结果：对Mbd5+/GT小鼠的三个脑区开展基因表达分析后发现，大脑皮层是受基因敲降影响最显著的区域，这表明MBD5的调控作用具有区域依赖性。基因共表达网络分析揭示了与MBD5敲降相关的基因簇，这些基因簇富集于G蛋白偶联受体（G protein-coupled receptor, GPCR）信号通路以及与纤毛功能相关的功能术语。本研究还在两种模型中均鉴定出了表达下调的基因，例如与神经元发育和神经胶质功能相关的EPHA3、OLIG1。
局限性：本研究仅对有限数量的脑区与神经元模型开展了分析，且基因敲降所产生的效应较为微弱。因此，本研究结果无法完整反映早期脑发育与ASD神经发育过程中，MBD5功能异常在全脑组织内的真实影响范围。
结论：本研究揭示了大脑中Mbd5功能异常所带来的转录组学改变，并提示MBD5与纤毛功能等通路存在关联，而这些通路已被证实与发育障碍及综合征相关。
样本信息：本研究共纳入10只Mbd5杂合缺失型小鼠，以及8只同窝野生型（wild type, WT）小鼠；每只小鼠的三种脑组织均进行了测序，分别为小脑（Cb）、大脑皮层（Cx）与纹状体（St）。