An exploratory study of candidate gene(s) for Delirium Tremens: Adding the new cholinergic dimension to the conundrum.

BACKGROUND AND AIMS: Delirium Tremens (DT) is the most severe form of alcohol withdrawal syndrome, with a potential risk of mortality. Search for the predictors of DT led to study of candidate genes, with inconsistent and inconclusive results. This study aimed to explore the association of various candidate gene polymorphisms and DT in a case-control design. METHODS: This was a genetic association study with a case control design. Two hundred ten Alcohol dependent (AD) male subjects and 200 age matched controls were recruited. DT was diagnosed with the help of Semi-structured Assessment for Genetics of Alcoholism. SNP genotyping was done using TaqMan assay by real time PCR (q-PCR). RESULTS: T allele carrying status (GT and TT) [rs1824024] of muscarinic cholinergic receptor 2 (CHRM2) was found to be significantly associated with DT. When compared to the general population, this genetic polymorphism was not found to be more common in alcohol dependence per se, which excludes the possibility of spurious association between CHRM2 and DT. Withdrawal seizure was more common in the DT group and came out to be one of the important predictors of DT. However, the genetic association was found to be specific for DT, not related to withdrawal seizures. CONCLUSION: The present research added a new cholinergic dimension in the genetic association and biological mechanism of DT.

The association between the SLC6A3 VNTR 9-repeat allele and alcoholism-a meta-analysis.

BACKGROUND: Dopamine transporter gene (SLC6A3) represents a promising candidate involved in the development of alcoholism. This study aimed to explore the association between the 9-repeat allele (A9) of a 40-bp variable number tandem repeat (VNTR) polymorphism in the 3' un-translated region (3' UTR) of the SLC6A3 gene and alcoholism. METHODS: The SLC6A3 VNTR was genotyped by PCR in unrelated Mexican Americans including 337 controls and 365 alcoholics. Pearson's chi-square test or Fisher's exact test was used to compare the genotype and allele distribution. Meta-analyses were performed for population-based case-control association studies of the SLC6A3 VNTR polymorphism with alcoholism. Data were analyzed under random effect models with the Comprehensive Meta-analysis (v.2) statistical software package. RESULTS: In Mexican Americans, no significant difference was found in allele and genotype distribution between controls and alcoholics or between controls and alcoholics with alcohol withdrawal seizure (AWS) or delirium tremens (DT) (unadjusted p > 0.05). A total of 13 research articles were included in the meta-analyses. No significant difference of the SLC6A3 VNTR A9 was noted between controls and alcoholics at the genotypic and allelic level when all ethnic populations, only Caucasian populations, or only Asian populations were considered (p > 0.05). Significant associations were observed between SLC6A3 VNTR A9 and alcoholics with AWS or DT at the genotypic as well as allelic level when all ethnic populations or only Caucasian populations were considered (p < 0.05, OR 1.5-2.1). CONCLUSIONS: Meta-analyses suggest a possible association between the SLC6A3 VNTR A9 and alcoholic subgroup with AWS or DT.

Association of ADH4 genetic variants with alcohol dependence risk and related phenotypes: results from a larger multicenter association study.

Genetic variants of the alcohol-metabolizing enzyme ADH4, located on chromosome 4q22-4q23, have been related to alcohol dependence (AD) risk in previous research. The aim of this association study in a large multicenter sample of alcohol-dependent individuals and controls is to confirm ADH4 single nucleotide polymorphism (SNP) and haplotype association with AD and relevant related phenotypes. One thousand, six hundred and twenty-two (1622) inpatient subjects and 1469 control subjects with DSM-IV. AD from four addiction treatment centres were included. Characteristics of AD and related phenotypes including alcohol withdrawal, Cloninger's type I and II and first ages of drinking, regular drinking and AD onset were obtained using standardized structured interviews. After subjects were genotyped for 2 ADH4 polymorphisms, single SNP case-control and haplotype analyses were conducted. Both variants--rs1800759 and rs1042364--and the A-A and C-G haplotypes were significantly related to AD across samples. Furthermore, associations with AD-related phenotypes and subtypes revealed a potential protective influence of this haplotype. This study confirms the significant relationship of ADH4 variants with AD and related phenotypes. While the rs1800759 and rs1042364 A-A haplotype had a potential protective influence on the risk for several AD-related phenotypes, this effect is rather small compared to functional variants of other alcohol or acetaldehyde-metabolizing enzymes like ALDH2*2 or ADH1B*2.

