Does the extreme variability of methadone metabolism have an epigenetic cause?

简介（由申请人提供）：海洛英是最容易上瘾和最危险的药物之一。事实上，美国疾病控制与预防中心最近的一份报告表明，海洛因和其他鸦片类药物占美国所有无意中毒死亡的约30%。美沙酮治疗是治疗海洛因成瘾者为数不多的有效方法之一，既能防止他们复吸，又能让他们过正常的生活。不幸的是，个体之间的美沙酮代谢率差异很大，这使得维持美沙酮治疗的剂量决定变得复杂。此外，许多处方药和其他物质可以极大地改变美沙酮的代谢，从而导致戒断症状和再次滥用海洛因。美沙酮代谢的个体间差异也可能导致最近因非法使用美沙酮而导致的死亡急剧上升。美沙酮代谢的大部分变异性是由于药物代谢酶细胞色素P450 3A4 （CYP3A4）在肝脏和肠道中的个体间表达变异性超过40倍。在过去的二十年里，研究人员系统地研究了CYP3A4基因，试图确定可以解释CYP3A4 mRNA产生的变异的遗传多态性。然而，虽然发现了一些多态性，但它们只能解释CYP3A4变异的一小部分。这表明，其他一些“表观遗传”机制（与不直接基于DNA序列的遗传机制不同）可能导致CYP3A4 mRNA的产生变化。在本文所述的工作中，我们将通过检查54个人类肝脏样本库中通常与mRNA产生减少相关的三种表观遗传化学修饰是否与CYP3A4 mRNA水平降低相关来验证这一新的假设。这些表观遗传标记中的第一个是基因组DNA中CG二核苷酸C碱基的共价甲基化。另外两种是细胞核内作为DNA包装的组蛋白的化学修饰（H3K9的低乙酰化和三甲基化）。在其他系统中，这些修饰中的每一种都已被证明可以促进减少mRNA的产生。例如，这些修饰通常与重要肿瘤抑制基因mRNA的异常丢失有关，包括视网膜母细胞瘤蛋白（pRB）和乳腺癌相关蛋白1 （BRCA1）。然而，令人惊讶的是，它们与药物代谢基因mRNA的可变生产之间的联系从未被测试过。公共卫生相关性：如果这些研究表明CYP3A4 mRNA水平是由这些表观遗传修饰控制的，它将为预测个体患者对美沙酮的反应提供新的方法（例如，通过检测CYP3A4基因是否显示与抑制相关的表观遗传修饰）。在更大的背景下，它将为理解其他药物代谢基因以及其他变异性与人类疾病相关的基因的可变mRNA表达的原因建立一个新的范式。如果我们发现表观遗传修饰在CYP3A4 mRNA变异性中不起作用，这也将是一个重要的发现，这将鼓励进一步研究这种变异性的遗传或长期环境原因。

DESCRIPTION (provided by applicant): Heroin is one of the most addictive and dangerous drugs of abuse. Indeed, a recent CDC report indicated that heroin and other opiates account for ~30% of all inadvertent poisoning deaths in the United States. Treatment with methadone is one of the few effective ways to treat heroin addicts, keeping them from relapsing while allowing them to carry on mostly normal lives. Unfortunately, the rate of methadone metabolism differs hugely between individuals, which complicates dosing decisions in maintenance methadone treatment. Moreover, many prescription drugs and other substances can greatly alter methadone metabolism, which can lead to withdrawal symptoms and relapse to heroin abuse. Interindividual variation in methadone metabolism also likely contributes to the dramatic, recent rise in deaths due to illicit methadone use. Much of the variability in methadone metabolism is due to the greater than 40-fold range of inter-individual expression variability, in the liver and intestines, of the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). Over the past two decades, researchers have systematically examined the CYP3A4 gene to try to identify genetic polymorphisms that could explain the observed variability in CYP3A4 mRNA production. While some polymorphisms have been found, however, they could only explain a small fraction of the CYP3A4 variability. This has suggested that some other "epigenetic" mechanism (as distinguished from genetic mechanisms by not being directly based on DNA sequence) might result in variable CYP3A4 mRNA production. In the work described here, we will test this novel hypothesis by examining whether three epigenetic chemical modifications that are commonly associated with reduced mRNA production correlate with reduced CYP3A4 mRNA levels in a bank of 54 human liver samples. The first of these epigenetic marks is the covalent methylation of the C base in CG dinucleotides in genomic DNA. The other two are chemical modifications of the histone proteins that serve as packaging for DNA inside cell nuclei (hypoacetylation and trimethylation of H3K9). Each of these modifications have been shown to promote reduced mRNA production in other systems. For instance, these modifications are often associated with the aberrant loss of mRNA for important tumor suppressor genes, including retinoblastoma protein (pRB) and breast cancer associated protein 1 (BRCA1). Surprisingly, however, their connection to variable production of drug metabolizing gene mRNA has never been tested. PUBLIC HEALTH RELEVANCE: If these studies show that CYP3A4 mRNA levels are controlled by these epigenetic modifications, it will suggest new ways to predict an individual patient's response to methadone (e.g. by testing whether the CYP3A4 gene shows epigenetic modifications associated with repression). In a larger context, it will establish a new paradigm for understanding the causes of variable mRNA expression for other drug metabolizing genes, as well as for other genes whose variability is associated with human disease. If we find that epigenetic modifications do not play a role in CYP3A4 mRNA variability, this will also be an important discovery, that will encourage further research into the genetic or long-term environmental causes of this variability.