Targeting Persistent Changes in Neuroimmune and Epigenetic Signaling in Adolescent Drinking to Treat Alcohol Use Disorder in Adulthood.

Studies universally find early age of drinking onset is linked to lifelong risks of alcohol problems and alcohol use disorder (AUD). Assessment of the lasting effect of drinking during adolescent development in humans is confounded by the diversity of environmental and genetic factors that affect adolescent development, including emerging personality disorders and progressive increases in drinking trajectories into adulthood. Preclinical studies using an adolescent intermittent ethanol (AIE) exposure rat model of underage binge drinking avoid the human confounds and support lifelong changes that increase risks. AIE increases adult alcohol drinking, risky decision-making, reward-seeking, and anxiety as well as reductions in executive function that all increase risks for the development of an AUD. AIE causes persistent increases in brain neuroimmune signaling high-mobility group box 1 (HMGB1), Toll-like receptor, receptor for advanced glycation end products, and innate immune genes that are also found to be increased in human AUD brain. HMGB1 is released from cells by ethanol, both free and within extracellular vesicles, that act on neurons and glia, shifting transcription and cellular phenotype. AIE-induced decreases in adult hippocampal neurogenesis and loss of basal forebrain cholinergic neurons are reviewed as examples of persistent AIE-induced pathology. Both are prevented and reversed by anti-inflammatory and epigenetic drugs. Findings suggest AIE-increased HMGB1 signaling induces the RE-1 silencing transcript blunting cholinergic gene expression, shifting neuronal phenotype. Inhibition of HMGB1 neuroimmune signaling, histone methylation enzymes, and galantamine, the cholinesterase inhibitor, both prevent and reverse AIE pathology. These findings provide new targets that may reverse AUD neuropathology as well as other brain diseases linked to neuroimmune signaling. SIGNIFICANCE STATEMENT: Adolescent underage binge drinking studies find that earlier adolescent drinking is associated with lifelong alcohol problems including high levels of lifetime alcohol use disorder (AUD). Preclinical studies find the underage binge drinking adolescent intermittent ethanol (AIE) model causes lasting changes in adults that increase risks of developing adult alcohol problems. Loss of hippocampal neurogenesis and loss of basal forebrain cholinergic neurons provide examples of how AIE-induced epigenetic and neuroimmune signaling provide novel therapeutic targets for adult AUD.

Genetic factors associated with suicidal behaviors and alcohol use disorders in an American Indian population.

American Indians (AI) demonstrate the highest rates of both suicidal behaviors (SB) and alcohol use disorders (AUD) among all ethnic groups in the US. Rates of suicide and AUD vary substantially between tribal groups and across different geographical regions, underscoring a need to delineate more specific risk and resilience factors. Using data from over 740 AI living within eight contiguous reservations, we assessed genetic risk factors for SB by investigating: (1) possible genetic overlap with AUD, and (2) impacts of rare and low-frequency genomic variants. Suicidal behaviors included lifetime history of suicidal thoughts and acts, including verified suicide deaths, scored using a ranking variable for the SB phenotype (range 0-4). We identified five loci significantly associated with SB and AUD, two of which are intergenic and three intronic on genes AACSP1, ANK1, and FBXO11. Nonsynonymous rare and low-frequency mutations in four genes including SERPINF1 (PEDF), ZNF30, CD34, and SLC5A9, and non-intronic rare and low-frequency mutations in genes OPRD1, HSD17B3 and one lincRNA were significantly associated with SB. One identified pathway related to hypoxia-inducible factor (HIF) regulation, whose 83 nonsynonymous rare and low-frequency variants on 10 genes were significantly linked to SB as well. Four additional genes, and two pathways related to vasopressin-regulated water metabolism and cellular hexose transport, also were strongly associated with SB. This study represents the first investigation of genetic factors for SB in an American Indian population that has high risk for suicide. Our study suggests that bivariate association analysis between comorbid disorders can increase statistical power; and rare and low-frequency variant analysis in a high-risk population enabled by whole-genome sequencing has the potential to identify novel genetic factors. Although such findings may be population specific, rare functional mutations relating to PEDF and HIF regulation align with past reports and suggest a biological mechanism for suicide risk and a potential therapeutic target for intervention.

Cell-type brain-region specific changes in prefrontal cortex of a mouse model of alcohol dependence.

