Neural mechanisms of 3q29 deletion syndrome

项目概述：已知神经发育障碍如自闭症和精神分裂症具有 强烈的遗传成分，但驱动临床表型的基因和分子决定因素不是 明白重复性基因组拷贝数变异（CNVs）已成为重要的，高度渗透的 这些疾病的危险因素。3q 29缺失是这样一种CNV，其导致21个拷贝的丢失。 蛋白质编码基因，与自闭症密切相关，据估计， 与精神分裂症相关的遗传变异在>40。 表型上，现有的证据表明，3q 29缺失可能会损害大脑发育。 临床病例报告描述了小头畸形，两个独立的3q 29缺失小鼠模型， 降低大脑重量。脑生长的改变在特发性和CNV相关的 神经发育障碍，并已假设，失调的增殖和成熟， 神经祖细胞（NPC）可能是这种现象的基础。在这里，我们建议严格测试 从3q 29缺失研究参与者和同基因诱导多能干细胞分化的NPC的增殖 通过ELISA、免疫荧光和多参数流式细胞术检测iPSC细胞系。此外，我们将 生成背侧和腹侧前脑3D类器官，以评估两者的增殖和分化。 谷氨酸能和GABA能祖细胞。 此外，我们将通过转录组学研究缩小潜在的表型驱动基因的名单， 人iPSC衍生的NPC和前脑神经元。目前，3q 29缺失基因座中没有一个基因是 与孤独症或精神分裂症相关，这促使人们假设多个基因的单倍不足 在该区间内，会导致神经发育障碍的风险。初步测算， 表达网络分析表明，21个编码蛋白的3q 29缺失基因聚集在7个共 这意味着多个3q 29基因参与重叠的生物学途径。我们将 对NPC和FACS分离的神经元进行RNA-seq，以鉴定人类中差异表达的基因 3q 29缺失神经细胞。然后，我们将重新引入候选3q 29驱动基因到NPC和神经元中， 确定哪些3q 29基因负责下游靶点的失调。 遗传和新出现的表型证据强烈表明，3q 29缺失是一个高优先级的 机械研究的目标。Emory 3q 29项目已经从3q 29缺失中产生了12个iPSC系 携带者沿着年龄、性别和种族/民族匹配的对照。工具已经到位， 研究可归因于这种高风险变异的细胞和分子改变。这些发现将推动 我们对这种疾病的理解，将是理解遗传和 神经发育障碍的分子驱动因素。

PROJECT SUMMARY: Neurodevelopmental disorders such as autism and schizophrenia are known to have a strong hereditary component but the genes and molecular determinants driving clinical phenotypes are not understood. Recurrent genomic copy number variants (CNVs) have emerged as important, highly-penetrant risk factors for these disorders. 3q29 deletion is one such CNV that results in the loss of one copy of 21 protein-coding genes, is strongly associated with autism, and is estimated to have the highest odds ratio of any genetic variant linked to schizophrenia at >40. Phenotypically, the available evidence suggests that 3q29 deletion may compromise brain development. Clinical case reports have described microcephaly and two independent mouse models of 3q29 deletion have reduced brain weight. Alterations in brain growth are relatively common in idiopathic and CNV-associated neurodevelopmental disorders and it has been hypothesized that dysregulated proliferation and maturation of neural progenitor cells (NPCs) may underlie this phenomenon. Here, we propose to rigorously test the proliferation of NPCs differentiated from 3q29 deletion study participant and isogenic induced pluripotent stem cell (iPSC) lines by ELISA, immunofluorescence, and multiparametric flow cytometry. Furthermore, we will generate both dorsal and ventral forebrain 3D organoids to assess the proliferation and differentiation of both glutamatergic and GABAergic progenitors. In addition, we will narrow the list of potential phenotypic driver genes through transcriptomic studies of human iPSC-derived NPCs and forebrain neurons. No single gene within the 3q29 deletion locus is currently associated with autism or schizophrenia prompting the hypothesis that haploinsufficiency of multiple genes within the interval contribute to risk for neurodevelopmental disorders. Indeed, preliminary weighted co- expression network analysis indicates that the 21 protein-coding 3q29 deletion genes congregate in 7 co- expression modules implying that multiple 3q29 genes participate in overlapping biological pathways. We will conduct RNA-seq on NPCs and FACS-isolated neurons to identify differentially-expressed genes in human 3q29 deletion neural cells. We will then re-introduce candidate 3q29 driver genes to NPCs and neurons and determine which 3q29 genes are responsible for dysregulation of downstream targets. The genetic and emerging phenotypic evidence strongly suggests that the 3q29 deletion is a high-priority target for mechanistic investigation. The Emory 3q29 Project has generated 12 iPSC lines from 3q29 deletion carriers along with age, sex, and race/ethnicity-matched controls. The tools are in place to rigorously investigate cellular and molecular alterations attributable to this high-risk variant. These findings will advance our understanding of this disorder and will be an important step toward understanding the genetic and molecular drivers of neurodevelopmental disorders more broadly.