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

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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.