Methylation of the Oxytocin, Oxytocin Receptor, and Vasopressin Gene Promoters in Tobacco Use Disorder during Cessation.

INTRODUCTION: Vasopressin (AVP) and oxytocin (OT) exert sex-specific effects on social pair bonding and stress reactions while also influencing craving in substance use disorders. In this regard, intranasal oxytocin (OT) and AVP antagonists present potential treatments for tobacco use disorder (TUD). Since transcription of both hormones is also regulated by gene methylation, we hypothesized sex-specific changes in methylation levels of the AVP, OT, and OT receptor (OXTR) gene during nicotine withdrawal. METHODS: The study population consisted of 49 smokers (29 males, 20 females) and 51 healthy non-smokers (25 males, 26 females). Blood was drawn at day 1, day 7, and day 14 of smoking cessation. Craving was assessed with the questionnaire on smoking urges (QSU). RESULTS: Throughout cessation, mean methylation of the OT promoter gene increased in males and decreased in females. OXTR receptor methylation decreased in females, while in males it was significantly lower at day 7. Regarding the AVP promoter, mean methylation increased in males while there were no changes in females. Using mixed linear modeling, CpG position, time point, sex, and the interaction of time point and sex as well as time point, sex, and QSU had a significant fixed effect on OT and AVP gene methylation. The interaction effect suggests that sex, time point, and QSU are interrelated, meaning that, depending on the sex, methylation could be different at different time points and vice versa. There was no significant effect of QSU on mean OXTR methylation. DISCUSSION: We identified differences at specific CpGs between controls and smokers in OT and AVP and in overall methylation of the AVP gene. Furthermore, we found sex-specific changes in mean methylation levels of the mentioned genes throughout smoking cessation, underlining the relevance of sex in the OT and vasopressin system. This is the first study on epigenetic regulation of the OT promoter in TUD. Our results have implications for research on the utility of the AVP and OT system for treating substance craving. Future studies on both targets need to analyze their effect in the context of sex, social factors, and gene regulation.

Cytosine methylation in GABA B1 receptor identifies alcohol-related changes for men in blood and brain tissues.

AIMS: Alcohol use alters the reward signaling processes contributing to the development of addiction. We studied the effects of alcohol use disorder (AUD) on brain regions and blood of deceased women and men to examine sex-dependent differences in epigenetic changes associated with AUD. We investigated the effects of alcohol use on the gene promoter methylation of GABBR1 coding for GABAB receptor subunit 1 in blood and brain. METHODS: We chose six brain regions associated with addiction and the reward pathway (nucleus arcuatus, nucleus accumbens, the mamillary bodies, amygdala, hippocampus and anterior temporal cortex) and performed epigenetic profiling of the proximal promoter of the GABBR1 gene of post-mortem brain and blood samples of 17 individuals with AUD pathology (4 female, 13 male) and 31 healthy controls (10 female, 21 male). RESULTS: Our results show sex-specific effects of AUD on GABBR1 promoter methylation. Especially, CpG -4 showed significant tissue-independent changes and significantly decreased methylation levels for the AUD group in the amygdala and the mammillary bodies of men. We saw prominent and consistent change in CpG-4 across all investigated tissues. For women, no significant loci were observed. CONCLUSION: We found sex-dependent differences in GABBR1 promoter methylation in relation to AUD. CpG-4 hypomethylation in male individuals with AUD is consistent for most brain regions. Blood shows similar results without reaching significance, potentially serving as a peripheral marker for addiction-associated neuronal adaptations. Further research is needed to discover more contributing factors in the pathological alterations of alcohol addiction to offer sex-specific biomarkers and treatment.

Comparing DRD2 Promoter Methylation Between Blood and Brain in Alcohol Dependence.

