Dissecting the role of NOTCH2NL genes in human brain development and neurological disorders associated with chromosome 1q21.1 distal duplications and deletions.

剖析 NOTCH2NL 基因在人类大脑发育和与染色体 1q21.1 远端重复和缺失相关的神经系统疾病中的作用。

• 批准号: 10333371
• 负责人: Sofie Reda Salama
• 金额: $ 72.7万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10333371
• 关键词: 1q21; Affect; Alleles; Automobile Driving; Biological; Biological Assay; Brain; Cell Culture Techniques; Cell Line; Cell Maintenance; Cell Proliferation; Cerebral cortex; Cerebrum; Chromosome 1; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complex; Copy Number Polymorphism; DNA; DNA Resequencing; DNA Sequence Rearrangement; Defect; Development; Disease; Distal; Engineering; Equilibrium; Event; Evolution; Family; Gene Conversion; Gene Dosage; Gene Duplication; Gene Expression; Gene Family; Generations; Genes; Genome; Genomic Segment; Genomics; Histology; Human; Human Cell Line; Human Chromosomes; Individual; Lead; Link; Location; Measurement; Measures; Methods; Modeling; Molecular; Molecular Weight; Mutation; Neurodevelopmental Disorder; Neurologic; Neurons; Organoids; Pan Genus; Pathogenicity; Pathologic; Patients; Phenotype; Play; Pluripotent Stem Cells; Population; Predisposition; Recurrence; Reporter; Role; Sampling; Schizophrenia; Sequence Analysis; Signal Transduction; Technology; Testing; Tissues; Variant; autism spectrum disorder; base; brain size; cognitive function; cost effective; experimental study; genome sequencing; genome wide association study; genomic locus; human embryonic stem cell; human embryonic stem cell line; human pluripotent stem cell; improved; innovation; mutant; nerve stem cell; nervous system disorder; novel; premature; prevent; relating to nervous system; single-cell RNA sequencing; stem; stem cell proliferation; stem cells

Genomic copy number variation (CNV) in a part of human chromosome 1 emerged recently in the human lineage by repeated segmental duplications and rearrangements. CNVs here have been associated with autism, schizophrenia and other neurodevelopmental disorders in multiple genome-wide association studies. However, the gene(s) in this interval that contribute to these phenotypes have not been established. We recently identified a family of 3 genes, ​NOTCH2NLA​, -​B and -​C that reside in this locus, are highly expressed during early brain development and are capable of promoting cortical neuron stem cell maintenance and proliferation. In this proposal ​we test the hypothesis that NOTCH2NL genes are required for normal human brain development and alterations in their gene dosage contribute the neurological phenotypes observed in patients with 1q21.1 distal deletions and duplications. This will be accomplished in Aim 1 by ​resequencing this genomic interval ​in diverse human population samples using new long genomic fragment sequencing technologies that will enable us ​to identify the NOTCH2NL alleles present in the human population ​and structural variation present in this highly repetitive genomic region​. This information will inform ​subsequent sequence analysis of 14 samples harboring pathogenic 1q21.1 CNV events that may implicate specific NOTCH2NL loci and variants in these disorders. This information will be used to develop ​high throughput, cost-effective assays to identify the specific NOTCH2NL alleles present in large genomic DNA collections from patients with 1q21.1-associated neurological disorders. In Aim 2, we will ​test the activity of the various NOTCH2NL alleles identified in 2 ways. First, examine the ability of each NOTCH2NL allele ​to promote NOTCH signaling using reporter based assays​. Second, we will test the activity of specific NOTCH2NL alleles ​to promote normal cortical organoid formation using isogenic CRISPR engineered pluripotent stem cell lines that differ only by the specific NOTCH2NL alleles present. The ability of specific alleles to rescue defects in the balance of neural stem cells and cortical neurons associated with loss of NOTCH2NL will be measured by single-cell RNA Sequencing, bulk gene expression measurements and histology. Finally, in Aim 3 we will test whether ​heterozygous, large-scale 1q21.1 deletions similar to those observed in patients have similar or more severe defects in early brain development as assayed by our human pluripotent stem cell cerebral cortex organoid assay using the analysis methods described for Aim 2. We will then ​test the ability of NOTCH2NL and other genes in locus to rescue any defects observed. The experiments outlined here will improve our understanding of the specific biological defects resulting from 1q21.1 CNVs that ultimately lead to complex neurological disorders. These methods can be applied to other repetitive genomic loci implicated in neurodevelopmental diseases.

最近在人类染色体1的一部分中的基因组拷贝数变异（CNV）最近出现在 通过反复的节段重复和重排来进行人类谱系。这里的CNV已关联 自闭症，精神分裂症和其他全基因组关联中的神经发育障碍 研究。但是，在此间隔中尚未建立有助于这些表型的基因。 我们最近确定了一个存在于该基因座的3个基因的家族，即Notch2nla，-b和-c 在早期大脑发育中表达，能够促进皮质神经元干细胞维持 和增殖。在此提案中，我们检验了正常需要Notch2nl基因的假设 人脑发育及其基因剂量的变化促进了神经表型 在1q21.1患者中观察到不同的缺失和重复。这将在AIM 1中通过使用新的长基因组碎片测序技术重新陈述潜水员人口样本中的基因组间隔，从而实现这一目标，这将使我们能够鉴定人类中存在的Notch2NL等位基因和在这个高度重复的基因组区域中存在的结构变异。此信息将为14个样本的后续序列分析提供信息 具有致病性1q21.1 CNV事件，可能暗示特定的Notch2nl基因座和变体 这些疾病。此信息将用于开发高吞吐量，具有成本效益的测定 确定来自大型基因组DNA收集中的特定Notch2NL等位基因 1Q21.1相关神经系统疾病。在AIM 2中，我们将测试各种Notch2NL的活动 等位基因以两种方式识别。首先，检查每个Notch2NL等位基因促进Notch信号的能力 使用基于记者的测定法。其次，我们将测试特定Notch2NL等位基因的活性以促进 正常的皮质器官形成，使用Isenic CRIS工程性多能干细胞系 仅与存在的特定Notch2NL等位基因不同。特定等位基因挽救缺陷的能力 神经元细胞和与缺失Notch2NL相关的皮质神经元的平衡将通过 单细胞RNA测序，大量基因表达测量和组织学。最后，在AIM 3中，我们将测试 杂合，大规模1q21.1缺失是否类似于患者中观察到的缺失 或更严重的大脑发育中的缺陷，由我们的人类多能干细胞分配 使用针对AIM 2的分析方法，我们将测试能力 notch2nl和其他基因中的其他基因，以挽救观察到的任何缺陷。此处概述的实验将提高我们对1 Q21.1 CNV产生的特定生物缺陷的理解，最终导致复杂的神经系统疾病。这些方法可以应用于神经发育疾病中实施的其他重复的基因组基因局。