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Papers by Alexandra Doehring

PAIN, 2013

Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenot... more Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenotypic changes by altering gene transcription are summarized as epigenetics. A major epigenetic mechanism is methylation or demethylation at CpG-rich DNA islands. DNA methylation triggered by drugs has largely unexplored therapeutic consequences. Here we report increased methylation at a CpG rich island in the OPRM1 gene coding for l-opioid receptors and at a global methylation site (LINE-1) in leukocytes of methadone-substituted former opiate addicts compared with matched healthy controls. Higher DNA methylation associated with chronic opioid exposure was reproduced in an independent cohort of opioid-treated as compared to non-opioid-treated pain patients. This suggests that opioids may stimulate DNA methylation. The OPRM1 methylation had no immediate effect on l-opioid receptor transcription and was not associated with opioid dosing requirements. However, the global DNA methylation at LINE-1 was significantly correlated with increased chronic pain. This suggests inhibitory effects on the transcription of still unspecified nocifensive gene products. It further implies that opioids may be causally associated with increased genome-wide DNA methylation, although currently there is no direct evidence of this. This has phenotypic consequences for pain and may provide a new, epigenetics-associated mechanism of opioid-induced hyperalgesia. The results indicate a potential influence of opioid analgesics on the patients' epigenome. They emphasize the need for reliable and costeffective screening tools and may imply that high-throughput screening for lead compounds in artificial expression systems may not provide the best tools for identifying new pain medications.

Pharmacogenomics, 2013

The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genet... more The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genetic variants in the pain field. At the molecular level, the variant reduces opioid receptor signaling efficiency and expression, the latter probably via a genetic-epigenetic interaction. In experimental settings, the variant was reproducibly associated with decreased effects of exogenous opioids. However, this translates into very small clinical effects (meta-ana lysis of 14 studies: Cohen's d = 0.096; p = 0.008), consisting of slightly higher opioid dosing requirements in peri-and post-operative settings. An effect can neither be maintained for chronic analgesic therapy nor for opioid side effects. It seems unlikely that further studies will reveal larger effect sizes and, therefore, further analyses appear unwarranted. Thus, due to its small effect size, the SNP is without major clinical relevance as a solitary variant, but should be regarded as a part of complex genotypes underlying pain and analgesia.

PLoS ONE, 2013

Background and Aims: Mutations reducing the function of Na v 1.7 sodium channels entail diminishe... more Background and Aims: Mutations reducing the function of Na v 1.7 sodium channels entail diminished pain perception and olfactory acuity, suggesting a link between nociception and olfaction at ion channel level. We hypothesized that if such link exists, it should work in both directions and gain-of-function Na v 1.7 mutations known to be associated with increased pain perception should also increase olfactory acuity.

Human Molecular Genetics, 2012

Genetic and epigenetic mechanisms play important roles in protein expression, although at differe... more Genetic and epigenetic mechanisms play important roles in protein expression, although at different levels. Genetic variations can alter CpG sites and thus influence the epigenetic regulation of mRNA expression, providing an increasingly recognized mechanism of functional consequences of genetic polymorphisms. One of those genetic effects is the association of reduced m-opioid receptor expression with the functional genetic variant N40D (OPRM1 118A>G, rs1799971) that causes an amino acid exchange in the extracellular terminal of the m-opioid receptor. We report that the nucleotide exchange at gene position 1118 introduces a new CpG-methylation site into the OPRM1 DNA at position 1117. This leads to an enhanced methylation of the OPRM1 DNA at this site and downstream. This epigenetic mechanism impedes m-opioid receptor upregulation in brain tissue of Caucasian chronic opiate addicts, assessed postmortem. While in wildtype subjects, a reduced signalling efficiency associated with chronic heroin exposure was compensated by an increased receptor density, this upregulation was absent in carriers of the 118G receptor variant due to a diminished OPRM1 mRNA transcription. Thus, the OPRM1 118A>G SNP variant not only reduces m-opioid receptor signalling efficiency, but, by a genetic -epigenetic interaction, reduces opioid receptor expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure. This demonstrates that a change in the genotype can cause a change in the epigenotype with major functional consequences.

PLoS ONE, 2014

Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorou... more Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorous substances. Based on reported associations of TRPA1 genetics with increased sensitivity to thermal pain stimuli, we therefore hypothesized that this association also exists for increased olfactory sensitivity.