The prefrontal cortex is a crucial regulator of alcohol drinking, and dependence, and other behavioral phenotypes associated with AUD. Comprehensive identification of cell-type specific transcriptomic changes in alcohol dependence will improve our understanding of mechanisms underlying the excessive alcohol use associated with alcohol dependence and will refine targets for therapeutic development. We performed single nucleus RNA sequencing (snRNA-seq) and Visium spatial gene expression profiling on the medial prefrontal cortex (mPFC) obtained from C57BL/6 J mice exposed to the two-bottle choice-chronic intermittent ethanol (CIE) vapor exposure (2BC-CIE, defined as dependent group) paradigm which models phenotypes of alcohol dependence including escalation of alcohol drinking. Gene co-expression network analysis and differential expression analysis identified highly dysregulated co-expression networks in multiple cell types. Dysregulated modules and their hub genes suggest novel understudied targets for studying molecular mechanisms contributing to the alcohol dependence state. A subtype of inhibitory neurons was the most alcohol-sensitive cell type and contained a downregulated gene co-expression module; the hub gene for this module is Cpa6, a gene previously identified by GWAS to be associated with excessive alcohol consumption. We identified an astrocytic Gpc5 module significantly upregulated in the alcohol-dependent group. To our knowledge, there are no studies linking Cpa6 and Gpc5 to the alcohol-dependent phenotype. We also identified neuroinflammation related gene expression changes in multiple cell types, specifically enriched in microglia, further implicating neuroinflammation in the escalation of alcohol drinking. Here, we present a comprehensive atlas of cell-type specific alcohol dependence mediated gene expression changes in the mPFC and identify novel cell type-specific targets implicated in alcohol dependence.

Associations of ADH1B and ALDH2 genotypes and alcohol flushing with drinking history, withdrawal symptoms, and ICD-10 criteria in Japanese alcohol-dependent men.

OBJECTIVES: Given the high prevalence of fast-metabolizing alcohol dehydrogenase-1B*2 (ADH1B*2 ) and inactive aldehyde dehydrogenase-2*2 (ALDH2*2 ) alleles in East Asians, we evaluated how the ADH1B / ALDH2 genotypes and alcohol flushing might affect the development of alcohol dependence (AD). METHODS: We evaluated how the ADH1B / ALDH2 genotypes and self-reported alcohol flushing affected history of drinking events and withdrawal symptoms and ICD-10 criteria in 4116 Japanese AD men. RESULTS: The ADH1B*1/*1 group and ALDH2*1/*1 group were 1-5 years younger than the ADH1B*2 (+) and ALDH2*1/*2 groups, respectively, for all of the ages at onset of habitual drinking, blackouts, daytime drinking, uncontrolled drinking, withdrawal symptoms, and first treatment for AD, and the current age. Blackouts were more common in the ADH1B*1/*1 group and ALDH2*1/*1 group. Daytime drinking, uncontrolled drinking, and withdrawal symptoms, such as hand tremor, sweating, convulsions, and delirium tremens/hallucinations were more common in the ADH1B*1/*1 group. The ADH1B*1/*1 was positively associated with the ICD-10 criteria for 'tolerance' and 'withdrawal symptoms'. The ADH1B*1/*1 group and ALDH2*1/*2 group had a larger ICD-10 score. Never flushing was reported by 91.7% and 35.2% of the ALDH2*1/*1 and ALDH2*1/*2 carriers, respectively. After a 1-2-year delay in the onset of habitual drinking in the former-/current-flushing group, no differences in the ages of the aforementioned drinking milestones were found according to the flushing status. CONCLUSION: The ADH1B*1/*1 and ALDH2*1/*1 accelerated the development of drinking events and withdrawal symptoms in Japanese AD patients. ICD-10 score was larger in the ADH1B*1/*1 group and ALDH2*1/*2 group. The effects of alcohol flushing on drinking events were limited.

A dual-omics approach on the effects of fibroblast growth factor-2 (FGF-2) on ventral tegmental area dopaminergic neurons in response to alcohol consumption in mice.

