Alexandra Doehring

Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients

BACKGROUND & AIMS Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.

Impact of donor and recipient IL28B rs12979860 genotypes on hepatitis C virus liver graft reinfection

BACKGROUND & AIMS Recent studies have described a major impact of genetic variations near the IL28B gene on the natural course and outcome of antiviral therapy in chronic hepatitis C. We therefore, aimed to explore the impact of donor and recipient genotypes of these polymorphisms on hepatitis C virus (HCV) liver graft reinfection. METHODS Donor and recipient genotypes of IL28B rs12979860C>T single nucleotide polymorphism were determined in 91 patients with HCV liver graft reinfection, 47 of whom were treated with pegylated interferon-α (PEG-IFN-α) and ribavirin. IL28B genetic polymorphisms were correlated with the natural course and treatment outcome of recurrent hepatitis C. RESULTS Patients requiring liver transplantation due to end-stage chronic hepatitis C appeared to be selected toward the adverse genotypes rs12979860 CT/TT compared to non-transplanted HCV-infected patients (p=0.046). Patients with the donor genotype rs12979860 CC had higher peak ALT and HCV RNA serum concentrations than those with CT/TT (p=0.04 and 0.06, respectively). No association was observed between ALT/HCV RNA serum concentrations and recipient genotypes (p>0.3). More important, donor IL28B rs12979860 CC vs. CT/TT genotypes were associated with rapid, complete early, and sustained virologic response (RVR, cEVR, SVR) to treatment with PEG-IFN-α and ribavirin (p=0.003, 0.0012, 0.008, respectively), but weaker associations of recipient genotypes with RVR, cEVR, and SVR were observed as well (p=0.0046, 0.115, 0.118, respectively). CONCLUSIONS We provide evidence for a dominant, but not exclusive impact of the donor rather than the recipient IL28B genetic background on the natural course and treatment outcome of HCV liver graft reinfection.

Epigenetics in pain and analgesia: An imminent research field

Heritable phenotypes resulting from environment‐caused changes in a chromosome without alterations in the DNA sequence are increasingly recognized as a basis of personalized therapy. Epigenetic mechanisms include covalent modifications of the DNA (methylation) or of the DNA‐packaging histones (e.g., deacetylation or phosphorylation). In addition, regulatory non‐coding RNA molecules (micro‐RNAs) exert epigenetic actions. This leads to disruption or otherwise modified expression of genes. Environmental influences such as nutritional factors, exposure to chemicals or drugs, but also social factors appear to exert epigenetic actions. Histone modifications and DNA methylation are associated with the subjects age. Epigenetic mechanisms can silence the expression of pro‐ or antinociceptive genes. To the epigenetic control of nociception adds its control of the pharmacodynamics or pharmacokinetics of analgesics by epigenetic control of drug targets and analgesics metabolizing enzymes. Although epigenetics‐based strategies for pain therapy are not yet available, experiments in rodents suggest that RNA interference may become a new therapy approach for neuropathic and other pain. Another epigenetic approach to analgesic treatment employs inhibitors of histone deacetylase that act on the epigenome by indirectly remodeling the spatial conformation of the chromatin. Finally, epigenetic techniques such as RNA interference have been employed in pain research to proof the contribution of certain proteins to nociception. Thus, the new field of epigenetics becomes increasingly used in research and management of pain and will complement genetics. This article introduces epigenetics to pain and summarizes the current and future utility.

Chronic opioid use is associated with increased DNA methylation correlating with increased clinical pain

Summary Chronic opioid exposure is associated with OPRM1 and global DNA hypermethylation. Global DNA methylation correlates with chronic pain in opioid‐treated but not non‐opioid‐treated patients. Abstract 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 μ‐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 μ‐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 cost‐effective 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.

Genetic–epigenetic interaction modulates µ-opioid receptor regulation

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 μ-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 μ-opioid receptor. We report that the nucleotide exchange at gene position +118 introduces a new CpG-methylation site into the OPRM1 DNA at position +117. This leads to an enhanced methylation of the OPRM1 DNA at this site and downstream. This epigenetic mechanism impedes μ-opioid receptor upregulation in brain tissue of Caucasian chronic opiate addicts, assessed postmortem. While in wild-type 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 µ-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.

Can extremely low or high morphine formation from codeine be predicted prior to therapy initiation

ABSTRACT Activation of codeine by O‐demethylation into morphine is a prerequisite for its analgesic effects and severe toxicity. Identifying patients in whom morphine is formed either at extremely low or at extremely high amounts may improve efficacy and safety of codeine therapy. To assess how well this identification is possible, we compared the performance of current CYP2D6 phenotype association systems (traditional genotype‐based classification, a recently proposed CYP2D6 activity score, and the plasma dextromethorphan metabolic ratio) in 57 healthy Caucasians after oral administration of 30 mg dextromethorphan hydrobromide or 50 mg codeine. Most subjects (87.5%) at the lower 15% of morphine formation from codeine and thus likely to not to respond to codeine therapy were correctly identified by CYP2D6 genotype‐ or phenotype‐based systems. In contrast, in subjects at the upper 15% of morphine formation being at risk for opioid toxicity, CYP2D6 genotyping predicted only the 50% who carried gene duplication, whereas dextromethorphan‐based phenotyping identified 67.5% of the subjects with high morphine formation. However, satisfactory prediction (87.5%) of high morphine formation was only achieved when combining genotyping with phenotyping. In conclusion, insufficient morphine formation from codeine and thus likely failure of analgesia can currently be well predicted. However, to make codeine therapy safe, extremely high morphine formation has to be predicted as well, which has to be obtained at the effort of combining genotyping with phenotyping.