AIMS: The dopamine receptor D2 (DRD2) is substantially involved in several forms of addiction. In addition to genetic polymorphisms, epigenetic mechanisms have emerged as an important means of regulation. Previously, DRD2 hypo- and hyper-methylation have been observed in alcohol use disorder (AUD). Blood samples are commonly used as a surrogate marker of epigenetic alterations in epigenetic research, but few specific comparisons between blood and brain tissue samples in AUD exist. METHODS: We used post-mortem brain tissue samples of 17 deceased patients with AUD and 31 deceased controls to investigate the relationship between blood and brain methylation of the DRD2 promoter. RESULTS: When investigating individual cytosine methylation sites (CpG), several significant differences were found in the nucleus accumbens and hippocampus in the study population. Investigating binding sites with significant differences in methylation levels revealed hypomethylated CpGs targeting mainly activating transcription factors. CONCLUSION: These findings support an altered transcription of the DRD2 gene in AUD specimens with a consecutively changed reward response in the brain. While methylation between specific brain regions and blood is comparable, our study further suggests that blood methylation cannot provide meaningful perspectives on DRD2 promoter methylation in the brain.

Chronic Voluntary Alcohol Consumption Alters Promoter Methylation and Expression of Fgf-2 and Fgfr1.

Alcohol abuse accounts for 3.3 million deaths annually, rendering it a global health issue. Recently, fibroblast growth factor 2 (FGF-2) and its target, fibroblast growth factor receptor 1 (FGFR1), were discovered to positively regulate alcohol-drinking behaviors in mice. We tested whether alcohol intake and withdrawal alter DNA methylation of Fgf-2 and Fgfr1 and if there is a correlation regarding mRNA expression of these genes. Blood and brain tissues of mice receiving alcohol intermittently over a six-week period were analyzed using direct bisulfite sequencing and qRT-PCR analysis. Assessment of Fgf-2 and Fgfr1 promoter methylation revealed changes in the methylation of cytosines in the alcohol group compared with the control group. Moreover, we showed that the altered cytosines coincided with binding motives of several transcription factors. We also found that Fgf-2 and Fgfr1 gene expression was significantly decreased in alcohol-receiving mice compared with control littermates, and that this effect was specifically detected in the dorsomedial striatum, a brain region involved in the circuitry of the reward system. Overall, our data showed alcohol-induced alterations in both mRNA expression and methylation pattern of Fgf-2 and Fgfr1. Furthermore, these alterations showed a reward system regional specificity, therefore, resembling potential targets for future pharmacological interventions.

Sudden infant death syndrome revisited: serotonin transporter gene, polymorphisms and promoter methylation.

BACKGROUND: Based on findings in the brain stems of SIDS victims, the serotonin transporter (5-HTT) gene has been discussed to be associated with SIDS. METHODS: In the largest study to date, we investigated the promoter length (5-HTTLPR) and intron 2 VNTR polymorphisms in 274 cases and 264 controls and the Ile425Val polymorphism in 65 cases and 64 controls. Moreover, the methylation of the internal promoter region was investigated in 35 cases and 14 controls. RESULTS: For 5-HTTLPR, we observed a trend towards an association of allele L (58.8% vs. 53.4%) with SIDS and significant results were observed after stratifying for age, season at death, and prone position. Nevertheless, when pooling all published data, a significant association of allele L with SIDS is confirmed (p: 0.001). For the intron 2 VNTR polymorphism, no significant differences were observed. After pooling, a significant accumulation of the rare allele 9 was observed in SIDS (2.1% vs. 0.6%; p: 0.018). For the Ile425Val polymorphism, no differences were observed. CONCLUSION: We conclude that genetic variation at this gene might be of some importance in SIDS. Epigenetic analysis of the internal promoter, however, revealed no influence on the relative risk to succumb to SIDS. IMPACT: This is the largest study published up to now on 5-HTT gene polymorphisms and SIDS. Polymorphisms in the 5-HTT gene appear to contribute (although to a small degree) to the risk to die from SIDS. There is no evidence that a methylation of the promoter region is of impact for the etiology of SIDS.

Epigenetic Regulation of Neural Transmission after Cerebellar Fastigial Nucleus Lesions in Juvenile Rats.