Harmful alcohol consumption is a major socioeconomic burden to the health system, as it can be the cause of mortality of heavy alcohol drinkers. The dopaminergic (DAergic) system is thought to play an important role in the pathogenesis of alcohol drinking behaviour; however, its exact role remains elusive. Fibroblast growth factor 2 (FGF-2), a neurotrophic factor, associated with both the DAergic system and alcohol consumption, may play an important role in DAergic neuroadaptations during alcohol abuse. Within this study, we aimed to clarify the role of endogenous FGF-2 on the DAergic system and whether there is a possible link to alcohol consumption. We found that lack of FGF-2 reduces the alcohol intake of mice. Transcriptome analysis of DAergic neurons revealed that FGF-2 knockout (FGF-2 KO) shifts the molecular fingerprint of midbrain dopaminergic (mDA) neurons to DA subtypes of the ventral tegmental area (VTA). In line with this, proteomic changes predominantly appear also in the VTA. Interestingly, these changes led to an altered regulation of the FGF-2 signalling cascades and DAergic pathways in a region-specific manner, which was only marginally affected by voluntary alcohol consumption. Thus, lack of FGF-2 not only affects the gene expression but also the proteome of specific brain regions of mDA neurons. Our study provides new insights into the neuroadaptations of the DAergic system during alcohol abuse and, therefore, comprises novel targets for future pharmacological interventions.

Genetic susceptibility to optic neuropathy in patients with alcohol use disorder.

BACKGROUND: The pathophysiology of toxico-nutritional optic neuropathies remains debated, with no clear understanding of the respective roles played by the direct alcohol toxicity, smoking and the often associated vitamin deficiencies, which are risk factors for optic neuropathy. Our aim was to investigate genetic susceptibility in patients with bilateral infraclinical optic neuropathy associated with chronic alcohol use disorder. METHODS: This retrospective cohort study included 102 visually asymptomatic patients with documented alcohol use disorder from a French reference center. Optic neuropathy was identified with optical coherence tomography (OCT), after which genetic susceptibility in the group of affected patients was investigated. Genetic testing was performed using panel sequencing of 87 nuclear genes and complete mitochondrial DNA sequencing. RESULTS: Optic neuropathy was detected in 36% (37/102) of the included patients. Genetic testing of affected patients disclosed two patients (2/30, 6.7%) with optic neuropathy associated with pathogenic variants affecting the SPG7 gene and five patients (5/30, 16.7%) who harbored variants of uncertain significance close to probable pathogenicity in the genes WFS1, LOXL1, MMP19, NR2F1 and PMPCA. No pathogenic mitochondrial DNA variants were found in this group. CONCLUSIONS: OCT can detect presence of asymptomatic optic neuropathy in patients with chronic alcohol use disorder. Furthermore, genetic susceptibility to optic neuropathy in this setting is found in almost a quarter of affected patients. Further studies may clarify the role of preventative measures in patients who might be predisposed to avoidable visual loss and blindness.

Examining associations between genetic and neural risk for externalizing behaviors in adolescence and early adulthood.

BACKGROUND: Researchers have identified genetic and neural risk factors for externalizing behaviors. However, it has not yet been determined if genetic liability is conferred in part through associations with more proximal neurophysiological risk markers. METHODS: Participants from the Collaborative Study on the Genetics of Alcoholism, a large, family-based study of alcohol use disorders were genotyped and polygenic scores for externalizing (EXT PGS) were calculated. Associations with target P3 amplitude from a visual oddball task (P3) and broad endorsement of externalizing behaviors (indexed via self-report of alcohol and cannabis use, and antisocial behavior) were assessed in participants of European (EA; N = 2851) and African ancestry (AA; N = 1402). Analyses were also stratified by age (adolescents, age 12-17 and young adults, age 18-32). RESULTS: The EXT PGS was significantly associated with higher levels of externalizing behaviors among EA adolescents and young adults as well as AA young adults. P3 was inversely associated with externalizing behaviors among EA young adults. EXT PGS was not significantly associated with P3 amplitude and therefore, there was no evidence that P3 amplitude indirectly accounted for the association between EXT PGS and externalizing behaviors. CONCLUSIONS: Both the EXT PGS and P3 amplitude were significantly associated with externalizing behaviors among EA young adults. However, these associations with externalizing behaviors appear to be independent of each other, suggesting that they may index different facets of externalizing.

The joint effects of genetic liability and the death of close relatives on risk for major depression and alcohol use disorder in a Swedish national sample.

