Mobile DNA in Drug Abuse

 描述(申请人提供)：可卡因成瘾(CA)是一种常见的慢性大脑疾病，发病率和死亡率显著增加(Degenhardt等人，2011年)。据报道，过去一个月，美国约有150万成年人使用可卡因(SAMSHA，2013)。尽管双胞胎研究与中度遗传成分一致，但通过研究外周DNA很难识别风险增加的等位基因。建议从CA患者死后脑组织中研究神经元DNA，寻找与CA相关的体细胞神经元突变。中枢神经系统中一种常见的体细胞突变是由LINE1(L1)反转录转座子(RTPs)介导的，它占人类基因组的17%(Beck等人，2010年)。L1的长度为6 kb。它们编码逆转录酶和内切酶，在转录活跃的基因组区域发挥复制和粘贴机制的作用，以在人类基因组中传播(Richardson等人，2014)。虽然大多数这些体细胞新生L1对神经元功能几乎没有影响，但有些可能会干扰神经元的活动，因为它们插入了特定神经元正常功能所需的基因。如果功能性L1出现在中枢神经系统发育的早期，来自该神经元前体的所有子代神经元也将携带L1插入，可能导致注定会增加CA风险的神经元功能障碍。反复使用可卡因也有可能导致中枢神经系统神经元转录活动的波动，为L1s的扩散创造机会之窗，导致进行性 这些L1基因对神经元基因的破坏。在CA患者的mPFC中的初步结果表明，从头L1可能扰乱激酶/磷酸化途径(Bonferroni校正p=10-6-10-7)，这些途径在对照组中没有明显影响。这项建议将采用来自90名CA患者和90名匹配对照的mPFC。从mPFC神经元核中提取的DNA将用于半生成聚合酶链式反应，以丰富L1序列的DNA。DNA将被测序并与参考基因组比对，以检测从头开始L1 RTP插入神经元表达的基因。以这种方式，预计将发现与CA相关联的从头开始的L1 RTP。这些新发的L1可能是反复使用可卡因的结果，也可能是可卡因使用的先兆，是CA的危险因素。如果从头开始的L1是反复使用可卡因的结果，这将是一个范式转换的发现，即滥用药物扰乱了神经元基因组。如果新L1是可卡因使用的先兆，并作为CA的危险因素发挥作用，它们将识别与CA相关的新基因和途径，这可能为药物开发和遗传研究创造靶点。利用CRISPR/Cas9基因编辑技术，将通过在神经元前体细胞系中重新创建L1插入来研究来自CA组织的新L1的功能影响。通过这种方式，与CA相关的神经元DNA变异将被定义。

 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.