Human Chromosome 14 Analysis in Neuronal Cells

项目摘要/摘要 人类环状染色体是由染色体内融合形成的异常结构，形成环状 染色体。环染色体综合征是一种衰弱的疾病，会导致严重的抗药性癫痫， 智力残疾(IQ&lt；70)和发育迟缓。环状染色体患者和家人应对 长期癫痫发作，学习和记忆问题，严重的心理障碍，以及 无能为力，造成了极大的压力和紧张。在临床上，这种疾病会影响多个 器官系统，包括大脑(小头症、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专注于绘制和表征 环染色体结构的分子和细胞遗传学；然而，到目前为止，还没有发表过的研究 由于缺乏合适的模型，重点研究了细胞的神经病理。来调查这件事 我们从14号环状染色体(r(14))患者和家庭中创建了一个巨大的细胞库 会员。从这些细胞系中，我们已经产生了唯一可用的模型，通过产生针对患者的 诱导的多能干细胞，这为研究这种缺乏的破坏性疾病提供了基础 另一种模式。在我们的初步工作中，我们发现通过将环染色体ipscs分化为 前脑命运我们可以产生可进一步分化的神经前体细胞群 转化为有丝分裂后神经元。含有14号环状染色体的神经元前体细胞减少了细胞 端脑关键神经元标志物的生长和表达减少。有趣的是，一些基因 位于14号染色体上的基因参与了神经元的分化或维持，这让我们假设 环结构改变了这一关键基因的表达，该基因负责在体外产生皮质细胞。我们有 也开始建立大脑皮层器官的体外模型，这将对早期研究有重要意义 胚胎事件在R(14)发育模型中受到扰动。哺乳动物的基因组是以不同的方式组织起来的 允许协调适当的基因和表观遗传调控的体系结构。我们建议的研究 将确定14号环状染色体在细胞核中的定位，14号染色体上的表观遗传标记，并建立 用于研究14号环染色体表型的大脑皮层三维发育模型 综合症。我们假设由环引起的改变的高阶染色体结构 形成扰乱了正常的染色质压缩、环状、在细胞核内的定位和 归根结底是基因调控。拟议的工作将为研究R(14)综合征提供重要工具，以及 全面了解环染色体在DNA定位、染色质修饰、 以及体外神经元发育。此外，这一创新模式应该为 未来的工作完全表征R(14)中3D有机化合物的发育，并提供必要的试剂(神经元 组织)研究R(14)患者样本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.