A GTP cyclohydrolase 1 genetic variant delays cancer pain.

&NA; Pain is a major symptom in 70% of patients with advanced cancer. We analyzed data of 251 cancer patients (142 men and 109 women aged 29–89 years, Karnofsky status 10–90, 65.7 ± 13.9) for association of a reduced‐function haplotype in the GTP cyclohydrolase 1 (GCH1) gene with cancer pain therapy. The interval between cancer diagnosis and opioid therapy initiation was significantly (p = 0.002) longer in homozygous carriers of these genetic variants (78 ± 65.2 months) than in heterozygous (37 ± 46.5 months) and non‐carriers (30.4 ± 43.8 months). Thus, reduced GCH1 upregulation, here conferred by non‐coding and non‐splice site GCH1 variants known to lead to decreased tetrahydrobiopterin expression, delays the need for opioid therapy in cancer. This suggests the future possibility of using partial GCH1 blockade or BH4 inhibition as a prophylactic to prevent or delay the development of cancer pain.

A KCNJ6 (Kir3.2, GIRK2) gene polymorphism modulates opioid effects on analgesia and addiction but not on pupil size.

Aim KCNJ6 coding for potassium inwardly rectifying channels (Kir3.2, GIRK2) is important for opioid receptor transmission. The KCNJ6 rs2070995 AA genotype has been associated with increased opioid analgesic requirements in Japanese. We analyzed its consequences for other opioid effects. Methods Genotyping was done in 85 methadone-substituted former heroin addicts, 352 opioid-treated chronic pain patients, and in 51 healthy volunteers where miotic effects of levomethadone had been measured. Expression of Kir3.2 in the Edinger–Westphal nucleus of rat brains was analyzed by means of immunohistochemistry. Results Average daily methadone substitution doses during the first therapy year were larger in the AA genotype (n=4, 119.7±49.6 mg/day) than in other rs2070995 genotypes (77.5±26.2 mg/day, P=0.003) whereas AA carriers lacked opioid withdrawal symptoms. A similar tendency toward less opioid effectiveness was observed toward higher opioid dosing demands for analgesia in the AA genotype (n=17, opioid dose 2.03±0.45 log mg oral morphine equivalents per day, controls: 1.81±0.52 log mg oral morphine equivalents/day, P=0.093). In contrast, no pharmacogenetic effects were observed on miotic opioid effects. This could be traced back to the absence of Kir3.2 from the Edinger–Westphal nucleus in rat brains, a key cerebral structure governing pupil constriction. Conclusion The association of the KCNJ6 rs2070995 AA genotype with increased opioid requirements extends from analgesia to opiate substitution therapy. Opioid induced miosis is exempted for molecular histological reasons.

Kinetics of serum brain-derived neurotrophic factor following low-intensity versus high-intensity exercise in men and women.

Physical activity has been shown to enhance circulating brain-derived neurotrophic factor (BDNF) in animals and humans. However, the exact time course and sex-specific modulation of peripheral BDNF in response to exercise are still poorly understood. We examined the kinetics of BDNF serum concentrations in response to perceived high-intensity and low-intensity exercise, and during a subsequent recovery period by taking several blood samples during each phase. Furthermore, we compared the BDNF concentration between young men and women taking oral contraceptives. We found transient BDNF elevations during physical activity only for the high-intensity condition. Here, BDNF reached its maximum serum concentration after 20 min of exercise, and returned to baseline after approximately 10 min of recovery. Although there were no sex differences during baselineor recovery, the increase in the BDNF concentration during the exercise phase was more pronounced in men than in women.

Functional genomics of pain in analgesic drug development and therapy

Advances in genomic research have led to the clarification of the detailed involvement of gene products in biological pathways and these are being increasingly exploited in strategies for drug discovery and repurposing. Concomitant developments in informatics have resulted in the acquisition of complex gene information through the application of computational analysis of molecular interaction networks. This approach enables the acquired knowledge on hundreds of genes to be used to view molecular disease mechanisms from a genetic point of view. By analyzing 410 genes which control the complex process of pain, we show by computational analysis, based on functional annotations to pain-related genes, that 12 clearly circumscribed functional areas are essential for pain perception and thus for analgesic drug development. The genetics perspective revealed that future development strategies should focus on substances modulating intracellular signal transduction, ion transport and anatomical structure development. These processes are involved in the genetic-based absence of pain and therefore, provide promising fields for curative or preventive treatments. In contrast, interactions with G-protein coupled receptor pathways seem merely to provide symptomatic, not preventative relief of pain. In addition, biological functions accessed either by analgesic drugs or microRNAs suggest that synergistic therapies may be a future direction for drug development. With modern computational functional genomics, it is possible to exploit genetic information from increasingly available data sets on complex diseases, such as pain, and offers a new insight into drug development and therapy which is complementary to pathway-centered approaches.