BACKGROUND: To determine whether genetic risk factors for major depression (MD) and alcohol use disorder (AUD) interact with a potent stressor - death of spouse, parent, and sibling - in predicting episodes of, respectively, MD and AUD. METHODS: MD and AUD registrations were assessed from national Swedish registries. In individuals born in Sweden 1960-1970, we identified 7586, 388 459, and 34 370 with the loss of, respectively, a spouse, parent, and sibling. We started following subjects at age 18 or the year 2002 with end of follow-up in 2018. We examined time to event - a registration for MD within 6 months or AUD within a year - on an additive scale, using the Nelson-Aalen estimator. Genetic risk was assessed by the Family Genetic Risk Score (FGRS). RESULTS: In separate models controlling for the main effects of death of spouse, parent, and sibling, FGRS, and sex, significant interactions were seen in all analyses between genetic risk for MD and death of relative in prediction of subsequent MD registration. A similar pattern of results, albeit with weaker interaction effects, was seen for genetic risk for AUD and risk for AUD registration. Genetic risk for bipolar disorder (BD) and anxiety disorders (AD) also interacted with event exposure in predicting MD. CONCLUSIONS: Genetic risk for both MD and AUD act in part by increasing the sensitivity of individuals to the pathogenic effects of environmental stressors. For prediction of MD, similar effects are also seen for genetic risk for AD and BD.

IL17RB genetic variants are associated with acamprosate treatment response in patients with alcohol use disorder: A proteomics-informed genomics study.

Acamprosate is a Food and Drug Administration (FDA) approved medication for the treatment of alcohol use disorder (AUD). However, only a subset of patients achieves optimal treatment outcomes. Currently, no biological measures are utilized to predict response to acamprosate treatment. We applied our established pharmaco-omics informed genomics strategy to identify potential biomarkers associated with acamprosate treatment response. Specifically, our previous open-label acamprosate clinical trial recruited 442 patients with AUD who were treated with acamprosate for three months. We first performed proteomics using baseline plasma samples to identify potential biomarkers associated with acamprosate treatment outcomes. Next, we applied our established "proteomics-informed genome-wide association study (GWAS)" research strategy, and identified 12 proteins, including interleukin-17 receptor B (IL17RB), associated with acamprosate treatment response.​ A GWAS for IL17RB concentrations identified several genome-wide significant signals. Specifically, the top hit single nucleotide polymorphism (SNP) rs6801605 with a minor allele frequency of 38% in the European American population mapped 4 kilobase (Kb) upstream of IL17RB, and intron 1 of the choline dehydrogenase (CHDH) gene on chromosome 3 (p: 4.8E-20). The variant genotype (AA) for the SNP rs6801605 was associated with lower IL17RB protein expression. In addition, we identified a series of genetic variants in IL17RB that were associated with acamprosate treatment outcomes. Furthermore, the variantgenotypes for all of those IL17RB SNPs were protective for alcohol relapse. Finally, we demonstrated that the basal level of mRNA expression of IL17RB was inversely correlated with those of nuclear factor-κB (NF-κB) subunits, and a significantly higher expression of NF-κB subunits was observed in AUD patients who relapsed to alcohol use. In summary, this study illustrates that IL17RB genetic variants might contribute to acamprosate treatment outcomes. This series of studies represents an important step toward generating functional hypotheses that could be tested to gain insight into mechanisms underlying acamprosate treatment response phenotypes. (The ClinicalTrials.gov Identifier: NCT00662571).

A Developmentally-Informative Genome-wide Association Study of Alcohol Use Frequency.

Contemporary genome-wide association study (GWAS) methods typically do not account for variability in genetic effects throughout development. We applied genomic structural equation modeling to combine developmentally-informative phenotype data and GWAS to create polygenic scores (PGS) for alcohol use frequency that are specific to developmental stage. Longitudinal cohort studies targeted for gene-identification analyses include the Collaborative Study on the Genetics of Alcoholism (adolescence n = 1,118, early adulthood n = 2,762, adulthood n = 5,255), the National Longitudinal Study of Adolescent to Adult Health (adolescence n = 3,089, early adulthood n = 3,993, adulthood n = 5,149), and the Avon Longitudinal Study of Parents and Children (ALSPAC; adolescence n = 5,382, early adulthood n = 3,613). PGS validation analyses were conducted in the COGA sample using an alternate version of the discovery analysis with COGA removed. Results suggest that genetic liability for alcohol use frequency in adolescence may be distinct from genetic liability for alcohol use frequency later in developmental periods. The age-specific PGS predicts an increase of 4 drinking days per year per PGS standard deviation when modeled separately from the common factor PGS in adulthood. The current work was underpowered at all steps of the analysis plan. Though small sample sizes and low statistical power limit the substantive conclusions that can be drawn regarding these research questions, this work provides a foundation for future genetic studies of developmental variability in the genetic underpinnings of alcohol use behaviors and genetically-informed, age-matched phenotype prediction.

