Impact of Long‐Term Alcohol Consumption and Relapse on Genome‐Wide DNA Methylation Changes in Alcohol‐Dependent Subjects: A Longitudinal Study

Background: Genetic factors play an important role in the development and maintenance of alcohol use disorder (AUD). Significant and widespread differences in methylation levels of multiple regions within the genome have been reported between AUD patients and healthy controls in large epigenome-wide association studies (EWASs). Also, within patient populations, methylation changes over time (both during and after withdrawal) have been identified as sensitive indicators for disease activity. The detection of changes in methylation levels is a powerful tool to further explore and understand the biological correlates and underpinnings of AUD. Although there is strong and convincing evidence for differences in methylation of various sites between AUD patients and controls, only few studies assessed changes within patients over longer periods of time while taking into account alcohol consumption, relapse, and abstinence. So far, the longest period assessed as a within-subject design using EWASs was 4 weeks. Methods: Here, we investigated changes in whole-genome methylation levels within a sample of 69 detoxified AUD patients over a period as long as 12 months for the first time, comparing patients that relapsed within the follow-up period to those that remained abstinent. Results: Whole-genome methylation patterns of individual CpG sites over time did not differ between abstinent and relapsing patients. However, there was a negative association between global mean methylation at the 12-month follow-up and alcohol consumption within our sample. Conclusion: Although the present study represents the largest study of methylation levels in a sample of AUD patients with a follow-up period of 1 year and accounting for alcohol consumption and relapse to date, the sample size might still not be large enough to detect genome-wide significant effects. Therefore, large-scale, long-term studies with AUD subjects are needed to determine the utility of DNA methylation for the assessment and monitoring of persons with alcohol use disorders

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 patient

Neuropathological and Reelin Deficiencies in the Hippocampal Formation of Rats Exposed to MAM; Differences and Similarities with Schizophrenia

Adult rats exposed to methylazoxymethanol (MAM) at embryonic day 17 (E17) consistently display behavioral characteristics similar to that observed in patients with schizophrenia and replicate neuropathological findings from the prefrontal cortex of psychotic individuals. However, a systematic neuropathological analysis of the hippocampal formation and the thalamus in these rats is lacking. It is also unclear if reelin, a protein consistently associated with schizophrenia and potentially involved in the mechanism of action of MAM, participates in the neuropathological effects of this compound. Therefore, a thorough assessment including cytoarchitectural and neuromorphometric measurements of eleven brain regions was conducted. Numbers of reelin positive cells and reelin expression and methylation levels were also studied.Compared to untreated rats, MAM-exposed animals showed a reduction in the volume of entorhinal cortex, hippocampus and mediodorsal thalamus associated with decreased neuronal soma. The entorhinal cortex also showed laminar disorganization and neuronal clusters. Reelin methylation in the hippocampus was decreased whereas reelin positive neurons and reelin expression were unchanged.Our results indicate that E17-MAM exposure reproduces findings from the hippocampal formation and the mediodorsal thalamus of patients with schizophrenia while providing little support for reelin's involvement. Moreover, these results strongly suggest MAM-treated animals have a diminished neuropil, which likely arises from abnormal neurite formation; this supports a recently proposed pathophysiological hypothesis for schizophrenia

BDNF Plasma Levels and BDNF Exon IV Promoter Methylation as Predictors for Antidepressant Treatment Response

