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.

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.

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.

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.

Transporter gene expression in human head and neck squamous cell carcinoma and associated epigenetic regulatory mechanisms.

Expression levels of membrane transporters may affect the disposition, and thereby treatment efficacy, of anticancer drugs in human head and neck squamous cell carcinoma (HNSCC). Herein, we analyzed the gene expression profile of membrane transporters in HNSCC. In addition, we evaluated the mechanisms of transporter regulation in HNSCC and focused on the role of the nuclear pregnane X receptor (or NR1I2) and epigenetic mechanisms. Real-time RT-PCR revealed a significantly increased mRNA expression of membrane transporters SLCO1A2 and SLCO1B3 and a significantly decreased expression of transporters SLCO2B1, SLCO2A1, and ABCC3 in human HNSCC tumors compared with adjacent normal mucosa. An association between SLCO2B1 mRNA levels in tumors and 5-year survival of patients with HNSCC was observed (χ2 = 6.59, P = 0.010). Bisulfite sequencing revealed that promoter CpG islands of ABCC3 and SLCO2A1 were not hypermethylated, indicating that these genes were not epigenetically silenced in HNSCC tumors. In HNSCC-derived cell lines, transcript expression of transporters (e.g., ABCC3 or SLCO2A1; P < 0.001 for both) and NR1I2 (P < 0.001) was markedly induced by the DNA methyltransferase inhibitor, decitabine. Cotreatment with the prototypical pregnane X receptor activator, rifampicin, significantly reversed decitabine-induced ABCC3 and SLCO2A1 expression. In summary, the expression of drug transporters (i) is markedly changed in HNSCC tumor tissues compared with normal mucosa, (ii) might be predictive of the outcome of patients with HNSCC, and (iii) is affected by novel epigenetic therapies and is further modulated by nuclear receptor-mediated mechanisms.

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.

Epigenetic down regulation of nerve growth factor during alcohol withdrawal.

We investigated the Cytosin-phosphatidyl-Guanin (CpG) island promoter methylation (mean and methylation of individual CpG-sites) of the nerve growth factor (NGF) gene in the blood of alcohol-dependent patients (57 male patients) during withdrawal (days 1, 7 and 14). Methylation and NGF serum levels did not change significantly from days 1-7. From days 7-14, mean methylation increased (F = 30.55, P < 0.001), whereas the NGF serum levels decreased significantly (days 7-14: F = 17.95, P < 0.001). The NGF serum levels were significantly associated with the mean methylation of the investigated CpG-sites (F = 1.55, P < 0.001). These results imply an epigenetic regulation of the NGF gene during alcohol withdrawal.

Alterations of homocysteine serum levels during alcohol withdrawal are influenced by folate and riboflavin: results from the German Investigation on Neurobiology in Alcoholism (GINA).

AIMS: Various studies have shown that plasma homocysteine (HCY) serum levels are elevated in actively drinking alcohol-dependent patients a during alcohol withdrawal, while rapidly declining during abstinence. Hyperhomocysteinemia has been associated not only with blood alcohol concentration (BAC), but also with deficiency of different B-vitamins, particularly folate, pyridoxine and cobalamin. METHODS: Our study included 168 inpatients (110 men, 58 women) after admission for detoxification treatment. BAC, folate, cobalamin, pyridoxine, thiamine and riboflavin were obtained on admission (Day 1). HCY was assessed on Days 1, 7 and 11. RESULTS: HCY levels significantly declined during withdrawal. General linear models and linear regression analysis showed an influence of BAC, folate and riboflavin on the HCY levels on admission as well as on HCY changes occurring during alcohol withdrawal. No significant influence was found for thiamine, cobalamin and pyridoxine. CONCLUSIONS: These findings show that not only BAC and plasma folate levels, but also plasma levels of riboflavin influence HCY plasma levels in alcohol-dependent patients.

Smoking, but not malnutrition, influences promoter-specific DNA methylation of the proopiomelanocortin gene in patients with and without anorexia nervosa.