Investigating genetically stratified subgroups to better understand the etiology of alcohol misuse.

Alcohol misuse (AM) is highly prevalent and harmful, with theorized subgroups differing on internalizing and externalizing dimensions. Despite known heterogeneity, genome-wide association studies (GWAS) are usually conducted on unidimensional phenotypes. These approaches have identified important genes related to AM but fail to capture a large part of the heritability, even with recent increases in sample sizes. This study aimed to address phenotypic heterogeneity in GWAS to aid gene finding and to uncover the etiology of different types of AM. Genetic and phenotypic data from 410,414 unrelated individuals of multiple ancestry groups (primarily European) in the UK Biobank were obtained. Mixture modeling was applied to measures of alcohol misuse and internalizing/externalizing psychopathology to uncover phenotypically homogenous subclasses, which were carried forward to GWAS and functional annotation. A four-class model emerged with "low risk", "internalizing-light/non-drinkers", "heavy alcohol use-low impairment", and "broad high risk" classes. SNP heritability ranged from 3 to 18% and both known AM signals and novel signals were captured by genomic risk loci. Class comparisons showed distinct patterns of regional brain tissue enrichment and genetic correlations with internalizing and externalizing phenotypes. Despite some limitations, this study demonstrated the utility of genetic research on homogenous subclasses. Not only were novel genetic signals identified that might be used for follow-up studies, but addressing phenotypic heterogeneity allows for the discovery and investigation of differential genetic vulnerabilities in the development of AM, which is an important step towards the goal of personalized medicine.

Genetic nurture effects for alcohol use disorder.

We tested whether aspects of the childhood/adolescent home environment mediate genetic risk for alcohol problems within families across generations. Parental relationship discord and parental divorce were the focal environments examined. The sample included participants of European ancestry (N = 4806, 51% female) and African ancestry (N = 1960, 52% female) from the high-risk Collaborative Study on the Genetics of Alcoholism. Alcohol outcomes in the child generation included lifetime criterion counts for DSM-5 Alcohol Use Disorder (AUD), lifetime maximum drinks in 24 h, age at initiation of regular drinking, and age at first alcohol intoxication. Predictors in the parent generation included relationship discord, divorce, alcohol measures parallel to those in the child generation, and polygenic scores for alcohol problems. Parental polygenic scores were partitioned into alleles that were transmitted and non-transmitted to the child. The results from structural equation models were consistent with genetic nurture effects in European ancestry families. Exposure to parental relationship discord and parental divorce mediated, in part, the transmission of genetic risk for alcohol problems from parents to children to predict earlier ages regular drinking (ßindirect = -0.018 [-0.026, -0.011]) and intoxication (ßindirect = -0.015 [-0.023, -0.008]), greater lifetime maximum drinks (ßindirect = 0.006 [0.002, 0.01]) and more lifetime AUD criteria (ßindirect = 0.011 [0.006, 0.016]). In contrast, there was no evidence that parental alleles had indirect effects on offspring alcohol outcomes via parental relationship discord or divorce in the smaller number of families of African ancestry. In conclusion, parents transmit genetic risk for alcohol problems to their children not only directly, but also indirectly via genetically influenced aspects of the home environment. Further investigation of genetic nurture in non-European samples is needed.

Arc controls alcohol cue relapse by a central amygdala mechanism.

Alcohol use disorder (AUD) is a chronic and fatal disease. The main impediment of the AUD therapy is a high probability of relapse to alcohol abuse even after prolonged abstinence. The molecular mechanisms of cue-induced relapse are not well established, despite the fact that they may offer new targets for the treatment of AUD. Using a comprehensive animal model of AUD, virally-mediated and amygdala-targeted genetic manipulations by CRISPR/Cas9 technology and ex vivo electrophysiology, we identify a mechanism that selectively controls cue-induced alcohol relapse and AUD symptom severity. This mechanism is based on activity-regulated cytoskeleton-associated protein (Arc)/ARG3.1-dependent plasticity of the amygdala synapses. In humans, we identified single nucleotide polymorphisms in the ARC gene and their methylation predicting not only amygdala size, but also frequency of alcohol use, even at the onset of regular consumption. Targeting Arc during alcohol cue exposure may thus be a selective new mechanism for relapse prevention.