Major problems of current antidepressant pharmacotherapy are insufficient response rates and difficulties in response prediction. We recently provided preliminary evidence in a small study that patients with major depressive disorder (MDD) with a hypomethylation of the CpG-87 site of the promoter IV region of the brain-derived neurotrophic factor (BDNF) gene are less likely to benefit from antidepressants. Here, we aimed at replicating this finding in a secondary analysis of 561 MDD patients (mean age 40.0 ± 11.9 years, 56% female) included into the Early Medication Change study (EMC). We measured BDNF exon IV promoter and p11 gene methylation at Baseline (BL) as well as BDNF-plasma-levels (pBDNF) at BL and day 14 and related them to treatment outcome. Although we were not able to replicate the predictor function of hypomethylation of the BDNF exon IV promoter, a subgroup of patients with severe depression (Hamilton Depression Rating Scale [HAMD-17] ≥ 25) (n = 199) and hypermethylation at CpG-87 of the BDNF exon IV promoter had significantly higher remission rates than patients without a methylation (p = 0.032). We also found that 421 (75%) of 561 patients showed an early improvement (≥ 20% HAMD-17 reduction after 2 weeks), which was associated with a 4.24-fold increased likelihood to remit at study end compared to the 140 patients without early improvement. However, specificity of response prediction of early improvement was low (34%) and false positive rate high (66%). The combination of early improvement with a pBDNF increase between BL and day 14, however, increased the specificity of response prediction from 34 to 76%, and the combination with methylation of the CpG-87 site of the BDNF exon IV promoter from 34 to 62%. Thus, the combined markers reduced false positives rates from 66 to 24% and 38%, respectively. Methylation at other sites or p11 promoter methylation failed to increase specificity of early improvement prediction. In sum, the results add to previous findings that BDNF, BDNF promoter methylation and the combination of clinical and biological markers may be interesting candidates for therapy response prediction which has to be confirmed in further studies.Clinical Trial Registration:https://clinicaltrials.gov/ct2/show/NCT00974155, identifier: NCT0097415

Preeclampsia-Associated Alteration of DNA Methylation in Fetal Endothelial Progenitor Cells

ObjectiveThe pregnancy complication preeclampsia represents an independent risk factor for cardiovascular disease. Our previous research shows a diminished function of fetal endothelial colony-forming cells (ECFC), a proliferative subgroup of endothelial progenitor cells (EPC) in preeclampsia. The aim of this study was to further investigate whether DNA methylation of fetal EPC is affected in preeclampsia.MethodsThe genomic methylation pattern of fetal ECFC from uncomplicated and preeclamptic pregnancies was compared for 865918 CpG sites, and genes were classified into gene networks. Low and advanced cell culture passages were compared to explore whether expansion of fetal ECFC in cell culture leads to changes in global methylation status and if methylation characteristics in preeclampsia are maintained with increasing passage.ResultsA differential methylation pattern of fetal ECFC from preeclampsia compared to uncomplicated pregnancy was detected for a total of 1266 CpG sites in passage 3, and for 2362 sites in passage 5. Key features of primary networks implicated by methylation differences included cell metabolism, cell cycle and transcription and, more specifically, genes involved in cell-cell interaction and Wnt signaling. We identified an overlap between differentially regulated pathways in preeclampsia and cardiovascular system development and function. Cell culture passages 3 and 5 showed similar gene network profiles, and 1260 out of 1266 preeclampsia-associated methylation changes detected in passage 3 were confirmed in passage 5.ConclusionMethylation modification caused by preeclampsia is stable and detectable even in higher cell culture passages. An epigenetically modified endothelial precursor may influence both normal morphogenesis and postnatal vascular repair capacity. Further studies on epigenetic modifications in complicated pregnancies are needed to facilitate development of EPC based therapies for cardiovascular alterations

Treating schizophrenia with cariprazine : from clinical research to clinical practice. Real world experiences and recommendations from an International Panel

Altres ajuts: Supported by an unrestricted grant from Recordati.Management of schizophrenia is sub-optimal in many patients. Targeting negative symptoms, among the most debilitating aspects of schizophrenia, together with positive symptoms, can result in significant functional benefits and dramatically improve quality of life for patients and their carers. Cariprazine, a partial agonist of the dopamine receptors D2/D3 has demonstrated effectiveness across symptom domains in clinical trials, particularly on negative symptoms. To obtain a broader insight from clinicians with specific experience with cariprazine, on how it affects patient populations outside the clinical trial setting. The panel addressed a series of psychopharmacologic topics not comprehensively addressed by the evidence-based literature, including characteristics of patients treated, dosing and switching strategies, duration of therapy, role of concomitant medications and tolerability as well as recommendations on how to individualize cariprazine treatment for patients with schizophrenia. Patients recommended for cariprazine treatment are those with first episodes of psychosis, predominant negative symptoms (maintenance/acute phase) and significant side effects (metabolic side effects, hyperprolactinemia, sedation) with other antipsychotics. When the long-term treatment of a lifetime illness is adequately weighted, cariprazine becomes one of the first-line medications, not only for patients with predominant negative symptoms but also for those with relatively severe positive symptoms, especially if they are at the first episodes and if a specific medication is added for symptoms such as agitation or insomnia. For instance, patients with agitation may also benefit from the combination of cariprazine and a benzodiazepine or another sedating agent. Cariprazine may be prescribed as add-on to medications such as clozapine, when that medication alone is ineffective for negative symptoms, and sometimes the first may be discontinued or its dose lowered, after a period of stability, leaving the patient on a better tolerated antipsychotic regimen. Based on real-world clinical experience, the panel considered that cariprazine, with its distinct advantages including pharmacokinetics/pharmacodynamics, good efficacy and tolerability, represents a drug of choice in the long-term management of schizophrenia not only for patients with predominant negative symptoms but also for those with positive symptoms

