Mobile DNA in Drug Abuse

药物滥用中的移动 DNA

基本信息

批准号：
9128371
负责人：
Wade H Berrettini
金额：
$ 46.34万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9128371
关键词：
Adult Age Alleles Anterior Antibodies Beryllium Biological Assay Brain Brain Diseases Brain region Brodmann&apos s area CRISPR/Cas technology Cell Line Cell Nucleus Cell Separation Cell physiology Chromatin Chronic Cocaine Cocaine Dependence DNA Data Daughter Detection Development Dopamine Drug abuse Drug usage Elements Embryonic Development Event FDA approved Fluorescence Gene Frequency Genes Genetic Research Genetic Transcription Genome Genomic Segment Glutamates Goals Health Heritability High-Throughput Nucleotide Sequencing Human Human Genome Individual Inherited Label Length Medial Mediating Mendelian disorder Morbidity - disease rate Mutation Neuronal Differentiation Neurons Nuclear Nucleus Accumbens Paste substance Pathway Analysis Pathway interactions Patients Peripheral Persons Pharmaceutical Preparations Pharmacotherapy Phosphorylation Phosphotransferases Polynucleotide 5&apos -Hydroxyl-Kinase Population Prefrontal Cortex Psychotherapy Public Health Published Comment RNA RNA Polymerase II RNA-Directed DNA Polymerase Reading Reporting Retrotransposition Retrotransposon Rewards Risk Risk Factors Sampling Somatic Mutation Support Groups Surveys Synapses Technology Testing Tissues Twin Studies United States Substance Abuse and Mental Health Services Administration Variant Ventral Tegmental Area cocaine overdose cocaine use digital drug development drug of abuse endonuclease genome wide association study improved mortality neuron development novel peer precursor cell promoter public health relevance reference genome research and development response reuptake sex transposon/insertion element

项目摘要

DESCRIPTION (provided by applicant): Cocaine addiction (CA) is a common chronic brain disorder with markedly increased morbidity and mortality (Degenhardt et al, 2011). ~ 1,500,000 adults in the US reported using cocaine in the past month (SAMSHA, 2013). Although twin studies are consistent with a moderate inherited component, it has been difficult to identify risk-increasing alleles by studying peripheral DNA. It is proposed to study neuronal DNA from post-mortem brain of CA patients, to find somatic neuronal mutation associated with CA. One common somatic mutation in the CNS is mediated by LINE1 (L1) retrotransposons (RTPs), which constitute 17% of the human genome (Beck et al, 2010). L1s are 6 kb in length. They encode a reverse transcriptase and an endonuclease, which function in a copy and paste mechanism, in transcriptionally active genomic regions, to spread in the human genome (Richardson et al, 2014). While most of these somatic de novo L1s will have little effect on neuronal function, some may interfere with neuronal activity because they have inserted into a gene needed by that particular neuron for normal function. If functional L1s occur early in CNS development, all the daughter neurons that derive from that neuronal precursor will also carry the L1 insertion, perhaps leading to a dysfunctional population of neurons destined to increase risk for CA. It is also possible that repeated use of cocaine results in waves of CNS neuronal transcriptional activity, creating windows of opportunity for L1s to spread, leading to progressive disruption of neuronal genes by those L1s. Preliminary results in mPFC of CA patients indicate that de novo L1s may disrupt kinase/phosphorylation pathways (Bonferroni corrected p = 10-6-10-7), pathways which are not significantly impacted among controls. This proposal will employ mPFC from 90 CA patients and 90 matched controls. DNA extracted from mPFC neuronal nuclei will be used in hemidegenerate PCR to enrich the DNA for L1 sequences. DNA will be sequenced and aligned to the reference genome to detect de novo L1 RTP insertions into neuronally-expressed genes. In this manner, it is expected that de novo L1 RTPs, associated with CA, will be discovered. These de novo L1s may be a consequence of repeated cocaine use or they may be antecedent to cocaine use, acting as risk factors for CA. If the de novo L1s are a consequence of repeated cocaine use, this would be a paradigm-shifting discovery that a drug of abuse disrupts neuronal genomes. If the de novo L1s are antecedent to cocaine use, and function as CA risk factors, they would identify novel genes and pathways related to CA, which may create targets for drug development and genetic research. Confirmed de novo L1s from CA tissue will be studied for functional impact by re-creation of the L1 insertion in a neuronal precursor cell line, using CRISPR/Cas9 gene editing technology. In this manner neuronal DNA variation associated with CA will be defined.

描述（由适用提供）：可卡因成瘾（CA）是一种常见的慢性脑疾病，发病率和死亡率明显增加（Degenhardt等，2011）。在美国，在过去一个月中使用可卡因的〜1,500,000名成年人（Samsha，2013年）。尽管双胞胎研究与现代遗传成分是一致的，但很难通过研究外围DNA来识别风险增加等位基因。建议研究CA患者验尸后大脑的神经元DNA，以发现与Ca相关的体细胞神经元突变。中枢神经系统中的一种常见的体细胞突变是由构成人类基因组17％的Line1（L1）逆转座子（RTP）介导的（Beck等，2010）。 L1的长度为6 kb。他们在转录活跃的基因组区域中编码逆转录酶和一个在复制和粘贴机制中起作用的内切酶，以扩散在人基因组中（Richardson等，2014）。尽管这些从头l1的大多数对神经元功能的影响很小，但有些可能会干扰神经元活性，因为它们已插入该特定神经元所需的基因以实现正常功能。如果功能性L1在中枢神经系统发育的早期发生，那么从该神经元前体衍生的所有女儿神经元也将带来L1插入，可能导致神经元功能失调的神经元，注定会增加CA的风险。重复使用可卡因也可能导致CNS神经元转录活动的波浪，从而为L1s提供了机会窗口，从而导致了进步这些L1对神经元基因的破坏。 CA患者MPFC的初步结果表明，从头L1可能会破坏激酶/磷酸化途径（Bonferroni校正后的P = 10-6-10-7），这些途径在对照组之间没有显着影响。该提案将采用来自90名CA患者和90个匹配对照的MPFC。从MPFC神经元核中提取的DNA将用于Hemidegerator PCR，以富集L1序列的DNA。 DNA将进行测序并与参考基因组对齐，以检测从头L1 RTP插入到神经元表达的基因中。通过这种方式，可以发现与CA相关的Novo L1 RTP。这些从头使用可卡因的结果可能是可卡因的重复使用的结果，或者可能是可卡因使用的前提，是CA的危险因素。如果从头使用可卡因是反复使用的结果，那将是一种范式转移发现，滥用药物会破坏神经元基因组。如果从头使用可卡因的前提是使用可卡因，并且起着CA风险因素的作用，他们将确定与CA相关的新型基因和途径，这可能为药物开发和遗传研究带来靶标。使用CRISPR/CAS9基因编辑技术，将研究从CA组织中确认的从CA组织中确认的从CA组织中的NR1 L1s进行功能影响。以这种方式，将定义与CA相关的神经DNA变异。