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)是最近在人类基因组中出现的 人类通过重复的节段性复制和重排而形成的血统。此处的CNV已关联 自闭症、精神分裂症和其他神经发育障碍与多个基因组范围的关联 学习。然而，在这个区间内影响这些表型的基因(S)还没有建立起来。 我们最近发现了一个由3个基因组成的家族，​NOTCH2NLA​，-​B和-​C，它们位于这个基因座上，高度依赖于 在大脑发育早期表达，能够促进皮质神经元干细胞的维持 和扩散。在这个提案​中，我们检验了NOTCH2NL基因是正常所必需的假设 人脑发育及其基因剂量的变化对神经学表型有贡献 观察有1q21.1远端缺失和重复的患者。在目标1中，这将通过​使用新的长基因组片段测序技术在不同的人类群体样本中对该基因组间隔​进行重新测序来实现，该技术将使我们的​能够识别存在于人类群体​中的NOTCH2N1等位基因以及在这个高度重复的基因组区域​中存在的结构变异。该信息将为​随后对14个样本进行序列分析提供信息 携带致病的1q21.1 CNV事件，可能与特定的NOTCH2NL基因座和变异有关 这些障碍。这些信息将被用于开发​高通量、成本效益高的分析方法 鉴定患者大量基因组DNA中存在的特异性NOTCH2NL等位基因 1q21.1-相关神经疾病。在目标2中，我们将​测试各种NOTCH2NL的活性 等位基因鉴定有两种方法。首先，检查每个NOTCH2NL等位基因​促进NOTCH信号传递的能力 使用基于报告的分析​。第二，我们将测试特定的NOTCH2NL等位基因​促进 使用等基因CRISPR工程多能干细胞系形成正常皮质器官 唯一的区别是存在特定的NOTCH2NL等位基因。特定等位基因修复缺陷的能力 与NOTCH2NL丢失相关的神经干细胞和皮质神经元的平衡将通过 单细胞RNA测序、批量基因表达测量和组织学。最后，在目标3中，我们将测试 ​杂合子、大规模1q21.1缺失是否与患者观察到的相似 或更严重的脑发育缺陷，通过我们的人类多能干细胞检测 使用目标2所述的分析方法进行大脑皮层有机体分析。然后我们将进行​测试 NOTCH2NL和其他基因在基因座上修复任何观察到的缺陷。这里概述的实验将提高我们对1q21.1 CNV引起的特定生物学缺陷的理解，这些缺陷最终会导致复杂的神经疾病。这些方法也可以应用于其他与神经发育疾病有关的重复基因座。

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.