Testing the Hypothesis of Somatic Cell Retrotransposition in Human Brain

描述(申请人提供)：这个项目将使用全基因组测序来回答一个关键问题，这个问题与所有神经精神疾病基本相关，可能与一般人脑的发育和功能基本相关：脑组织基因组中是否发生基因组移动元素的换位？它们已经在癌细胞中观察到，并在体细胞组织中表达。Gage的团队证明了L1s可以在人类神经前体细胞和胚胎干细胞中逆转录转座，并提出了基于聚合酶链式反应的证据，表明人类大脑区域的L1序列总数增加。3目前还没有基于测序的直接证明证实在大脑中插入了其他组织中没有的ME。人类基因组约40%至50%由重复序列组成，称为移动元件(MES)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)是细胞或灭活的逆转录病毒序列的残留物，它们可以单独或相互配合转录，然后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，每20个活产婴儿中就有一个)。大约有8,000个已知的多态位点像其他多态一样传递，并似乎受到选择的影响。生殖系ME插入可通过多种机制发挥致病作用。ME的正常和致病功能的大部分方面仍不清楚。如果这种基因组移动元件转位事件确实发生在大脑中，那么将需要深入的研究(对更大的脑组织收集)来确定它们的功能和致病作用。大脑中L1或其他MES的任何大幅增加都表明在正常的脑发育过程中或对于正常的大脑功能(考虑到存在优雅的ME抑制机制)1、8、9可能存在致病缺陷。或者，可能有罕见的(异常的)致病逆位事件(很像罕见的生殖系CNV)。但是，如果在大脑中没有发生功能相关的体细胞基因组反转录转座(例如，只有罕见的基因间事件被观察到)，那么可以通过对从非脑组织(即主要从血液或面颊拭子)获得的基因组DNA的大规模研究来确定MES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重大影响。我们将把有精神分裂症病史的受试者作为一种已知结构变异具有实质性致病影响的疾病的例子，尽管这一假设的确认并不取决于在这项研究中找到病例对照差异。因此，我们建议对同一个体的死后脑和肝DNA进行全基因组测序(Illumina HiSeq2000,100bp成对末端读取，400-500bp片段长度)，以确定脑中是否存在肝脏中不存在的MES，这表明存在体细胞转位事件。我们将检验组织间一致差异(提示早期胚胎事件)与组织内嵌合体(提示后期事件)的替代假说。这项研究设计将结合对50名精神分裂症患者和50名对照患者的DNA进行高覆盖率和中覆盖率测序，斯坦利医学研究所为他们提供了这两种组织。用于计算检测MES的全面管道(由我们的顾问Stewart7博士在1000基因组计划中开发，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和安装，将在那里进行这项研究)将使用来自配对末端差异(一端位于唯一可映射区，另一端代表ME数据库中的重复序列)和分离读取(单个片段中的唯一和ME序列)的信息。二次分析将考虑ME插入和其他结构变异之间的关系，以及MES对基因表达的影响(利用一些Stanley受试者可用的脑表达微阵列数据)。我们首先将重点放在颞叶上回(有数量的前脑组织)上。在稍后的研究中，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存在的差异。 公共卫生相关性：这个项目将使用全基因组测序来回答一个关于人脑中正常和与疾病相关的机制的关键问题：被称为“移动元件”的常见DNA序列(古代病毒和细胞序列的残留物，占基因组的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组织和肝脏组织进行全基因组测序。这些结果将提供关键信息，即脑细胞基因组中的移动元素序列是否与其他组织的相同，或者是否受到后来在脑细胞内新位置插入的移动元素序列的影响。这个问题的答案与所有神经精神疾病都有根本关系，也可能与整个人脑的发育和功能有关。

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.