Structural and functional abnormalities in the cerebellar midline region, including the fastigial nucleus, have been reported in neuropsychiatric disorders, also comprising the cerebellar cognitive affecting syndrome. In rats, early fastigial lesions reduce social interaction during development and lead to cognitive and emotional deficits in adults, accompanied by compromised neuronal network activity. Since epigenetic mechanisms are implicated in the etiology of neuropsychiatric disorders, we investigated whether fastigial nucleus lesions in juvenile rats would impact epigenetic regulation of neural transmission. The fastigial nucleus was lesioned bilaterally in 23-day-old male rats. Sham-lesion and naïve rats served as controls. DNA methylation was investigated for target genes of the GABAergic, dopaminergic, glutamatergic and oxytocinergic systems in brain regions with anatomic connections to the fastigial nucleus, i.e., medial prefrontal cortex, nucleus accumbens, striatum, thalamus, and sensorimotor cortex. Protein expression was examined for the respective target genes in case of altered DNA methylation between lesion and control groups. Lesioning of the fastigial nucleus led to significant differences in the epigenetic regulation of glutamate decarboxylase 1 and the oxytocin receptor in the nucleus accumbens and the prefrontal cortex. No differences were found for the other target genes and brain regions. Our findings indicate that epigenetic dysregulation after lesioning of the fastigial nucleus may influence long-term recovery and the emergence of behavioral changes. Together with previous behavioral and electrophysiological investigations of this rat model, these observations can play a role in the cerebellar cognitive affective syndrome and other neuropsychiatric disorders.

Trauma Severity in Early Childhood Correlates with Stress and Satiety Hormone Levels in a Pilot Cohort Receiving Diamorphine Maintenance Treatment.

AIMS: Childhood trauma is of importance for the manifestation of substance-related disorders and maintenance of hypothalamic-pituitary-adrenal (HPA)-axis disorders. Since stress plays a crucial role in opioid compliance and craving, we investigated the immediate effects of diacetylmorphine application on the HPA axis. In particular, adrenocorticotropic hormone (ACTH) and cortisol secretion, as well as satiety regulating proopiomelanocortin peptides α-melanocyte-stimulating hormone (MSH) and ß-endorphin (END) in a cohort of opioid-dependent patients in diamorphine maintenance treatment concerning the clinical severity of their childhood trauma. METHODS: We compared the serum levels of ACTH, cortisol, MSH, and END in 15 opioid-dependent patients. All participants received treatment with diamorphine and were observed at 5 timepoints before and after injection. We split the cohort into 2 subgroups concerning childhood trauma measured by the Childhood Trauma Questionnaire. RESULTS: Splitting in 2 subgroups for mild (5) and severe trauma (10), we found that while both groups show a significant reduction of ACTH and cortisol levels over time, slopes display different progressions over time for cortisol (F[1.6] = 9.38, p = 0.02), while remaining identical for ACTH (F[1.6] = 1.69, p = 0.24). Also, levels of both MSH and END were significantly lower in severely traumatized patients. CONCLUSIONS: For the first time, we present a detailed representation of stress- and addiction-related proteins for the first 5 h after diamorphine application, demonstrating the interrelationship between stress hormones and childhood trauma as well as its potential effects on the progression of addictions such as opioid dependence.

Alcohol Withdrawal and Proopiomelanocortin Neuropeptides in an Animal Model of Alcohol Dependence.

BACKGROUND: Alcohol is one of the leading threats to health worldwide. Craving for alcohol makes abstinence a difficult challenge by maintaining alcohol dependence. Many studies suppose the hypothalamic-pituitary-adrenal axis, especially the proopiomelanocortin (POMC)-derived neuropeptides, to mediate craving during withdrawal in alcohol dependence. Evidence is available that the two POMC proteins, α-melanocyte-stimulating hormone (α-MSH) and ß-endorphin (ß-END) are altered by alcohol consumption and influence alcohol consumption, respectively. OBJECTIVES: We investigated the dynamics of α-MSH and ß-END during alcohol withdrawal and the influence of intraperitoneal administration of either α-MSH or ß-END in an established rodent model (Wistar rats) for alcohol dependence. RESULTS: After long-term alcohol self-administration over 12 months and repeated deprivation periods for 3 days, we found a significant decrease in α-MSH levels during withdrawal in rodents (p = 0.006) compared to controls, while ß-END levels remained unchanged. Treatment with intraperitoneally administered α-MSH and ß-END did not affect alcohol drinking behavior after deprivation. CONCLUSION: We demonstrate the effects of alcohol deprivation on α-MSH in alcohol-dependent rodents, which appear to mimic α-MSH alteration found after fasting periods during appetite regulation. Therefore, low α-MSH levels are a possible indicator for craving in alcohol-dependent individuals and hence would be a potential target for anti-craving treatment.

