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

人类1号染色体部分区域的基因组拷贝数变异（CNV）最近出现在 通过重复的片段复制和重排形成人类谱系。这里的CNVs与 与自闭症、精神分裂症等神经发育障碍的多基因组关联 问题研究然而，在这一区间内导致这些表型的基因尚未确定。 我们最近发现了一个由3个基因组成的家族，NOTCH 2 NLA，- B和- C，它们位于该基因座，与其他基因高度同源。 在早期脑发育过程中表达，并能够促进皮质神经元干细胞的维持 和扩散。在这项提议中，我们检验了NOTCH 2NL基因是正常人生长所必需的这一假设。 人类大脑的发育和基因剂量的改变有助于神经系统的表型 在1q21.1远端缺失和重复的患者中观察到。这将在目标1中通过使用新的长基因组片段测序技术对不同人群样本中的该基因组间隔进行重测序来实现，该技术将使我们能够鉴定人群中存在的NOTCH 2NL等位基因和该高度重复的基因组区域中存在的结构变异。该信息将为随后的14个样本的序列分析提供信息 携带致病性1q21.1 CNV事件，可能涉及特定的NOTCH 2NL基因座和变异， 这些紊乱。这些信息将用于开发高通量、成本效益高的检测方法， 鉴定存在于来自患有以下疾病的患者的大量基因组DNA集合中的特异性NOTCH 2NL等位基因： 1q21.1相关的神经系统疾病。在目标2中，我们将测试各种NOTCH 2NL的活性， 以两种方式鉴定等位基因。首先，检查每个NOTCH 2NL等位基因促进NOTCH信号传导的能力 使用基于报告基因的测定。第二，我们将测试特定NOTCH 2NL等位基因的活性，以促进 使用等基因CRISPR工程化多能干细胞系形成正常皮质类器官， 不同之处仅在于存在的特定NOTCH 2NL等位基因。特异性等位基因挽救基因缺陷的能力， 将通过以下方法测量与NOTCH 2NL缺失相关的神经干细胞和皮质神经元的平衡： 单细胞RNA测序、大量基因表达测量和组织学。最后，在目标3中，我们将测试 是否杂合子，大规模1q21.1缺失与患者中观察到的相似， 或更严重的缺陷，在早期的大脑发育，如我们的人类多能干细胞检测 使用针对目标2所述的分析方法进行大脑皮质类器官测定。然后我们将测试 以挽救观察到的任何缺陷。这里概述的实验将提高我们对1q21.1 CNVs导致的特定生物学缺陷的理解，这些缺陷最终导致复杂的神经系统疾病。这些方法可以应用于其他涉及神经发育疾病的重复基因组位点。

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.