Genomic mosaicism in developing human brain

描述(申请人提供)：新出现的证据表明，并不是所有人体细胞都有相同的DNA序列，这一现象被称为体细胞嵌合体。分裂细胞可以积累单核苷酸变异(SNV)以及更大的结构变异(SVS)，如拷贝数变异(CNV)。我们最近的研究表明，体细胞嵌合体通常发生在至少30%的人皮肤成纤维细胞中。人类大脑皮层在个体发育过程中表现出高度的有丝分裂扩张，在发育过程中可能特别容易积累体细胞变异。体细胞嵌合体可能是一种适应性或非适应性现象，解释了人类个体间的遗传变异，并塑造了个体对神经精神疾病的敏感性和恢复力。然而，正常人脑中的体细胞嵌合体的程度尚不清楚。在这项建议中，我们将使用死后的胎儿人体组织来研究发育中的人脑的体细胞变异程度。研究体细胞嵌合体的理想方法是对单个细胞的基因组进行测序，然而，所需的极端程度的扩增会产生不可避免的人工制品。我们的主要任务将是对来自单个脑细胞的克隆细胞群体的基因组进行排序，识别在每个克隆中表现出来的基因组变异，并验证存在和 为了验证这些变体在原始脑组织中的频率，我们将使用这个全面的数据集来验证它确实是镶嵌的，然后我们将评估和改进通过以下方式获得的变体呼叫 单个脑细胞的全基因组扩增。在目标1中，我们将构建人脑前体细胞的体细胞变异图谱，并估计它们在发育中的大脑皮层和基底节中的频率。我们将比较克隆细胞群体和从脑组织中提取的单个细胞的基因组，然后进行高分辨率分析，以验证它们在原始脑组织和血液中的存在和等位基因频率。在目标2中，我们将通过评估克隆表现的基因组变异是否在基因转录水平上产生后果和/或对可能赋予细胞适应优势的生物功能产生影响，来确定体细胞嵌合体对基因表达的影响。在目标3中，我们将研究体细胞最可能的生物学起源。 通过分析变异位点的序列特征，将变异与重组热点、CpG岛和组蛋白标记相关联。总而言之，这些特定的目标将首次全面估计脑体细胞基因组变异的数量和等位基因频率，并将产生关于它们产生的机制以及它们对大脑发育的意义的假说。

DESCRIPTION (provided by applicant): Emerging evidence suggest that not all cells of the human body have identical DNA sequence, a phenomenon called somatic mosaicism. Dividing cells can accumulate single nucleotide variations (SNVs) as well as larger structural variants (SVs), such as copy number variations (CNVs). Our recent studies suggest that somatic mosaicism normally occurs in at least 30% of human skin fibroblasts. The human cerebral cortex displays a very high degree of mitotic expansion during ontogenesis and may be particularly susceptible to accumulating somatic variation during development. Somatic mosaicism could be an adaptive or maladaptive phenomenon, accounting for inter-individual human genetic variability and shaping individual susceptibility and resilience to neuropsychiatric disorders. Yet, the extent of somatic mosaicism in the normal human brain is unknown. In this proposal we will investigate the degree of somatic variation in the developing human brain, using postmortem fetal human tissue. The ideal way to study somatic mosaicism would be to sequence the genome of single cells, however, the extreme degree of amplification that is required creates inevitable artifacts. Our principal appoach will be to sequence the genome of clonal cell populations derived from single brain cells, identify genomic variants manifested in each clone, and verify the presence and frequency of these variants in the original brain tissue to verify that it is, indeed, mosaic Using this comprehensive dataset, we will then evaluate and refine variant calls obtained by whole genome amplification of single brain cells. In Aim 1, we will construct a map of somatic variations in human brain progenitor cells and estimate their frequency in the developing cerebral cortex and basal ganglia. We will compare the genomes of clonal cell populations and single cells extracted from brain tissue, followed by high resolution analyses to verify their presence and allele frequency in the original brain tissue as well as in th blood. In Aim 2, we will determine the impact of somatic mosaicism on gene expression by assessing whether clone-manifested genomic variants have consequences at the level of gene transcription and/or have effects on biological functions that may confer adaptve advantage to the cells. In Aim 3, we will investigate the most likely biological origin of somatic variants by analyzing sequence features at variation sites, correlating variants with recombination hotspots, CpG islands and histone marks. Together, these specific aims will provide the first comprehensive estimate of the number and allelic frequency of genomic variation in somatic cells of the brain and will yield hypotheses about mechanisms responsible for their creation as well as their significance for brain development.