Testing the Hypothesis of Somatic Cell Retrotransposition in Human Brain

检验人脑体细胞逆转座的假设

• 批准号: 8179895
• 负责人: DOUGLAS Frederick LEVINSON
• 金额: $ 31.6万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-25 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8179895
• 关键词: Base Pairing; Base Sequence; Benchmarking; Blood; Brain; Brain region; Cheek structure; DNA; DNA Insertion Elements; DNA Sequence; Data; Databases; Defect; Detection; Development; Disease; Elements; Event; Gene Expression; Genetic Polymorphism; Genome; Genomics; Germ Cells; Human; Human Genome; Indium; Individual; L1 Elements; Length; Live Birth; Liver; Location; Malignant Neoplasms; Medical Research; Medicine; Mosaicism; Positioning Attribute; Prosencephalon; Reading; Recording of previous events; Repetitive Sequence; Research; Research Design; Research Institute; Retrotransposition; Role; Schizophrenia; Site; Somatic Cell; Superior temporal gyrus; Swab; Testing; Tissue Banks; Tissues; Variant; Viral; brain cell; brain tissue; case control; embryonic stem cell; evidence base; genome sequencing; hindbrain; nerve stem cell; neurodevelopment; neuropsychiatry

DESCRIPTION (provided by applicant): This project will use whole-genome sequencing to answer a critical question with fundamental relevance to all neuropsychiatric diseases and possibly with fundamental relevance to development and functioning of the human brain in general: do genomic mobile element transpositions occur in the genome of brain tissue? They have been observed in cancer cells1 and are expressed in somatic tissues2. Gage's group demonstrated that L1s could retrotranspose in human neural progenitor and embryonic stem cells, and also presented PCR- based evidence suggesting an increased total number of L1 sequences in human brain regions.3 There has been no direct, sequencing-based demonstration of confirmed ME insertions in brain that are absent in other tissues. About 40% to 50% of the human genome consists of repetitive sequences known as mobile elements (MEs)4, 5, with ~33% consisting of retrotransposable elements (LINE-1, Alu, SVA). 6 These sequences (hundreds or thousands of base pairs long) are remnants of cellular or inactivated retroviral sequences which, alone or in cooperation with each other, can be transcribed and then reverse transcribed and inserted in a different location. This usually occurs in germ cells (~1 in 20 live births according to new 1000 Genomes data7). There are ~ 8,000 known polymorphic sites which are transmitted like other polymorphisms and appear subject to selection. Germline ME insertions can exert pathogenic effects by numerous mechanisms. Most aspects of normal and pathogenic ME functions remain unknown. If such genomic mobile element transposition events do occur in brain, then intensive study (of much larger brain tissue collections) will be needed to determine their functional and pathogenic effects. Any substantial increase in L1 or other MEs in brain would suggest a positively-selected functional role during normal brain development or for normal brain function (given the elegant ME inhibitory mechanisms which exist)1, 8, 9, with pathogenic defects likely to exist. Or, there could be rare (abnormal) pathogenic retroposition events (much like rare germline CNVs). But if no functionally relevant somatic cell genomic retrotransposition occurs in brain (e.g., only rare intergenic events are observed), then the role of MEs in disease can be confidently pursued with large-scale studies of genomic DNA obtained from non-brain tissue (i.e. mostly from blood or cheek swabs). Thus the answer to this question could have dramatic effects on the course of research into neural development and neuropsychiatric disease. We will include subjects with histories of schizophrenia as an example of a disease in which structural variants are known to have substantial pathogenic effects, although confirmation of the hypothesis does not depend on finding case-control differences in this study. We therefore propose to carry out whole-genome sequencing (Illumina HiSeq2000, 100bp paired-end reads, 400-500bp fragment lengths) of post-mortem brain vs. liver DNA from the same individuals to determine whether there are MEs (validated by PCR) in brain which are absent in liver, suggesting somatic cell transposition events. We will examine alternative hypotheses of uniform differences between tissues (suggesting early embryological events) vs. mosaicism within tissues (suggesting later events). The study design will utilize a combination of high- and medium-coverage sequencing of DNA from 50 individuals with schizophrenia and 50 control individuals for whom both tissues are available from the Stanley Medical Research Institute. A comprehensive pipeline for computational detection of MEs (developed in the 1000 Genomes Project by our consultant, Dr. Stewart7, and currently being benchmarked for use and installation at the Stanford Center for Genomics and Personalized Medicine where this study will be carried out) will use information from paired-end differences (one end in a unique mappable region and the other end representing repetitive sequence from a ME database) and from split reads (unique and ME sequence within a single fragment). Secondary analyses will consider issues such as the relationship between ME insertions and other structural variants, and effects of MEs on gene expression (utilizing brain expression microarray data available for some Stanley subjects). We will focus initially on superior temporal gyrus (forebrain tissue that is available in quantity). Later in the study we will study cerebellar (hindbrain) tissue from the same subjects to evaluate possible differences between brain regions. PUBLIC HEALTH RELEVANCE: This project will use whole-genome sequencing to answer a critical question about normal and disease-related mechanisms in the human brain: do common DNA sequences called "mobile elements" (remnants of ancient viral and cellular sequences which make up 40-50% of the genome and can move within the human genome) move in brain cells in way that are potentially relevant to brain function? We will carry out whole-genome sequencing of brain and liver post-mortem tissues from 50 individuals with and 50 individuals without schizophrenia. The results will provide critical information about whether mobile element sequences in brain cell genomes are identical to those of other tissues, or are influenced by later insertions of mobile element sequences in new positions within brain cells. The answer to this question has fundamental relevance to all neuropsychiatric diseases and possibly to development and functioning of the human brain in general.