OBJECTIVE: Our pilot study evaluates the impact of environmental factors, such as nutrition and smoking status, on epigenetic patterns in a disease-associated gene. METHOD: We measured the effects of malnutrition and cigarette smoking on proopiomelanocortin (POMC) promoter-specific DNA methylation in female patients with and without anorexia nervosa (AN). POMC and its derived peptides (alpha melanocyte stimulating hormone and adrenocorticotropic hormone) are implicated in stress and feeding response. Promoter-specific DNA methylation of the POMC gene was determined in peripheral blood mononuclear cells of 54 healthy female control subjects, 40 underweight patients with AN, and 21 weight-restored patients with AN using bisulfite sequencing. Malnutrition was characterized by plasma leptin. RESULTS: POMC promoter-specific DNA methylation was not affected by diagnosis or nutritional status but significantly negatively associated with cigarette smoking. CONCLUSIONS: Although malnutrition may be expected to reduce DNA methylation through its effects on one-carbon metabolism, our negative results are in line with several in vitro and clinical studies that did not show a direct relation between gene-specific DNA methylation and folate levels. In contrast, smoking has been repeatedly reported to alter DNA methylation of specific genes and should be controlled for in future epigenetic studies.

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.

DNA methylation of the POMC gene promoter is associated with craving in alcohol dependence.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction has been implicated in the pathogenesis of addictive behaviour and especially in alcohol craving. The pro-opiomelanocortin gene (POMC), encoding a 241 amino acids stretching polypeptide hormone precursor, plays an important role in the regulation of the HPA, and is prone to epigenetic regulation due to promoter-related DNA methylation. Aim of the present study therefore was to investigate possible differences in promoter-related DNA methylation in patients suffering from alcohol dependence compared to healthy controls. We analysed the DNA methylation of the 5' promoter of the POMC gene that is embedded in a CpG island using bisulfite sequencing in 145 alcohol-dependent patients and 37 healthy controls taken from the Franconian Alcoholism Research Studies. We found only marginal, hence significant differences at single CpG sites between patients and controls. We identified a cluster of CpGs showing a significant association with alcohol craving in the patients group. These results implicate that epigenetic changes possibly due to alcohol intake may contribute to craving via promoting HPA-axis dysfunction. Further studies should more closely investigate the impact of these changes on the several derivatives of the POMC gene.

Serum levels of vascular endothelial growth factor A increase during alcohol withdrawal.

Vascular endothelial growth factor A (VEGF-A) is a key regulator of angiogenesis. This study investigated VEGF-A serum levels during alcohol withdrawal (days 1, 7 and 14, 76 male patients, 38 healthy controls). Patients showed significantly higher VEGF-A serum levels (t = 2.620, P = 0.010), which increased significantly during withdrawal (F = 4.484, P = 0.014, mean difference = -36.835, P = 0.037). The increase of VEGF-A serum levels was significantly associated with initial breath alcohol concentration and the sumscore of the severity scale of alcohol dependence (SESA questionnaire, F = 5.252, P = 0.008). Increase of VEGF-A serum levels is closely associated to alcohol intoxication and severity of alcohol dependence.

Epigenetic regulation and gene expression of vasopressin and atrial natriuretic peptide in alcohol withdrawal.

Disturbances of volume regulating peptides like vasopressin and atrial natriuretic peptide (ANP) have been described in early abstinent patients. Aim of the present study was to evaluate possible alterations of the promoter-related DNA methylation of the ANP and vasopressin precursor genes and the related mRNA-expression of these genes in early alcohol withdrawal. We analyzed blood samples of 57 healthy controls and of 111 patients suffering from alcohol dependence that were admitted for detoxification treatment. Promoter-related DNA methylation and mRNA-expression of vasopressin and ANP genes were assessed using real-time PCR. Vasopressin mRNA-expression was not statistically different between patients and controls. However, we found a significantly elevated promoter-related DNA methylation of the vasopressin gene in patients with alcohol dependence (Mann-Whitney U-test: Z=-2.178, p=0.029). ANP mRNA-expression was significantly elevated in alcoholic patients (Z=-6.240, p<0.001) while promoter-related DNA methylation of ANP was significantly decreased (Z=-2.282, p=0.023). Furthermore, promoter-related DNA methylation of ANP was significantly correlated to the extent of craving measured with the OCDS (r=-0.197, p=0.040). The findings of the present study show significant alterations of the mRNA-expression and promoter-related DNA methylation of vasopressin and especially ANP precursor genes in patients with alcohol dependence. Further studies focusing on longitudinal changes of epigenetic regulation and gene expression of both peptides are needed to clarify the pathophysiological role of these findings.