Characterizing patient-specific TBR1 mutations: Understanding a master regulator of autism risk.

项目总结/摘要 孤独症谱系障碍（ASD）是一种常见的神经发育障碍，其特征是 社交和限制性的重复行为。到目前为止，有效的治疗方法有限， 帮助个人管理ASD的核心症状。ASD风险遗传因素的多样性 强调需要将我们的重点从一刀切的治疗方法转移到更定制的个性化治疗方法。 最近，下一代测序（NGS）使研究人员能够重新鉴定（新出现在 许多重要的大脑发育基因的功能缺失突变，包括与儿童相关的基因， 在无关儿童中的多重突变。这些发现为反复突变的基因提供了强有力的证据 在ASD风险中起着重要作用，并表明， 大脑发育足以导致自闭症TBR 1，作为“主调节器”的转录因子 在大脑发育中，是一个特别感兴趣的基因。TBR 1在约0.2%的ASD儿童中发生突变， 使其成为最常见的风险因素之一。使用生物网络方法的计算分析 表明，尽管遗传复杂性，在特定的发育窗口， 大脑区域可能在ASD的遗传子集中起作用。具体而言，高置信风险的共同表达 基因在皮质发育的胎儿中期聚集，在此，TBR 1被认为在皮质发育中起关键作用。 分化、迁移和功能的研究。而且是 现在很清楚，TBR 1也结合并调节约1/3的其他高自信自闭症风险基因，使其成为一个重要的基因。 潜在的“主调节器”的至少一个新兴的共同自闭症病因。 评估特定突变的功能后果代表了验证和分析的关键步骤。 了解基因型和表型之间的因果关系，并设计合理的靶向 治疗/干预。我们假设，TBR 1单拷贝的丢失破坏了人类大脑皮层 通过改变TBR 1调节的网络来促进神经元的发育，而TBR 1调节的网络是正确的神经元身份和迁移所必需的。 此外，在ASD风险的这一关键发育窗口期间破坏TBR 1或其靶基因定义了 通往ASD的必经之路我们先前证实，从头突变严重影响了定位， 突变TBR 1蛋白调节靶基因的能力。在这里，我们将解决我们目前的差距， 了解患者特异性TBR 1突变如何通过利用尖端基因组影响发育中的神经元 编辑，功能基因组学和互补模型，利用患者衍生的诱导多能 干细胞（iPSC）转化为前脑样类器官（Aim 1）和小鼠遗传学（Aim 2）。这些研究 将为TBR 1患者特异性突变对神经元的影响提供前所未有的视角 大脑皮层发育的过程中。此外，该iPSC/小鼠遗传学平台可以扩展到其他风险 基因，并为ASD和相关疾病设计合理的靶向治疗/干预措施的基础。

PROJECT SUMMARY/ABSTRACT Autism spectrum disorder (ASD) is a common neurodevelopmental disorder characterized by impaired social communication and restricted, repetitive behaviors. As of now, there are limited effective therapies to help individuals manage the core symptoms of ASD. The diversity of genetic factors underlying ASD risk highlights the need to shift our focus from one-size fits all therapeutics to more tailored, individualized therapies. Recently, next-generation sequencing (NGS) has enabled researchers to identify de novo (newly appearing in the child) loss of function mutations in many important brain development genes, including genes with in multiple mutations in unrelated children. These findings provide strong evidence for recurrently mutated genes playing a significant role in ASD risk and indicate that heterozygous disruption of a single gene essential for brain development is sufficient to cause autism. TBR1, a transcription factor that serves as a `master regulator' in brain development, is one such gene of particular interest. TBR1 is mutated in ~0.2% of children with ASD, making it one of the most common risk factors. Computational analyses using biologic network approaches suggest that, despite the genetic complexity, converging biology at particular developmental windows and brain regions may be at play in genetic subsets of ASD. Specifically, the co-expression of high confidence risk genes converge at midfetal stages of cortical development, where TBR1 is thought to play a key role in the differentiation, migration, and function of deep layer glutamatergic cortical projection neurons. Moreover, it is now clear that TBR1 also binds to and regulates ~1/3 of other high confidence autism risk genes, making it a potential `master regulator' of at least one emerging common autism etiology. Evaluating the functional consequences of specific mutations represents a critical step in validating and understanding the causal link between genotype and phenotype and designing rational targeted therapies/interventions. We hypothesize that loss of a single copy of TBR1 disrupts human cortical development by altering the TBR1-regulated network required for proper neuronal identity and migration. Moreover, disrupting TBR1 or its target genes during this critical developmental window of ASD risk define a common route to ASD. We previously confirmed that de novo mutations severely impacted the localization and ability of mutant TBR1 proteins to regulate target genes. Here, we will address the current gaps in our knowledge of how patient-specific TBR1 mutations affect developing neurons by utilizing cutting-edge genome editing, functional genomics, and complementary models that leverage patient-derived induced pluripotent stem cells (iPSCs) converted to forebrain-like organoids (Aim 1) and mouse genetics (Aim 2). These studies will provide an unprecedented view into the consequences of TBR1 patient-specific mutations on neurons during cortical development. Moreover, this iPSC/mouse genetics platform can be expanded to other risk genes and be the basis for designing rational targeted therapies/interventions for ASD and related disorders.