DNA Methylation of the Leptin Gene Promoter is Altered by Chronic Alcohol Exposure in an Animal Model for Alcohol Dependence.

Appetite-regulating peptides, such as leptin, are linked to craving and have been in the focus of alcohol dependence research for years. The objective of our study was to investigate the dynamics of leptin gene promoter methylation during alcohol withdrawal and specific treatment in a rodent (rat) model for alcohol dependence. DNA methylation was measured using direct bisulfite sequencing at 0 h, 24 h, and 6 days of alcohol withdrawal as well as after treatment with alpha-melanocyte-stimulating hormone (alpha-MSH), Beta-Endorphin, or saline. We found significantly lower methylation levels in alcohol-consuming animals compared to alcohol-naïve animals. During 6 days of alcohol deprivation, this difference in methylation vanished. Leptin methylation of the alpha-MSH-treated group and 6 days alcohol-deprived animals was significantly higher than that in saline-treated animals, possibly indicating compensatory effects of the treatment. Our results further expand on previous findings from human studies that explain leptin's role in bridging the gap between alcohol consumption and appetite regulation.

Desacylghrelin but not acylghrelin is reduced during smoking cessation.

This study focused on the acetylation status of ghrelin examining acyl- and desacylghrelin and its effect on craving during 14 days of smoking abstinence. This is the first study demonstrating changes in desacylghrelin plasma levels in smokers compared to non-smokers while there was no difference of acylated ghrelin. Future studies should specifically refer to plasma ghrelin as either desacyl- or acylghrelin since both have different effects on tobacco-seeking behavior and the underlying physiology.

Epigenetic Effects of Intravenous Diacetylmorphine on the Methylation of POMC and NR3C1.

The administration of diacetylmorphine (DAM) reduces the activation of the hypothalamic-pituitary-adrenal (HPA) axis in opioid-maintained patients. However, the epigenetic effects of DAM on addiction-related genes have not been investigated yet. In a randomized controlled study, we examined the immediate effects of intravenous DAM versus placebo on the promoter methylation of the POMC (pro- opiomelanocortin) and NR3C1 (glucocorticoid receptor 1) genes. Twenty-eight heroin-dependent patients on DAM-assisted treatment received either DAM or saline in a randomized crossover design and 17 healthy participants received saline only. EDTA blood samples were taken 25 min before and 10 min after the injection of DAM or saline. We found reciprocal regulation effects for DAM versus saline application regarding the methylation of POMC; while DAM injection significantly increased methylation, saline injection led to a significant decrease in methylation for patients as well as controls. NR3C1 data did not show significant changes in methylation. Injection of DAM blunted stress hormone levels and the POMC promoter methylation of heroin-dependent patients. These findings provide first preliminary insights into the epigenetic mechanisms underlying the emotional regulation effects of DAM-assisted treatment in severe heroin-dependent patients.

Promoter Polymorphism rs886205 Genotype Interacts With DNA Methylation of the ALDH2 Regulatory Region in Alcohol Dependence.

