Human Chromosome 14 Analysis in Neuronal Cells

项目总结/摘要 人类环状染色体是由染色体内融合形成的异常结构， 染色体环状染色体综合征是一种导致严重耐药性癫痫的衰弱性疾病， 智力残疾（IQ <70）和发育迟缓。环状染色体患者及家属应对 长期癫痫发作，学习和记忆问题，严重的心理障碍，以及 无力感，导致大量的压力和紧张。临床上，这种疾病影响多种 器官系统，包括大脑（小头畸形，早发性癫痫和智力残疾），眼睛， 免疫系统和生长。我们的实验室和其他实验室一直致力于绘制和表征 环状染色体结构的分子和细胞遗传学;然而，迄今为止，还没有发表的研究 由于缺乏合适的模型，研究重点集中在细胞神经病理学上。调查这一 我们已经建立了一个大型的14号环状染色体（r（14））患者和家族细胞系生物库， 成员从这些细胞系中，我们已经通过产生患者特异性的 诱导多能干细胞，这为研究这种缺乏免疫力的破坏性疾病提供了基础。 替代模型。在我们的初步工作中，我们发现通过将环状染色体iPSCs分化为 前脑的命运，我们可以产生群体的神经元前体细胞（NPC），可以进一步分化 有丝分裂后的神经元。携带14号环状染色体的神经元前体细胞具有减少的细胞凋亡。 生长和关键端脑神经元标记物表达降低。有趣的是， 位于14号染色体上的基因参与神经元的分化或维持，这使我们假设， 该环结构改变了负责体外皮质细胞生成的关键基因的表达。我们有 也开始产生一个体外大脑皮层类器官模型，这将是重要的研究早期 在R（14）发育模型中受到干扰的胚胎事件。哺乳动物的基因组以不同的方式组织 这些结构允许适当的基因和表观遗传调节的编排。我们建议的研究 将确定环14号染色体在细胞核中的方向，14号染色体上的表观遗传标记， 用于研究14号环状染色体表型的三维（3D）大脑皮层发育模型 综合征我们推测，由于环的存在，染色体的高级结构发生了改变， 形成破坏了正常的染色质致密化、成环、核内定位， 最后是基因调控。本工作将为R（14）综合征的研究提供重要工具， 产生一个全面的看法，一个环形染色体的作用，对DNA定位，染色质修饰， 和体外神经元发育。此外，这一创新模式还将为以下方面奠定坚实的基础： 未来的工作充分表征R（14）中的3D类器官发育，并提供必要的试剂（神经元 组织）来研究R（14）患者样本中的神经回路和电生理模式。

Project Summary/Abstract Human ring chromosomes are abnormal structures formed by intrachromosomal fusions, creating a circular chromosome. Ring chromosome syndrome is a debilitating disorder resulting in severe drug-resistant epilepsy, intellectually disabilities (IQ <70), and developmental delay. Ring chromosome patients and families deal with life-long challenges of prolonged seizures, learning and memory issues, severe psychological disruption, and a feeling of powerlessness, resulting in a great deal of stress and strain. Clinically this disorder affects multiple organ systems including the brain (microcephaly, early onset epilepsy, and intellectual disabilities), the eye, the immune system, and growth. Our laboratory and others, have focused on mapping and characterizing the molecular and cytogenetics of ring chromosome structures; however, to date no published studies have focused on investigating the cellular neuropathology due to lack of appropriate models. To investigate this disorder, we have created a large biobank of cell lines from ring chromosome 14 (r(14)) patients and family members. From these cell lines, we have produced the only model available by generating patient-specific induced pluripotent stem cells, which provide the foundation for studying this devastating disorder that lacks alternative models. In our preliminary work, we find that by differentiating ring chromosome iPSCs toward a forebrain fate we can generate populations of neuronal precursor cells (NPCs) that can be further differentiated into post-mitotic neurons. Neuronal precursor cells harboring the ring chromosome 14 have reduced cellular growth and decreased expression of key telencephalon neuronal markers. Interestingly, a number of genes located on chromosome 14 are involved in neuronal differentiation or maintenance, leading us to postulate that the ring structure modifies expression of this key gene responsible for in vitro cortical cell generation. We have also begun to generate an in vitro cerebral cortex organoid model, which will be important for studying the early embryonic events perturbed in a R(14) developmental model. Mammalian genomes are organized in distinct architectures which allow for the orchestration of proper gene and epigenetic regulation. Our proposed studies will identify ring chromosome 14 orientation in the nucleus, epigenetic marks on chromosome 14, and establish a 3-dimensional (3D) cerebral cortex developmental model for studying phenotypes of Ring chromosome 14 syndrome. We hypothesize that the altered higher order chromosome structure resulting from ring formation disrupts normal chromatin compaction, looping, localization within the nucleus and ultimately gene regulation. The proposed work will provide important tools for studying R(14) syndrome, and yield a comprehensive view of the role a ring chromosome has on DNA positioning, chromatin modifications, and in vitro neuronal development. In addition, this innovative model should provide a strong foundation for future work fully characterizing 3D organoid development in R(14) and provide essential reagents (neuronal tissues) to study the neural circuits and electrophysiological patterns in R(14) patient samples.