Association of Leptin Gene DNA Methylation With Diagnosis and Treatment Outcome of Anorexia Nervosa

Epigenetic alterations are increasingly implicated in the pathophysiology of anorexia nervosa (AN) but are as yet poorly understood. We investigated possible associations between the leptin gene (LEP) and the leptin receptor gene (LEPR) DNA promoter methylation and (1) a diagnosis of AN and (2) outcome after a 10 months psychotherapeutic outpatient treatment. 129 (LEPR: n = 135) patients with AN were investigated during the large scale psychotherapeutic Anorexia Nervosa Treatment Outpatient Study (ANTOP) trial, compared to 117 (LEPR: n = 119) age and height matched, normal-weight healthy controls. Blood samples were taken at baseline, the end of therapy (40 weeks) and the 12-months follow-up and compared to controls. Methylation was measured in whole blood via bisulfite sequencing. Within the promoter region 32 (LEP) and 39 CpG sites (LEPR) were analyzed. Two key findings were observed. First, LEP and LEPR methylation at baseline were lower in patients compared to controls (LEP: [%] AN: 30.94 ± 13.2 vs. controls: 34.53 ± 14.6); LEPR ([%] AN: 3.73 ± 5.4 vs. controls: 5.22 ± 8.3, mixed linear models: both P < 0.001). Second, lower DNA methylation of the LEP promoter, with a dynamic upregulation during treatment, was associated with a full recovery in AN patients (% change from baseline to follow-up in full recovery patients: +35.13% (SD: 47.56); mixed linear model: P < 0.0001). To test for potential predictive properties of mean LEP DNA methylation a LEP DNA methylation cut-off (31.25% DNA methylation) was calculated, which significantly discriminated full recovery vs. full syndrome AN patients. This cut-off was then tested in a group of previously unclassified patients (missing follow-up data of the Structured Interview for Anorexic and Bulimic disorders; n = 33). Patients below the cut-off (31.25% LEP DNA methylation) showed an increase in BMI over time, while those above the cut-off had a decrease in BMI (ANOVA at the 12-months follow-up: P = 0.0142). To our knowledge, this is the first study investigating epigenetic alterations in AN over time. Our findings indicate that LEP DNA methylation might be involved in the disease course of AN

Mechanismen der Polyhexanid bedingten Vasodilatation während Peritoneallavage : Ergebnisse tierexperimenteller Untersuchungen

