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: 8461655
• 负责人: DOUGLAS Frederick LEVINSON
• 金额: $ 30.34万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461655
• 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.

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关键问题，该问题与所有神经精神疾病基本相关，并且可能与一般人脑的发育和功能基本相关：基因组移动的元件易位是否发生在脑组织的基因组中？它们在癌细胞中观察到1，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录转座，并且还提出了基于PCR的证据，表明人脑区域中L1序列的总数增加。3没有直接的、基于测序的证据证实脑中的ME插入在其他组织中不存在。约40%至50%的人类基因组由称为移动的元件（ME）的重复序列组成4、5，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留物，它们可以单独或相互协作转录，然后逆转录并插入不同的位置。这通常发生在生殖细胞中（根据新的1000个基因组数据7，约20个活产婴儿中有1个）。有大约8,000个已知的多态性位点，它们像其他多态性一样被传播，并且似乎受到选择的影响。生殖系ME插入可以通过多种机制发挥致病作用。正常和致病ME功能的大部分方面仍然未知。如果这样的基因组移动的元件转座事件确实发生在脑中，那么将需要深入研究（更大的脑组织集合）来确定其功能和致病作用。大脑中L1或其他ME的任何实质性增加都表明正常大脑发育期间或正常大脑功能（考虑到存在的优雅ME抑制机制）的积极选择功能作用1，8，9，可能存在致病缺陷。或者，可能存在罕见的（异常的）致病性逆转录事件（很像罕见的生殖系CNV）。但是如果在脑中没有发生功能相关的体细胞基因组逆转录转座（例如，仅观察到罕见的基因间事件），那么ME在疾病中的作用可以通过从非脑组织（即主要来自血液或脸颊拭子）获得的基因组DNA的大规模研究来确信地追求。因此，这个问题的答案可能对神经发育和神经精神疾病的研究进程产生巨大影响。我们将纳入有精神分裂症病史的受试者，作为已知结构变异具有实质性致病作用的疾病的一个例子，尽管该假设的证实并不依赖于在本研究中发现病例对照差异。因此，我们建议对来自相同个体的死后脑与肝脏DNA进行全基因组测序（Illumina HiSeq 2000，100 bp配对末端读取，400- 500 bp片段长度），以确定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。我们将检验组织间一致差异（提示早期胚胎学事件）与组织内嵌合现象（提示后期事件）的替代假设。研究设计将利用来自50名精神分裂症患者和50名对照个体的高覆盖率和中等覆盖率DNA测序的组合，这两种组织均可从斯坦利医学研究所获得。用于ME的计算检测的综合管道（由我们的顾问斯图尔特博士7在1000个基因组项目中开发，目前正在斯坦福大学基因组学和个性化医学中心进行基准测试，本研究将在该中心进行）将使用来自配对末端差异的信息在一个实施方案中，所述方法包括从序列（一端在独特的可映射区域中，另一端代表来自ME数据库的重复序列）和从分裂读段（单一片段内的独特和ME序列）中分离序列。二次分析将考虑ME插入和其他结构变异之间的关系，以及ME对基因表达的影响（利用斯坦利一些受试者的大脑表达微阵列数据）等问题。我们最初将重点关注上级颞回（大量可用的前脑组织）。在研究的后期，我们将研究来自相同受试者的小脑（后脑）组织，以评估大脑区域之间可能的差异。