Increased OPRM1 DNA methylation in lymphocytes of methadone-maintained former heroin addicts

Abstract

The mu-opioid receptor is the site of action of opiates and opioids. We examined whether there are differences in cytosine:guanine (CpG) dinucleotide methylation in the OPRM1 promoter between former heroin addicts and controls. We analyzed methylation at 16 CpG dinucleotides in DNA obtained from lymphocytes of 194 Caucasian former severe heroin addicts stabilized in methadone maintenance treatment and 135 Caucasian control subjects. Direct sequencing of bisulfite-treated DNA showed that the percent methylation at two CpG sites was significantly associated with heroin addiction. The level of methylation at the -18 CpG site was 25.4% in the stabilized methadone-maintained former heroin addicts and 21.4% in controls (p=0.0035, generalized estimating equations (GEE); p=0.0077, t-test; false discovery rate (FDR)=0.048), and the level of methylation at the +84 CpG dinucleotide site was 7.4% in cases and 5.6% in controls (p=0.0095, GEE; p=0.0067, t-test; FDR=0.080). Both the -18 and the +84 CpG sites are located in potential Sp1 transcription factor-binding sites. Methylation of these CpG sites may lead to reduced OPRM1 expression in the lymphocytes of these former heroin addicts.

Conflict of interest statement

DISCLOSURE/CONFLICT OF INTEREST

All the authors, except J.O., declare that, except for the income received from our primary employers, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interests. One author, J.O., wishes to declare that he personally receives book royalties from the Johns Hopkins University Press and that his laboratory receives funding from Hoffmann-La Roche Inc.

Figures

Figure 1

The OPRM1 promoter region. Schematic of the OPRM1 gene promoter region (from 400 nucleotides upstream to 1000 nucleotides downstream of the transcription start site) is presented in the upper diagram. The two CpG islands are boxed. The CpG dinucleotides are indicated as |. The major transcription start site () is located at −253 upstream of the ATG translation start site. The sequence of the amplified CpG island is displayed below with the sequences corresponding to the nested PCR primers in italics. The sixteen CpG sites analyzed for cytosine methylation (bold) with their position relative to the A (chr6:154,402,373; NCBI Build 36.1, March 2006) of the ATG translation start site (underlined) are indicated. The three putative Sp1 transcription factor binding sites are boxed.

Figure 2

Methylation at the sixteen CpG sites. (a) Analysis of cloned amplified bisulfite-treated DNA from five randomly selected controls and five randomly selected former heroin addicts. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified OPRM1 region. (b) Analysis of cloned bisulfite-treated DNA from five former heroin addicts selected for varying levels of methylation. (c) Correlation of percent cytosine methylation determined by sequencing/ESME (ordinate) and by cloning (abscissa). The correlation is derived from data using clones 1, 2, 4, and 6–10 of (a), and 1–3 and 5 of (b), as these samples had a forward to reverse correlation ≥ 0.7 by sequencing/ESME analysis.

Figure 3

Percent methylation of sixteen CpG dinucleotides in the OPRM1 promoter region in former severe heroin addicts and controls. CpG sites in Sp1 binding sites are indicated. ** represents p < 0.01. Error bars represent s.e.m.