Hintergrund: Die Peritonitistherapie beinhaltet häufig die Abdominallavage mit dem lokalen Antiinfektivum Polyhexanid, als 0,05% Lösung, die häufig zu einer Hypotension führt. Methode: Wir untersuchten in gesunden Ratten die Auswirkungen einer Peritoneallavage mit Polyhexanid oder NaCl (je 10ml über 10min; Polyhexanid n=5, NaCl n=5) auf den Blutdruck und registrierten bei 7 weiteren Tieren die Durchmesser submukosaler Blutgefäße einer Ileumschlinge vor und nach lokaler Superfusion mit Polyhexanid mittels intravitaler Fluoreszenzmikroskopie. In isometrischen Präparationen der thorakalen Rattenaorta mit und ohne Endothel, die mit Phenylephrin(PE) oder KCl 20 oder 40 mmol kontrahiert worden waren, wurden die Effekte von Polyhexanid auf den Gefäßtonus bestimmt. Resultate: Es wurde festgestellt, dass Peritoneallavage mit Polyhexanide zu einem Abfall des mittleren arteriellen Drucks führte, während Superfusion mit Polyhexanid eine lokaleVasodilatation der Gefäße der Darmwand bewirkte. In vitro relaxierte Polyhexanide Aortenringe, die mit Phenylephrin kontrahiert worden waren, Präparationen +End reagierten empfindlicher Inkubation mit dem NO-Synthetasehemmer N(G)-nitro-L-arginine methyl ester oder dem Guanylylcyclasehemmer 1H-(1,2,4)Oxodiazolo-(4,3-a)quinoxalin-1-one führte jeweils zu einer partiellen Hemmung der relaxierenden Wirkung. Die relaxierenden Effekte von Polyhexanid konnten in Präparationen ohne Endothel durch keinen der Kaliumkanalblocker Charibdotoxin, Tetraäthylammoniumchlorid, Glibenclamid oder 4-Aminopyrridine verhindert werden. Polyhexanid zeigte keine Wirkung auf Aortenpräparate, die mit 40mmol KCl kontrahiert worden waren. Schlussfolgerung: Es ist zu folgern, dass Polyhexanid NO freisetzen, Kaliumkanäle aktivieren und darüber zu einer Vasodilatation führen kann, die dann eine Hypotension bewirkt.Background and aims: Treatment of peritonitis may include abdominal lavage with a local disinfectant polihexanide, available as 0,04% solution, which is often accompanied by hypotension. Methods: We examined in healthy rats, the effects of peritoneal installation of polihexanide or NaCl 0,9% (10ml each, during 10min, polihexanide n=5, NaCl n=5) on mean arterial pressure and measured, in 7 other animals, using intravital microscopy, diameter of terminal ileum submucosal arterioles and venules before and after local superfusion with polihexanide. Furthermore, in an in vitro isometric preparation of rat thoracic aortal rings, with and without endothelium, we tested the effects of cumulative concentrations of polihexanide on vascular basic tension and on tension elicited by phenylephryne and KCl. Results: It was found that polihexanide peritoneal installation produced decrease of mean arterial pressure while superfusion with polihexanide caused local vasodilatation of intestinal wall blood vessels. In vitro, polihexanide produced endothelium dependent relaxation in the preparations precontracted with phenylephrine which (in aortas +endothelium) could by either N(G)-nitro-L-arginine methyl ester - nitric oxide generation inhibitor, or 1H-(1,2,4)Oxodiazolo-(4,3-a)quinoxalin 1-one - an inhibitor of guanylyl cyclase. The relaxing effect of polihexanide (aortas -endothelium) was not affected by k+-channel blocking agents charibdotoxin tetraethylammoniumchloride, glibenclamide, or 4-aminopyrridine, while polihexanide had no effects on 40mM KCl contractions. Conclusion: This implies that polihexanidemay promote nitric oxide liberation, potassium channel activation and vasodilatation that may result in hypertension

A targeted long-read sequencing approach questions the association of OXTR methylation with high-functioning autism

Abstract Background DNA sequence variation and altered epigenetic regulation of the oxytocin receptor gene (OXTR) have been implicated in autism and autistic-like behaviors. While previous studies have examined subsegments of OXTR, nanopore Cas9-targeted sequencing (nCATS) allows deep characterization of entire genes with simultaneous assessment of epigenetic 5-methylcytosine (5mC) modification and without the need for prior DNA amplification or bisulfite conversion. This pilot study uses an nCATS approach to sequence the entire OXTR gene and its regulatory construct and screen for 5mC modification to compare results between individuals with high-functioning autism (HFA) and neurotypical controls (NC). Methods Using DNA extracted from peripheral blood, OXTR (Hg38, chr3: 8750381–8770434, 20,054 base pairs) was analyzed by nCATS. 5mC modification probabilities were calculated and visualized across the gene and differential methylation analysis was performed. Results Twenty adults with HFA (10 males, 10 females) and 20 age- and sex-matched NC (± 5 years) were included. There were no apparent group differences in the entire OXTR gene sequence, except for the intron variant rs918316, which was clustered in the HFA group. However, differential methylation analysis did not reveal a single significant group-dependent differentially methylated site among the 412 CpG sites captured. Limitations Limitations of this study include the small number of samples due to the pilot nature of the study, which particularly limits the relevance of the sequence variants found. It should also be noted that the use of peripheral blood material limits the ability to draw conclusions about central processes. Conclusions Previous findings of autism-associated OXTR epigenetic alterations were not reproducible with our method. In our opinion, this may lead to a reconsideration of the relevance of altered methylation at individual OXTR CpG positions in autism research. However, given the pilot nature of the study, these results need to be replicated in independent cohorts and with larger sample sizes