AIMS: Aldehyde dehydrogenase 2 (ALDH2) protects cells from ethanol toxicity by metabolizing acetaldehyde. We studied the single nucleotide polymorphism (SNP) rs886205s located between a negative and a positive regulating promoter element in the ALDH2 gene. The negative regulatory region was already associated with differential DNA methylation in the two allele variations of rs886205 SNP. Another CpG island, in the positive regulatory region of the ALDH2 promoter, extends through the SNP rs886205 and a nuclear receptor response element. METHODS: We assessed rs886305 genotype and DNA methylation using bisulfite sequencing in a cohort of 83 male alcohol-dependent patients undergoing detoxification treatment (Days 1, 7 and 14) and in 33 male age-matched controls. Luciferase reporter assays were performed to address the functional significance of genotype and methylation. RESULTS: We observed a higher methylation in alcohol-dependent patients compared to controls. Patients with AA (n = 52) or GG/GA (n = 31) genotype differed significantly in baseline methylation levels as well as in methylation kinetics during withdrawal. AA carriers display an increase in methylation from low baseline levels while GG/GA showed the inverse pattern. The reporter gene assays corroborate these data by showing a significant effect of genotype on ALDH2 expression as well as an interaction between genotype and methylation. CONCLUSION: Our results describe a new regulatory role of rs886205 in the methylation of ALDH2 promoter region and provide additional insight into the complex regulation of ALDH2 under the condition of alcohol dependence. SHORT SUMMARY: Genetic variations have been described to influence DNA promoter methylation of various genes. We investigated the association between the polymorphism rs886205, located on ALDH2 promoter and methylation kinetics of the neighboring CpG island in alcohol-dependent patients. Luciferase reporter assays showed functional significance of genotype, methylation and a genotype-epigenotype interaction in vitro.

Smoking and Promoter-Specific Deoxyribonucleic Acid Methylation of the Atrial Natriuretic Peptide Gene: Methylation of Smokers and Non-Smokers Differs Significantly during Withdrawal.

BACKGROUND: Atrial natriuretic peptide (ANP) is well known in psychiatric disorders to modulate hypothalamic-pituitary-adrenal axis activity. Disturbances of ANP have been described in early abstinent alcohol-dependent patients. This is the first longitudinal investigation on cytosine-phosphatidyl-guanine (CpG)-island promoter methylation of the ANP gene in the blood of tobacco-dependent patients. METHODS: In a longitudinal approach, we investigated whether changes in ANP serum levels correlated to CpG methylation of the respective gene promoters on days 1, 7, and 14 of tobacco withdrawal. RESULTS AND CONCLUSION: Compared to non-smokers, promoter-related deoxyribonucleic acid methylation of the ANP promoter was significantly elevated on days 7 and 14 of withdrawal in tobacco-dependent patients. Baseline methylation status of the ANP promoter was not significantly different from controls, arguing for an impaired regulation during withdrawal.

The Epigenetic Regulation of GATA4-Dependent Brain Natriuretic Peptide Expression during Alcohol Withdrawal.

OBJECTIVE: Natriuretic peptides participate in the collection of metabolic effects during alcohol withdrawal. Having witnessed modulation of other natriuretic peptides in alcohol-dependent patients during alcohol withdrawal, we were interested in the relation of brain natriuretic peptide (BNP) methylation with protein expression and craving in this longitudinal study. METHODS: Ninety-nine male patients were compared to 101 healthy controls concerning epigenetic regulation and protein expression during detoxification treatment. RESULTS: With BNP expression being GATA4 dependent, we observed a correlation of GATA4 binding site methylation and protein expression. BNP serum levels and Obsessive Compulsive Drinking Scale scores are significantly decreased during withdrawal. Focusing on the two CpGs that are between GATA transcription factor binding sites, statistical analysis revealed a reversely proportional methylation pattern, significantly increasing with ongoing detoxification and thereby supporting the observed serum level changes. CONCLUSION: Without the functional knowledge about regulation of BNP expression via the GATA transcription factor, it would have been easy to take the mean results of the global CpG data and propose a direct relationship between methylation and expression. Thus, these findings are a voice for functionally and mechanistically approved results. There was no causal relationship between protein expression levels and epigenetic changes. Further research is needed which includes protein expression and other approaches.

Impaired Regulation of ALDH2 Protein Expression Revealing a Yet Unknown Epigenetic Impact of rs886205 on Specific Methylation of a Negative Regulatory Promoter Region in Alcohol-Dependent Patients.

