Testing the Hypothesis of Somatic Cell Retrotransposition in Human Brain

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以及可能与人类大脑发育和功能基本相关的关键问题：脑组织基因组中是否发生基因组移动元件转位？它们已在癌细胞中被观察到，并在体细胞组织中表达。Gage的研究小组证明了L1可以在人类神经祖细胞和胚胎干细胞中进行反转录，并提出了基于PCR的证据，表明L1序列在人类大脑区域的总数增加目前还没有直接的、基于测序的证明，证实ME在大脑中的插入在其他组织中是不存在的。大约40%至50%的人类基因组由称为可移动元件（MEs） 4,5的重复序列组成，其中约33%由反转录转座元件（LINE-1, Alu， SVA）组成。这些序列（数百或数千个碱基对长）是细胞或失活逆转录病毒序列的残余物，它们单独或相互合作，可以转录，然后逆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基因组数据，每20个活产婴儿中约有1个）。有大约8000个已知的多态性位点，它们像其他多态性一样传播，并且似乎受到选择的影响。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功能的大多数方面仍然未知。如果这样的基因组移动元件转位事件确实发生在大脑中，那么将需要对（更大的脑组织收集）进行深入研究，以确定其功能和致病作用。大脑中L1或其他MEs的任何显著增加都表明，在正常大脑发育或正常大脑功能（考虑到存在优雅的ME抑制机制）中，存在积极选择的功能作用1,8,9，可能存在致病性缺陷。或者，可能存在罕见的（异常的）致病性逆转录事件（很像罕见的种系CNVs）。但是，如果在大脑中没有发生与功能相关的体细胞基因组逆转录（例如，仅观察到罕见的基因间事件），则可以通过对非脑组织（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，自信地探索MEs在疾病中的作用。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，作为一种已知结构变异具有实质性致病作用的疾病的例子，尽管该假设的证实并不取决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对端reads， 400-500bp片段长度），以确定大脑中是否存在肝脏中缺失的MEs（通过PCR验证），这表明存在体细胞转位事件。我们将研究组织之间统一差异（提示早期胚胎事件）与组织内嵌合（提示后期事件）的其他假设。该研究设计将结合50名精神分裂症患者和50名对照患者的高覆盖率和中等覆盖率DNA测序，这些对照患者的两种组织均可从斯坦利医学研究所获得。一个全面的MEs计算检测管道（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的基准测试，该研究将在该中心进行)将使用来自成对端差异的信息（一端在一个独特的可映射区域，另一端代表来自ME数据库的重复序列）和来自分裂读取的信息（单个片段内的独特和ME序列）。二次分析将考虑ME插入与其他结构变异之间的关系，以及MEs对基因表达的影响等问题（利用一些Stanley受试者的大脑表达微阵列数据）。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，我们将研究来自同一受试者的小脑（后脑）组织，以评估大脑区域之间可能存在的差异。

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.