Reduced expression of circadian clock genes in male alcoholic patients.

BACKGROUND: There are clear interactions between chronic alcohol consumption and circadian rhythmicity that is regulated by several circadian clock genes. The altered expressions of these genes have been mainly described in animals. The mammalian master clock in the suprachiasmatic nuclei orchestrates the biological rhythms in peripheral tissues. As peripheral blood mononuclear cells (PBMCs) are the most accessible tissue clinically, we assessed the mRNA levels of these genes in patients with alcohol dependence (AD) undergoing alcohol-withdrawal (AW) treatment. METHODS: Twenty-two male patients fulfilled the DSM-IV diagnostic criteria of AD, and 12 comparison healthy control subjects were recruited. The patients with AD were further divided by the presence of delirium tremens (DTs), the most severe form of AW syndrome, into DT group and non-DT group. All the participants received blood withdrawal at 9 am, while the patients with AD had blood collection twice: on the next morning of admission (baseline) and on the seventh day. PBMCs were isolated from whole blood, and the mRNA expression profiles of hClock1, hBmal1, hPer1, hPer2, hCry1, and hCry2 were determined by quantitative real-time PCR. RESULTS: The baseline mRNA levels of the target circadian clock genes were markedly lower in patients with AD than in control subjects. After 1 week of alcohol detoxification, there were very limited restorations of discrete circadian gene expressions. DT group did not differ in the expression patterns of circadian clock genes from non-DT group. CONCLUSIONS: This is the first study demonstrating the overall lowering of circadian clock genes among patients with AD. The expression pattern is comparable between patients with and without DTs. Although preliminary with data at only one single time point, the observation of strikingly reduced mRNA levels supports the association between circadian clock gene dysregulation and chronic alcohol intake.

Interaction of SLC6A4 and DRD2 polymorphisms is associated with a history of delirium tremens.

Several genetic polymorphisms have been reported to be associated with alcohol withdrawal seizures (AWS) and delirium tremens (DT). To replicate and further explore these findings, we investigated the effects of 12 previously reported candidate genetic variations in two groups of alcohol-dependent European Americans with a history of withdrawal, which differed according to the presence (n = 112) or absence (n = 92) of AWS and/or DT. Associations of AWS and/or DT with the genomic and clinical characteristics and gene-gene interaction effects were investigated using logistic regression models. None of the polymorphisms were significantly associated with AWS/DT after correction for multiple testing. However, we found a significant interaction effect of the SLC6A4 promoter polymorphism (5-HTTLPR) and DRD2 exon 8 single nucleotide polymorphism rs6276 on AWS and/or DT history (P = 0.009), which became more significant after adjustment for lifetime maximum number of drinks consumed per 24 hours (P < 0.001). Subsequent analysis revealed an even stronger association of the SLC6A4-DRD2 interaction with DT (P < 0.0001), which remained significant after Bonferroni correction. Results reveal decreased likelihood of DT in alcoholics that carry the DRD2 rs6276 G allele and SLC6A4 LL genotype. This study provides the first evidence to implicate the interaction between serotonin and dopamine neurotransmission in the etiology of DT. Replication is necessary to verify this potentially important finding.

Mice lacking Gad2 show altered behavioral effects of ethanol, flurazepam and gabaxadol.

Gamma-aminobutyric acid (GABA) is synthesized in brain by two isoforms of glutamic acid decarboxylase (Gad), Gad1 and Gad2. Gad1 provides most of the GABA in brain, but Gad2 can be rapidly activated in times of high GABA demand. Mice lacking Gad2 are viable whereas deletion of Gad1 is lethal. We produced null mutant mice for Gad2 on three different genetic backgrounds: predominantly C57BL/6J and one or two generations of backcrossing to 129S1/SvimJ (129N1, 129N2). We used these mice to determine if actions of alcohol are regulated by synthesis of GABA from this isoform. We also studied behavioral responses to a benzodiazepine (flurazepam) and a GABAA receptor agonist (gabaxadol). Deletion of Gad2 increased ethanol palatability and intake and slightly reduced the severity of ethanol-induced withdrawal, but these effects depended strongly on genetic background. Mutant mice on the 129N2 background showed the above three ethanol behavioral phenotypes, but the C57BL/6J inbred background did not show any of these phenotypes. Effects on ethanol consumption also depended on the test as the mutation did not alter consumption in limited access models. Deletion of Gad2 reduced the effect of flurazepam on motor incoordination and increased the effect of extrasynaptic GABAA receptor agonist gabaxadol without changing the duration of loss of righting reflex produced by these drugs. These results are consistent with earlier proposals that deletion of Gad2 (on 129N2 background) reduces synaptic GABA but also suggest changes in extrasynaptic receptor function.