Clinical Pharmacokinetics, 2012

Arterial blood sampling is necessary when drugs such as the fast-acting opioid analgesic remifent... more Arterial blood sampling is necessary when drugs such as the fast-acting opioid analgesic remifentanil exhibit relevant differences between arterial and venous blood concentrations. Arterial cannulation is generally considered to be clinically safe and has thus become a standard procedure in pharmacokinetic-pharmacodynamic assessments. However, rare cases of arterial occlusions have to be considered in risk-benefit assessments of arterial sampling in pharmacokinetic studies, especially when including healthy volunteers. In an actual case, arterial occlusion requiring surgical repair was caused by a factor V Leiden thrombophilia associated genetic variant F5 1691G>A (rs6025) and aggravated by a hypoplastic radial artery. Neither risk factor had been identified prior to enrolment by routine laboratory tests such as the prothrombin time (international normalized ratio), partial thromboplastin time and the clinical Allen's test of arterial function. Re-assessment of the necessity of arterial sampling showed that none of the potential alternatives, target concentrations of computerized infusions or venous concentrations during non-steady-state and steady-state conditions could provide the arterial concentrations. Relying on venous concentrations may result in erroneous pharmacodynamic parameters. Accurate pharmacokinetic-pharmacodynamic studies relying on precisely measured blood concentrations require serial sampling techniques during both steady-state and non-steady-state conditions. However, as illustrated by the presented case, incidents involving the generally safe procedure of arterial sampling are possible, although rare. To further minimize the risks, screening of subjects for prothrombotic risks and careful assessment of the suitability of the artery should be considered in pharmacokinetic studies requiring arterial cannulation.

Pharmacogenomics, 2007

Genetic variants in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which codes for COX-2... more Genetic variants in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which codes for COX-2, have been identified to modulate the response to COX-2-inhibiting drugs and to be possible risk factors for the incidence or prognosis of cardiovascular or neoplastic diseases, Alzheimer's disease, multiple sclerosis, asthma or osteoarthritis. Clinical evidence thus suggests a clinical importance of COX-2 genetics reaching from disease risk or prognostics up to a personalized therapy with COX-2 inhibitors. The aim of this study was to develop rapid and reliable screening assays for PTGS2 mutations with reported clinical consequences. SNPs (dbSNP-IDs rs689465, rs689466, rs3918304, rs20415, rs20417, rs5270, rs2745557, rs5277, rs2066826, rs4648276, rs5273, rs5275, rs4648298, rs689469) and a nucleotide-deletion variant (rs20431) were chosen according to reported functional associations. For this selection of variants spanning the whole PTGS2 gene range, Pyrosequencing assays were established in DNA from 350 healthy unrelated Caucasians. In all 350 DNA samples, the 15 PTGS2 polymorphisms were identified correctly as verified by control samples obtained by conventional sequencing. In silico haplotype analysis based on ten SNPs of greater than 1% observed frequencies identified two haploblocks with a linkage disequilibrium of D' = 0.59. Approximately 50% of the reconstructed haplotypes consisted of non-mutated alleles. The presently developed Pyrosequencing assays allow for quick and reliable detection of PTGS2 genotypes and may promote further research toward personalized approaches to pathophysiological conditions involving COX-2.

PAIN, 2013

Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenot... more Environmentally caused changes in chromosomes that do not alter the DNA sequence but cause phenotypic changes by altering gene transcription are summarized as epigenetics. A major epigenetic mechanism is methylation or demethylation at CpG-rich DNA islands. DNA methylation triggered by drugs has largely unexplored therapeutic consequences. Here we report increased methylation at a CpG rich island in the OPRM1 gene coding for l-opioid receptors and at a global methylation site (LINE-1) in leukocytes of methadone-substituted former opiate addicts compared with matched healthy controls. Higher DNA methylation associated with chronic opioid exposure was reproduced in an independent cohort of opioid-treated as compared to non-opioid-treated pain patients. This suggests that opioids may stimulate DNA methylation. The OPRM1 methylation had no immediate effect on l-opioid receptor transcription and was not associated with opioid dosing requirements. However, the global DNA methylation at LINE-1 was significantly correlated with increased chronic pain. This suggests inhibitory effects on the transcription of still unspecified nocifensive gene products. It further implies that opioids may be causally associated with increased genome-wide DNA methylation, although currently there is no direct evidence of this. This has phenotypic consequences for pain and may provide a new, epigenetics-associated mechanism of opioid-induced hyperalgesia. The results indicate a potential influence of opioid analgesics on the patients' epigenome. They emphasize the need for reliable and costeffective screening tools and may imply that high-throughput screening for lead compounds in artificial expression systems may not provide the best tools for identifying new pain medications.

Pharmacogenomics, 2013

The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genet... more The human µ-opioid receptor variant 118 A>G (rs1799971) has become one of the most analyzed genetic variants in the pain field. At the molecular level, the variant reduces opioid receptor signaling efficiency and expression, the latter probably via a genetic-epigenetic interaction. In experimental settings, the variant was reproducibly associated with decreased effects of exogenous opioids. However, this translates into very small clinical effects (meta-ana lysis of 14 studies: Cohen's d = 0.096; p = 0.008), consisting of slightly higher opioid dosing requirements in peri-and post-operative settings. An effect can neither be maintained for chronic analgesic therapy nor for opioid side effects. It seems unlikely that further studies will reveal larger effect sizes and, therefore, further analyses appear unwarranted. Thus, due to its small effect size, the SNP is without major clinical relevance as a solitary variant, but should be regarded as a part of complex genotypes underlying pain and analgesia.

PLoS ONE, 2013

Background and Aims: Mutations reducing the function of Na v 1.7 sodium channels entail diminishe... more Background and Aims: Mutations reducing the function of Na v 1.7 sodium channels entail diminished pain perception and olfactory acuity, suggesting a link between nociception and olfaction at ion channel level. We hypothesized that if such link exists, it should work in both directions and gain-of-function Na v 1.7 mutations known to be associated with increased pain perception should also increase olfactory acuity.