Acetaldehyde, the carcinogenic metabolite of ethanol known to provoke aversive symptoms of alcohol consumption, is predominantly eliminated by aldehyde dehydrogenase 2 (ALDH2). Reduced ALDH2 activity correlates with low alcohol tolerance and low risk for alcohol dependence. The ALDH2 promoter polymorphism rs886205 (A>G) is associated with decreased promoter activity, but a molecular mechanism and allele-dependent ALDH2 protein expression has not been described yet. On the basis of allele-dependent epigenetic effects, we analyzed the rs886205 genotype, methylation rates of cytosine-phosphatidyl-guanine (CpG)-sites within a regulatory promoter region and ALDH2 protein levels in 82 alcohol-dependent patients during a 2-week withdrawal and compared them to 34 matched controls. Patients without the G-allele of rs886205 showed higher methylation of the promoter region than controls and readily adapted epigenetically as well as on protein level during withdrawal, while patients with the G-allele displayed retarded methylation readjustment and no change in ALDH2 protein levels. Our data provide novel insights into an unknown genetic-epigenetic interaction, revealing impaired ALDH2 protein expression in patients with the G-allele of rs886205. Additionally, we checked for an association between rs886205 and protection against alcohol dependence and found a trend association between the G-allele and protection against alcohol dependence that needs replication in a larger Caucasian cohort.

Alcohol-induced changes in methylation status of individual CpG sites, and serum levels of vasopressin and atrial natriuretic peptide in alcohol-dependent patients during detoxification treatment.

Disturbances of volume-regulating peptides like vasopressin (AVP) and atrial natriuretic peptide (ANP) have been described in early abstinent alcohol-dependent patients. In a longitudinal approach, we investigated whether changes in AVP and ANP serum levels correlated to cytosine-phosphatidyl-guanine (CpG) methylation of the respective gene promoters on days 1, 7 and 14 of alcohol withdrawal. We analyzed the blood samples of 99 patients suffering from alcohol dependence alongside age- and BMI-matched controls. Concerning AVP promoter methylation, we observed an interaction between time of measurement and CpG loci with CpG 2 showing a significant increase in methylation from day 1 to 14. Serum levels of AVP were significantly decreased in the patient group. Compared to healthy controls, promoter-related DNA methylation of the ANP promoter was significantly reduced on days 7 and 14. Moreover, we detected a significant interaction between CpG position and group. In both cases the difference was mainly observed at CpG 1. The present study shows significant changes in the methylation status of individual CpG sites of AVP and ANP. Observing respective alterations of AVP serum protein levels in alcohol-dependent patients during detoxification treatment, we consider methylation as a possible mode of regulation for these proteins during alcohol detoxification.

Therapeutic Use of Cannabinoids in Critically Ill Patients: A Survey of Intensive Care Physicians in Germany.

Background: In the course of the legalization of cannabis for therapeutic purposes in Germany, there has been growing interest in the medical use of cannabinoids. To date, the therapeutic potential of cannabinoids for the treatment of critically ill patients has not been explored. Objectives: This study aims to understand better whether and how frequently cannabinoids have been administered to critically ill patients in recent years. Study Design: Initially, a survey was conducted among physicians working in intensive care units (ICUs) at the Hannover Medical School. Subsequently, 653 physicians working in ICUs throughout Germany were surveyed. The frequency and regimen of cannabinoid therapy initiated by the participating physicians in the last 2 years at the time of the survey were characterized. Results: Eight out of 9 physicians at Hannover Medical School and 59 out of 653 physicians in ICUs in Germany participated. At Hannover Medical School, 6 out of 8 physicians and in ICUs in Germany, 16 out of 59 physicians had used cannabinoids in some patients (mainly 9-10) during the 2-year period studied, with dronabinol in doses between 1 and 20 mg being their cannabinoid of choice. Metabolic and psychological distress and medication savings, followed by pain and nausea/vomiting, were the most frequently cited indications for cannabinoid therapy. No relevant safety issues arrived. Lack of personal experience, limited evidence, and gaps in knowledge were the most commonly cited reservations about cannabinoid use. Conclusions: During a 2-year period, dronabinol is used in a few critically ill patients in ICUs. The main indications are to reduce metabolic and psychological distress and to save medication. The majority of participating physicians indicated that the use of cannabinoids in the context of critical care medicine needs further exploration.

