DNA Methylation Profiling of Chronic Pain

慢性疼痛的 DNA 甲基化分析

基本信息

批准号：
7687453
负责人：
ANDREAS S. BEUTLER
金额：
$ 20.43万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-16 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7687453
关键词：
Adult Affect American Animals Axotomy Binding Bioinformatics Biological Assay Characteristics Conserved Sequence Control Animal CpG Islands Cytosine DNA DNA Methylation DNA amplification Development Digestion Disease Disease model Electrophysiology (science)Functional RNA Future Gene Expression Genes Genome Genomics Glass Harvest Healed Healthcare Hour Individual Injury Knowledge Lasers Ligation Literature Malignant Neoplasms Mass Spectrum Analysis Mediating Memory Methods Methylation Modeling Molecular Mus Nerve Nervous system structure Neurobiology Neuroglia Neurons Nucleic Acid Regulatory Sequences Outcomes Research Pain Pathology Patients Pattern Peripheral Plastics Population Positioning Attribute Process Productivity Promoter Regions Quantitative Reverse Transcriptase PCR Rattus Recovery Research Resolution Resources Rodent Rodent Model Site Slide Spinal Cord Spinal Ganglia Techniques Testing Time Tissues Transcript Transcription Initiation Site Validation base chronic pain cost design ganglion cell gene function genome wide association study genome-wide genome-wide analysis healing nerve injury new technology operation painful neuropathy public health relevance research study

项目摘要

DESCRIPTION (provided by applicant): DNA cytosine methylation is a keeper of "cellular memory", stable and able to (co-)determine gene expression. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause disease. Chronic pain often arises without visible insult or persists after tissue injury has healed. In the rodent single nerve ligation (SNL) pain model, L4 and L5 dorsal root ganglion (DRG) neurons and glia become dysfunctional with altered electrophysiology, gene expression, and cellular plasticity. The basic molecular force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to known pain mechanisms. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic; that it is altered by experimental nerve injury; that it (co-)determines gene expression patterns characteristic of the pain state; and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. The HELP assay (=HpaII tiny fragment Enrichment by Ligation-mediated PCR) will be used to perform a genome-wide analysis of 1 million HpaII tiny fragments (HTF) reflecting the methylation status of ~1.4 million unique HpaII/MspI sites in the L4 and L5 DRGs harvested 14 days after SNL or sham operation. Analyses will identify genomic regions where cytosine methylation is altered and sets of individual loci and/or of large gene regulatory regions where methylation changes are most marked. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using MassArray, a method of methylation analysis combining base-specific cleavage and mass-spectrometry. Research outcomes of Aims 1 and 2 will be validated in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain. PUBLIC HEALTH RELEVANCE: Cytosine is one out of the four building blocks that make up DNA. Unlike the other three building blocks, cytosine can be permanently chemically modified through a process called DNA cytosine methylation. This process leads to "methyl-cytosine", which is often referred to as the "fifth base" of the genome. Methyl cytosine is a keeper of "cellular memory", stable and able to (co-)determine how genes function. De novo cytosine methylation occurs in development and, if activated inappropriately in the adult, can cause diseases like cancer. Chronic pain often arises without a clear cause and can persist after tissue injury has healed. In the rodent model, two small structures of the nervous system located near the spinal cord, the L4 and L5 dorsal root ganglion (DRG) become dysfunctional. The basic force underlying these alterations is unknown. Here we propose that chronic pain is reflected in - and possibly driven by - DNA cytosine methylation changes in dorsal root ganglion cells. We imply that cytosine methylation may constitute a new conceptual layer to be added to our current knowledge about how chronic pain arises. The hypothesis of this proposal is that cytosine methylation in adult DRG neurons and/or glia is plastic; that it is altered by experimental nerve injury; that it (co- )determines gene expression patterns characteristic of the pain state; and that it is a cause of poor patient recovery from chronic pain. Our proposal has two aims. Aim 1: To test if cytosine methylation is globally altered in DRGs of neuropathic pain-rats. We will answer this question using a new technology, which we have developed, the HELP assay. This method combines digestion of DNA, amplification, and binding to 2.1 million probes on a glass slide. It allows us to obtain a picture of cytosine methylation for the entire genome in a single experiment. Aim 2: To test if cytosine methylation is locally altered in predicted regulatory regions. 70 pain genes will be chosen from the literature and from HELP result for in-depth methylation analysis of every CpG around each transcription start site using another method termed MassArray, which has specific advanteages when results with high resolution are desired. Research outcomes of Aims 1 and 2 will be confirmed in an independent set of animals. If conducted now, the proposed study would be the first to investigate DNA cytosine methylation in pain.