RNA alternative splicing impacts the risk for alcohol use disorder.

Alcohol use disorder (AUD) is a complex genetic disorder characterized by problems arising from excessive alcohol consumption. Identifying functional genetic variations that contribute to risk for AUD is a major goal. Alternative splicing of RNA mediates the flow of genetic information from DNA to gene expression and expands proteome diversity. We asked whether alternative splicing could be a risk factor for AUD. Herein, we used a Mendelian randomization (MR)-based approach to identify skipped exons (the predominant splicing event in brain) that contribute to AUD risk. Genotypes and RNA-seq data from the CommonMind Consortium were used as the training dataset to develop predictive models linking individual genotypes to exon skipping in the prefrontal cortex. We applied these models to data from the Collaborative Studies on Genetics of Alcoholism to examine the association between the imputed cis-regulated splicing outcome and the AUD-related traits. We identified 27 exon skipping events that were predicted to affect AUD risk; six of these were replicated in the Australian Twin-family Study of Alcohol Use Disorder. Their host genes are DRC1, ELOVL7, LINC00665, NSUN4, SRRM2 and TBC1D5. The genes downstream of these splicing events are enriched in neuroimmune pathways. The MR-inferred impacts of the ELOVL7 skipped exon on AUD risk was further supported in four additional large-scale genome-wide association studies. Additionally, this exon contributed to changes of gray matter volumes in multiple brain regions, including the visual cortex known to be involved in AUD. In conclusion, this study provides strong evidence that RNA alternative splicing impacts the susceptibility to AUD and adds new information on AUD-relevant genes and pathways. Our framework is also applicable to other types of splicing events and to other complex genetic disorders.

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons.

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium channel subunit 2 (GIRK2), have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ("Affected: AF") and four control subjects without variants ("Unaffected: UN"). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single-cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

Genomic risk for post-traumatic stress disorder in families densely affected with alcohol use disorders.

Recent genome-wide association studies (GWAS) have identified genetic markers of post-traumatic stress disorder (PTSD) in civilian and military populations. However, studies have yet to examine the genetics of PTSD while factoring in risk for alcohol dependence, which commonly co-occur. We examined genome-wide associations for DSM-IV PTSD among 4,978 trauma-exposed participants (31% with alcohol dependence, 50% female, 30% African ancestry) from the Collaborative Study on the Genetics of Alcoholism (COGA). We also examined associations of polygenic risk scores (PRS) derived from the Psychiatric Genomics Consortium (PGC)-PTSD Freeze 2 (N = 3533) and Million Veterans Program GWAS of PTSD (N = 5200) with PTSD and substance dependence in COGA, and moderating effects of sex and alcohol dependence. 7.3% of COGA participants met criteria for PTSD, with higher rates in females (10.1%) and those with alcohol dependence (12.3%). No independent loci met genome-wide significance in the PTSD meta-analysis of European (EA) and African ancestry (AA) participants. The PGC-PTSD PRS was associated with increased risk for PTSD (B = 0.126, p < 0.001), alcohol dependence (B = 0.231, p < 0.001), and cocaine dependence (B = 0.086, p < 0.01) in EA individuals. A significant interaction was observed, such that EA individuals with alcohol dependence and higher polygenic risk for PTSD were more likely to have PTSD (B = 0.090, p < 0.01) than those without alcohol dependence. These results further support the importance of examining substance dependence, specifically alcohol dependence, and PTSD together when investigating genetic influence on these disorders.

Conserved role for PCBP1 in altered RNA splicing in the hippocampus after chronic alcohol exposure.

We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes encoding RNA splicing factors in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3' splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat and human hippocampus, which showed enriched PCBP1 association near the HAPLN2 exon 4 3' splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact hippocampal function in AUD.

Effect of MAOA rs1465108 polymorphism on susceptibility to substance/alcohol use disorder: a novel PCR-RFLP assay for the detection of MAOA rs1465108.