Human Molecular Genetics, 2012

Genetic and epigenetic mechanisms play important roles in protein expression, although at differe... more Genetic and epigenetic mechanisms play important roles in protein expression, although at different levels. Genetic variations can alter CpG sites and thus influence the epigenetic regulation of mRNA expression, providing an increasingly recognized mechanism of functional consequences of genetic polymorphisms. One of those genetic effects is the association of reduced m-opioid receptor expression with the functional genetic variant N40D (OPRM1 118A>G, rs1799971) that causes an amino acid exchange in the extracellular terminal of the m-opioid receptor. We report that the nucleotide exchange at gene position 1118 introduces a new CpG-methylation site into the OPRM1 DNA at position 1117. This leads to an enhanced methylation of the OPRM1 DNA at this site and downstream. This epigenetic mechanism impedes m-opioid receptor upregulation in brain tissue of Caucasian chronic opiate addicts, assessed postmortem. While in wildtype subjects, a reduced signalling efficiency associated with chronic heroin exposure was compensated by an increased receptor density, this upregulation was absent in carriers of the 118G receptor variant due to a diminished OPRM1 mRNA transcription. Thus, the OPRM1 118A>G SNP variant not only reduces m-opioid receptor signalling efficiency, but, by a genetic -epigenetic interaction, reduces opioid receptor expression and therefore, depletes the opioid system of a compensatory reaction to chronic exposure. This demonstrates that a change in the genotype can cause a change in the epigenotype with major functional consequences.

PLoS ONE, 2014

Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorou... more Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorous substances. Based on reported associations of TRPA1 genetics with increased sensitivity to thermal pain stimuli, we therefore hypothesized that this association also exists for increased olfactory sensitivity.

Clinical Pharmacokinetics, 2012

Arterial blood sampling is necessary when drugs such as the fast-acting opioid analgesic remifent... more Arterial blood sampling is necessary when drugs such as the fast-acting opioid analgesic remifentanil exhibit relevant differences between arterial and venous blood concentrations. Arterial cannulation is generally considered to be clinically safe and has thus become a standard procedure in pharmacokinetic-pharmacodynamic assessments. However, rare cases of arterial occlusions have to be considered in risk-benefit assessments of arterial sampling in pharmacokinetic studies, especially when including healthy volunteers. In an actual case, arterial occlusion requiring surgical repair was caused by a factor V Leiden thrombophilia associated genetic variant F5 1691G>A (rs6025) and aggravated by a hypoplastic radial artery. Neither risk factor had been identified prior to enrolment by routine laboratory tests such as the prothrombin time (international normalized ratio), partial thromboplastin time and the clinical Allen's test of arterial function. Re-assessment of the necessity of arterial sampling showed that none of the potential alternatives, target concentrations of computerized infusions or venous concentrations during non-steady-state and steady-state conditions could provide the arterial concentrations. Relying on venous concentrations may result in erroneous pharmacodynamic parameters. Accurate pharmacokinetic-pharmacodynamic studies relying on precisely measured blood concentrations require serial sampling techniques during both steady-state and non-steady-state conditions. However, as illustrated by the presented case, incidents involving the generally safe procedure of arterial sampling are possible, although rare. To further minimize the risks, screening of subjects for prothrombotic risks and careful assessment of the suitability of the artery should be considered in pharmacokinetic studies requiring arterial cannulation.

Pharmacogenomics, 2007

Genetic variants in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which codes for COX-2... more Genetic variants in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which codes for COX-2, have been identified to modulate the response to COX-2-inhibiting drugs and to be possible risk factors for the incidence or prognosis of cardiovascular or neoplastic diseases, Alzheimer's disease, multiple sclerosis, asthma or osteoarthritis. Clinical evidence thus suggests a clinical importance of COX-2 genetics reaching from disease risk or prognostics up to a personalized therapy with COX-2 inhibitors. The aim of this study was to develop rapid and reliable screening assays for PTGS2 mutations with reported clinical consequences. SNPs (dbSNP-IDs rs689465, rs689466, rs3918304, rs20415, rs20417, rs5270, rs2745557, rs5277, rs2066826, rs4648276, rs5273, rs5275, rs4648298, rs689469) and a nucleotide-deletion variant (rs20431) were chosen according to reported functional associations. For this selection of variants spanning the whole PTGS2 gene range, Pyrosequencing assays were established in DNA from 350 healthy unrelated Caucasians. In all 350 DNA samples, the 15 PTGS2 polymorphisms were identified correctly as verified by control samples obtained by conventional sequencing. In silico haplotype analysis based on ten SNPs of greater than 1% observed frequencies identified two haploblocks with a linkage disequilibrium of D' = 0.59. Approximately 50% of the reconstructed haplotypes consisted of non-mutated alleles. The presently developed Pyrosequencing assays allow for quick and reliable detection of PTGS2 genotypes and may promote further research toward personalized approaches to pathophysiological conditions involving COX-2.