描述（由申请人提供）：DNA 胞嘧啶甲基化是“细胞记忆”的保持者，稳定且能够（共同）决定基因表达。胞嘧啶从头甲基化发生在发育过程中，如果在成人中激活不当，可能会导致疾病。慢性疼痛通常在没有明显损伤的情况下出现，或者在组织损伤愈合后持续存在。在啮齿类单神经结扎 (SNL) 疼痛模型中，L4 和 L5 背根神经节 (DRG) 神经元和神经胶质细胞出现功能障碍，电生理学、基因表达和细胞可塑性发生改变。这些改变背后的基本分子力尚不清楚。在这里，我们提出慢性疼痛反映在背根神经节细胞中的 DNA 胞嘧啶甲基化变化中，并且可能是由这种变化驱动的。我们暗示胞嘧啶甲基化可能构成一个新的概念层，添加到已知的疼痛机制中。该提议的假设是，成人 DRG 神经元和/或神经胶质细胞中的胞嘧啶甲基化是可塑的；它会因实验性神经损伤而改变；它（共同）决定疼痛状态特征的基因表达模式；并且它是患者慢性疼痛恢复不良的一个原因。我们的建议有两个目标。目标 1：测试神经性疼痛大鼠 DRG 中胞嘧啶甲基化是否发生整体改变。 HELP 检测（=通过连接介导的 PCR 进行 HpaII 微小片段富集）将用于对 100 万个 HpaII 微小片段 (HTF) 进行全基因组分析，反映 L4 中约 140 万个独特 HpaII/MspI 位点的甲基化状态SNL 或假手术后 14 天收获 L5 DRG。分析将识别胞嘧啶甲基化发生改变的基因组区域以及甲基化变化最明显的单个基因座组和/或大基因调控区域。目标 2：测试预测的调控区域中胞嘧啶甲基化是否发生局部改变。将从文献和HELP结果中选择70个疼痛基因，使用MassArray（一种结合碱基特异性切割和质谱的甲基化分析方法）对每个转录起始位点周围的每个CpG进行深入的甲基化分析。目标 1 和 2 的研究成果将在一组独立的动物中得到验证。如果现在进行，拟议的研究将是第一个研究疼痛中 DNA 胞嘧啶甲基化的研究。公共卫生相关性：胞嘧啶是构成 DNA 的四个组成部分之一。与其他三个构建模块不同，胞嘧啶可以通过称为 DNA 胞嘧啶甲基化的过程进行永久化学修饰。这个过程产生“甲基胞嘧啶”，通常被称为基因组的“第五碱基”。甲基胞嘧啶是“细胞记忆”的守护者，稳定且能够（共同）决定基因如何发挥作用。胞嘧啶从头甲基化发生在发育过程中，如果在成人体内激活不当，可能会导致癌症等疾病。慢性疼痛通常没有明确的原因而出现，并且在组织损伤愈合后可能持续存在。在啮齿动物模型中，位于脊髓附近的神经系统的两个小结构，即 L4 和 L5 背根神经节 (DRG) 出现功能障碍。这些改变背后的基本力量尚不清楚。在这里，我们提出慢性疼痛反映在背根神经节细胞中的 DNA 胞嘧啶甲基化变化中，并且可能是由这种变化驱动的。我们暗示胞嘧啶甲基化可能构成一个新的概念层，以添加到我们目前关于慢性疼痛如何产生的知识中。该提议的假设是，成人 DRG 神经元和/或神经胶质细胞中的胞嘧啶甲基化是可塑的；它会因实验性神经损伤而改变；它（共同）决定疼痛状态特征的基因表达模式；并且它是患者慢性疼痛恢复不良的一个原因。我们的建议有两个目标。目标 1：测试神经性疼痛大鼠 DRG 中胞嘧啶甲基化是否发生整体改变。我们将使用我们开发的新技术 HELP 检测来回答这个问题。该方法结合了 DNA 消化、扩增以及与载玻片上 210 万个探针的结合。它使我们能够在一次实验中获得整个基因组的胞嘧啶甲基化图片。目标 2：测试预测的调控区域中胞嘧啶甲基化是否发生局部改变。将从文献和 HELP 结果中选择 70 个疼痛基因，使用另一种称为 MassArray 的方法对每个转录起始位点周围的每个 CpG 进行深入的甲基化分析，当需要高分辨率结果时，该方法具有特定的优势。目标 1 和 2 的研究结果将在一组独立的动物中得到证实。如果现在进行，这项拟议的研究将是第一个研究疼痛中 DNA 胞嘧啶甲基化的研究。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain.

具有不可知剪接位点发现的 mRNA-seq，用于在慢性疼痛中测试的神经系统转录组学。

DOI：
10.1101/gr.101204.109
发表时间：
2010-06-01
期刊：
Genome research
影响因子：
7
作者：
P. Hammer;M. Banck;Ronny Amberg;Cheng Wang;G. Petznick;Shujun J Luo;I. Khrebtukova;G. Schroth
通讯作者：
G. Schroth

Plasticity of DNA methylation in a nerve injury model of pain.

疼痛神经损伤模型中 DNA 甲基化的可塑性。

DOI：
发表时间：
2015
期刊：
影响因子：
3.7
作者：
Gölzenleuchter, Meike;Kanwar, Rahul;Zaibak, Manal;Al Saiegh, Fadi;Hartung, Theresa;Klukas, Jana;Smalley, Regenia L;Cunningham, Julie M;Figueroa, Maria E;Schroth, Gary P;Therneau, Terry M;Banck, Michaela S;Beutler, Andreas S
通讯作者：
Beutler, Andreas S

AAV provides an alternative for gene therapy of the peripheral sensory nervous system.

AAV 为周围感觉神经系统的基因治疗提供了一种替代方案。

DOI：
10.1038/mt.2010.41
发表时间：
2010-04-01
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
0
作者：
A. Beutler
通讯作者：
A. Beutler

Diametrically opposite methylome-transcriptome relationships in high- and low-CpG promoter genes in postmitotic neural rat tissue.

有丝分裂后神经大鼠组织中高和低 CpG 启动子基因的甲基化组-转录组关系截然相反。

DOI：
发表时间：
2012-05
期刊：
影响因子：
3.7
作者：
Hartung, Theresa;Zhang, Lu;Kanwar, Rahul;Khrebtukova, Irina;Reinhardt, Matthias;Wang, Cheng;Therneau, Terry M;Banck, Michaela S;Schroth, Gary P;Beutler, Andreas S
通讯作者：
Beutler, Andreas S

Targeted alignment and end repair elimination increase alignment and methylation measure accuracy for reduced representation bisulfite sequencing data.

靶向比对和末端修复消除提高了比对和甲基化测量的准确性，以减少亚硫酸氢盐测序数据的代表性。