描述（由申请人提供）：该项目将使用全基因组测序来回答一个与所有神经精神疾病根本相关的关键问题，并且可能与一般人脑的发育和功能基本相关：基因组移动元件转座是否发生在脑组织的基因组中？它们已在癌细胞1中观察到，并在体细胞组织中表达2。 Gage 的研究小组证明，L1 可以在人类神经祖细胞和胚胎干细胞中逆转座，并且还提供了基于 PCR 的证据，表明人脑区域中 L1 序列的总数有所增加。 3 目前还没有直接的、基于测序的证据证实大脑中存在其他组织中不存在的 ME 插入。人类基因组的约 40% 至 50% 由称为移动元件 (ME)4、5 的重复序列组成，其中约 33% 由逆转录转座元件（LINE-1、Alu、SVA）组成。 6 这些序列（数百或数千个碱基对长）是细胞或灭活逆转录病毒序列的残余物，它们可以单独或相互配合进行转录，然后进行反转录并插入到不同的位置。这通常发生在生殖细胞中（根据新的 1000 个基因组数据7，大约每 20 个活产婴儿中就有 1 个）。大约有 8,000 个已知的多态性位点，它们像其他多态性一样传播并且似乎受到选择。种系 ME 插入可通过多种机制发挥致病作用。正常和致病性 ME 功能的大部分方面仍然未知。如果这种基因组移动元件转座事件确实发生在大脑中，那么将需要进行深入研究（对更大的脑组织集合）来确定它们的功能和致病作用。大脑中 L1 或其他 ME 的任何实质性增加都表明在正常大脑发育或正常大脑功能期间有积极选择的功能作用（考虑到存在优雅的 ME 抑制机制）1,8,9，并且可能存在致病缺陷。或者，可能存在罕见（异常）的致病性逆转事件（很像罕见的种系 CNV）。但如果大脑中没有发生功能相关的体细胞基因组逆转座（例如，仅观察到罕见的基因间事件），那么可以通过对从非脑组织（即主要来自血液或脸颊拭子）获得的基因组DNA进行大规模研究，自信地探究ME在疾病中的作用。因此，这个问题的答案可能会对神经发育和神经精神疾病的研究过程产生巨大影响。我们将包括有精神分裂症病史的受试者作为已知结构变异具有重大致病作用的疾病的一个例子，尽管该假设的确认并不依赖于在本研究中发现病例对照差异。因此，我们建议对同一个体的死后大脑和肝脏 DNA 进行全基因组测序（Illumina HiSeq2000，100bp 双端读数，400-500bp 片段长度），以确定大脑中是否存在肝脏中不存在的 ME（通过 PCR 验证），这表明体细胞转座事件。我们将研究组织之间均匀差异（暗示早期胚胎事件）与组织内嵌合现象（暗示后期事件）的替代假设。该研究设计将结合对 50 名精神分裂症患者和 50 名对照个体的 DNA 进行高和中覆盖率测序，这两种组织均可从斯坦利医学研究所获得。用于计算检测 ME 的综合管道（由我们的顾问 Stewart 博士在 1000 基因组计划中开发，目前正在斯坦福基因组学和个性化医学中心进行使用和安装基准测试，这项研究将在该中心进行）将使用来自配对末端差异（一端位于唯一的可映射区域，另一端代表 ME 数据库中的重复序列）和分割读取（独特和 ME 序列）的信息 在单个片段内）。二次分析将考虑 ME 插入与其他结构变异之间的关系以及 ME 对基因表达的影响等问题（利用一些 Stanley 受试者可用的脑表达微阵列数据）。我们将首先关注颞上回（大量可用的前脑组织）。在研究的后期，我们将研究同一受试者的小脑（后脑）组织，以评估大脑区域之间可能存在的差异。 公共健康相关性：该项目将利用全基因组测序来回答有关人脑正常和疾病相关机制的一个关键问题：被称为“移动元件”的常见 DNA 序列（古代病毒和细胞序列的残余物，构成基因组的 40-50%，可以在人类基因组内移动）是否以与大脑功能潜在相关的方式在脑细胞中移动？我们将对 50 名精神分裂症患者和 50 名非精神分裂症患者的脑部和肝脏死后组织进行全基因组测序。结果将提供关于脑细胞基因组中的移动元件序列是否与其他组织的移动元件序列相同，或者是否受到后来在脑细胞内新位置插入移动元件序列的影响的关键信息。这个问题的答案与所有​​神经精神疾病以及可能与人脑的总体发育和功能具有根本相关性。