BACKGROUND: The health and social consequences of substance/alcohol use disorders are harmful. Most of the individuals cannot stop using them due to more likely their genetic background. The current study aimed both to develop a novel PCR-RFLP method for genotyping of MAOA rs1465108 and to analyze the effect of MAOA rs1465108 on the risk of alcohol (AUD), opioid (OUD) or methamphetamine (MUD) use disorders and on the depressive and anxiety symptoms in a Turkish population. METHODS AND RESULTS: A total of 353 individual with AUD (n = 154), OUD (n = 160) or MUD (n = 39) and 109 healthy subjects were included. The intensity of anxiety and depressive symptoms and craving and opioid withdrawal were measured by appropriate scales. Logistic regression analysis revealed no association between MAOA rs1465108 polymorphism and substance/alcohol use disorder (p > 0.05). Healthy subjects (3.0) had significantly lower levels of depressive symptoms than individuals with OUD (27.0), AUD (21.0) and MUD (25.5) groups. The severity of depressive symptoms was significantly higher in OUD as compared to AUD. There was a statistically significant difference between individuals with AUD, OUD and MUD in view of the average ages of first use (17, 19 and 20 years, respectively) (p < 0.05). CONCLUSIONS: The results presented here do not support the hypothesis that MAOA rs1465108 is associated with substance/alcohol use disorders. The intensity of depressive symptoms could be changed according to the abused substance type. A novel PCR-RFLP was developed for genotyping of MAOA rs1465108 polymorphism, which could be a better option for laboratories without high technology equipment.

A case control study investigating the methylation levels of GHRL and GHSR genes in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is a relapsing disease described as excessive use of alcohol. Evidence of the role of DNA methylation in addiction is accumulating. Ghrelin is an important peptide known as appetite hormone and its role in addictive behavior has been identified. Here we aimed to determine the methylation levels of two crucial genes (GHRL and GHSR) in ghrelin signaling and further investigate the association between methylation ratios and plasma ghrelin levels. METHODS: Individuals diagnosed with (n = 71) and without (n = 82) AUD were recruited in this study. DNA methylation levels were measured through methylation-sensitive high-resolution melting (MS-HRM). Acylated ghrelin levels were detected by ELISA. The GHRL rs696217 polymorphism was analyzed by the standard PCR-RFLP method. RESULTS: GHRL was significantly hypermethylated (P < 0.0022) in AUD between 25 and 50% methylation than in control subjects but no significant changes of GHSR methylation were observed. Moreover, GHRL showed significant positive correlation of methylation ratio between 25 and 50% with age. A significant positive correlation between GHSR methylation and ghrelin levels in the AUD group was determined (P = 0.037). The level of GHRL methylation and the ghrelin levels showed a significant association in the control subjects (P = 0.042). CONCLUSION: GHSR and GHRL methylation levels did not change significantly between control and AUD groups. However, GHRL and GHSR methylations seemed to have associations with plasma ghrelin levels in two groups. This is the first study investigating the DNA methylation of GHRL and GHSR genes in AUD.

Changes of neurofilament light chain in patients with alcohol dependence following withdrawal and the genetic effect from ALDH2 Polymorphism.

Neurofilament light chain (NFL), as a measure of neuroaxonal injury, has recently gained attention in alcohol dependence (AD). Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme which metabolizes the alcohol breakdown product acetaldehyde. An ALDH2 single nucleotide polymorphism (rs671) is associated with less ALDH2 enzyme activity and increased neurotoxicity. We examined the blood NFL levels in 147 patients with AD and 114 healthy controls using enzyme-linked immunosorbent assay and genotyped rs671. We also followed NFL level, alcohol craving and psychological symptoms in patients with AD after 1 and 2 weeks of detoxification. We found the baseline NFL level was significantly higher in patients with AD than in controls (mean ± SD: 264.2 ± 261.8 vs. 72.1 ± 35.6 pg/mL, p < 0.001). The receiver operating characteristic curve revealed that NFL concentration could discriminate patients with AD from controls (area under the curve: 0.85; p < 0.001). The NFL levels were significantly reduced following 1 and 2 weeks of detoxification, with the extent of reduction correlated with the improvement of craving, depression, and anxiety (p < 0.001). Carriers with the rs671 GA genotype, which is associated with less ALDH2 activity, had higher NLF levels either at baseline or after detoxification compared with GG carriers. In conclusion, plasma NFL level was increased in patients with AD and reduced after early abstinence. Reduction in NFL level corroborated well with the improvement of clinical symptoms. The ALDH2 rs671 polymorphism may play a role in modulating the extent of neuroaxonal injury and its recovery.