Project description:BackgroundA long-term opioid use has been associated with hypermethylation of the opioid receptor mu 1 (OPRM1) promoter. Very little is currently known about the early epigenetic response to therapeutic opioids. Here, we examine whether we can detect DNA methylation changes associated with a few days' use of prescribed opioids. Genome-wide DNA methylation was assayed in a cohort of 33 opioid-naïve participants who underwent standard dental surgery followed by opioid self-administration. Saliva samples were collected before surgery (visit 1), and at two postsurgery visits at 2.7 ± 1.5 days (visit 2), and 39 ± 10 days (visit 3) after the discontinuation of opioid analgesics.ResultsThe perioperative methylome underwent significant changes over the three visits that were primarily due to postoperative inflammatory response and cell heterogeneity. To specifically examine the effect of opioids, we started with a candidate gene approach and evaluated 10 CpGs located in the OPRM1 promoter. There was a significant cross-sectional variability in opioid use, and for participants who self-administered the prescribed drugs, the total dosage ranged from 5-210 morphine milligram equivalent (MME). Participants were categorized by cumulative dosage into three groups: < 25 MME, 25-90 MME, and ≥ 90 MME. Using mixed-effects modeling, 4 CpGs had significant positive associations with opioid dose at two-tailed p value < 0.05, and overall, 9 of the 10 OPRM1 promoter CpGs showed the predicted higher methylation in the higher dose groups relative to the lowest dose group. After adjustment for age, cellular heterogeneity, and past tobacco use, the promoter mean methylation also had positive associations with cumulative MME (regression coefficient = 0.0002, one-tailed p value = 0.02) and duration of opioid use (regression coefficient = 0.003, one-tailed p value = 0.001), but this effect was significant only for visit 3. A preliminary epigenome-wide association study identified a significant CpG in the promoter of the RAS-related signaling gene, RASL10A, that may be predictive of opioid dosage.ConclusionThe present study provides evidence that the hypermethylation of the OPRM1 promoter is in response to opioid use and that epigenetic differences in OPRM1 and other sites are associated with a short-term use of therapeutic opioids.

Project description:Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualised evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analysed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analysed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment. Bisulfite-converted DNA of PBMC from 24 patients before and after 4 days of rhGH treatment were hybridized to the Illumina Infinium HumanMethylation 450k BeadChip.

Project description:Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualised evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analysed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analysed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment. Bisulfite converted DNA of PB from 36 SGA born children treated long-term with rhGH and 18 SGA born children with no rhGH treatment (controls) were hybridized to the Illumina Infinium HumanMethylation 450k Bead Chip.

Project description:Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualised evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analysed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analysed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

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Effect of short-term prescription opioids on DNA methylation of the OPRM1 promoter

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