Stable-isotope dilution GC-MS method for ethanol in vapour ethanol and microdialysis systems based on carbonate-catalyzed extractive pentafluorobenzoylation.

Common ethanol detection methods are not applicable to cell culture media and microdialysates due to interference with medium constituents including amino acids and pH indicators. We present a novel GC-MS method for the accurate and precise analysis of ethanol in cell cultures and microdialysates. The method is based on the carbonate-catalyzed extractive pentafluorobenzoylation of ethanol and deuterium-labelled ethanol serving as the internal standard and on their GC-MS analysis in the electron-capture negative-ion chemical ionization mode. The method was used to optimize experimental conditions in a custom-made ethanol vapour system utilized for studies examining ethanol influences on neuronal cell lines and in microdialysis.

Leptin promoter methylation in female patients with painful multisomatoform disorder and chronic widespread pain.

BACKGROUND: Different functional somatic syndromes (FSS), fibromyalgia (FMS) and other unexplained painful conditions share many common clinical traits and are characterized by troubling and functionally disabling somatic symptoms. Chronic pain is most frequently reported and at the center of patients' level of disease burden. The construct of multisomatoform disorder (MSD) allows to subsume severely impaired patients suffering from FSS, FMS and other unexplained painful conditions to be examined for common underlying processes. Altered leptin levels and a pathological response of the HPA-axis as a result of chronic stress and childhood trauma have been suggested as one of the driving factors of disease development and severity. Previous studies have demonstrated that methylation of the leptin promoter can play a regulatory role in addiction. In this study, we hypothesized that methylation of the leptin promoter is influenced by the degree of childhood traumatization and differs between patients with MSD and controls. A cohort of 151 patients with MSD and 149 matched healthy volunteers were evaluated using clinical and psychometric assessment while methylation level analysis of the leptin promoter was performed using DNA isolated from whole blood. RESULTS: In female controls, we found CpG C-167 to be negatively correlated with leptin levels, whereas in female patients CpG C-289, C-255, C-193, C-167 and methylation cluster (C-291 to C-167) at putative bindings sites for transcription factors Sp1 and c/EBPalpha were negatively correlated with leptin levels. Methylation levels were significantly lower in female patients CpG C-289 compared with controls. When looking at female patients with chronic widespread pain methylation levels were significantly lower at CpG C-289, C-255 and methylation cluster (C-291 to C-167). CONCLUSION: Our findings support the hypothesis that epigenetic regulation of leptin plays a role in the regulation of leptin levels in patients with MSD. This effect is more pronounced in patients with chronic widespread pain.

Dysregulated Methylation Patterns in Exon IV of the Brain-Derived Neurotrophic Factor (BDNF) Gene in Nicotine Dependence and Changes in BDNF Plasma Levels During Smoking Cessation.

Introduction: Several studies reported dysregulated protein levels of brain-derived neurotrophic factor (BDNF) in smokers and during cessation. However, the epigenetic regulation of the BDNF gene has not yet been investigated. We measured the plasma levels of BDNF and the epigenetic regulation of exon IV of the BDNF gene in smokers compared to healthy controls over a cessation period of 14 days. Method: We measured BDNF plasma levels and BDNF promoter methylation in 49 smokers and 51 non-smokers at baseline, day 7, and day 14 of smoking cessation. Mean methylation levels of 11 Cytosine Guanosine dinucleotides of exon IV of the BDNF gene were determined via bisulfite sequencing. Results: BDNF plasma and methylation levels were significantly lower in healthy controls when compared with smokers across all time points. BDNF levels for smokers decreased significantly during the cessation period. Comparing the sexes, female smokers showed significantly lower plasma BDNF levels than healthy controls at baseline and over 14 days of cessation. Male and female smokers showed significantly higher mean methylation rates than non-smokers at baseline. In male smokers, mean methylation levels decreased significantly during the cessation period. Conclusion: Our findings replicate the findings of previous studies that BDNF plasma levels are altered in smokers. Furthermore, BDNF expression and gene methylation are altered during the first 14 days of cessation. Our novel findings of dysregulated methylation patterns in exon IV of the BDNF gene further support the thesis that BDNF plays a role in nicotine dependence.