Genome-wide association study of alcohol dependence.

CONTEXT: Alcohol dependence is a serious and common public health problem. It is well established that genetic factors play a major role in the development of this disorder. Identification of genes that contribute to alcohol dependence will improve our understanding of the mechanisms that underlie this disorder. OBJECTIVE: To identify susceptibility genes for alcohol dependence through a genome-wide association study (GWAS) and a follow-up study in a population of German male inpatients with an early age at onset. DESIGN: The GWAS tested 524,396 single-nucleotide polymorphisms (SNPs). All SNPs with P < 10(-4) were subjected to the follow-up study. In addition, nominally significant SNPs from genes that had also shown expression changes in rat brains after long-term alcohol consumption were selected for the follow-up step. SETTING: Five university hospitals in southern and central Germany. PARTICIPANTS: The GWAS included 487 male inpatients with alcohol dependence as defined by the DSM-IV and an age at onset younger than 28 years and 1358 population-based control individuals. The follow-up study included 1024 male inpatients and 996 age-matched male controls. All the participants were of German descent. MAIN OUTCOME MEASURES: Significant association findings in the GWAS and follow-up study with the same alleles. RESULTS: The GWAS produced 121 SNPs with nominal P < 10(-4). These, together with 19 additional SNPs from homologues of rat genes showing differential expression, were genotyped in the follow-up sample. Fifteen SNPs showed significant association with the same allele as in the GWAS. In the combined analysis, 2 closely linked intergenic SNPs met genome-wide significance (rs7590720, P = 9.72 x 10(-9); rs1344694, P = 1.69 x 10(-8)). They are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes. Nine SNPs were located in genes, including the CDH13 and ADH1C genes, that have been reported to be associated with alcohol dependence. CONCLUSIONS: This is the first GWAS and follow-up study to identify a genome-wide significant association in alcohol dependence. Further independent studies are required to confirm these findings.

Polymorphisms in the dopamine transporter gene (SLC6A3/DAT1) and alcohol dependence in humans: a systematic review.

Dopamine neurotransmission has been a key player in attempts to identify genetic factors involved in alcohol dependence. The dopamine transporter terminates dopaminergic neurotransmission, making the gene encoding the transporter (SLC6A3/DAT1) an attractive candidate in clinical studies on alcohol dependence. We conducted a systematic review of 18 studies examining associations between polymorphisms in DAT1 and alcohol dependence. The DAT1 variable number tandem repeat, the most frequent studied polymorphism in DAT1, did not show a direct association with alcohol dependence in general. Several, but not all, studies found that the DAT1 variable number tandem repeat (9-repeat allele) was associated with alcohol-withdrawal symptoms, such as seizures and delirium tremens. We discuss shortcomings, such as lack of power and disregarding moderating variables, as well as future challenges of gene association studies.

[Association studies of dopamine D4 receptor gene exon 3 in patients with alcohol dependence]. / Badanie zwiazku polimorfizmu VNTR genu receptora dopaminy DRD4 egzonu 3 z zespolem zaleznosci alkoholowej (ZZA).

AIM: The aim of this study was to evaluate the role of dopamine D4 receptor (DRD4) exon 3 polymorphisms (48 bp VNTR) in the pathogenesis of alcoholism. This polymorphism was investigated in the association study in a whole group of alcoholics (n = 122) and in homogenous overlapping subgroups: 1) with early age of onset of alcoholism (AOO < or = 26 years) (n = 65) and 2) with a co-occurrence of dissocial personality disorder (n = 38), and 3) in patients with a history of delirium tremens and/or alcohol seizures (n = 41). The control group consisted of healthy volunteers, gender and age matched, with excluded psychiatric disorders (n = 399). METHOD: The history of alcoholism was investigated using SSAGA (Semi-Structured Assessment for the Genetics of Alcoholism - Polish version). The DRD4 receptor exon 3 polymorphism was determined using PCR. RESULTS: We found significant differences in the short alleles (2-5 VNTR) frequencies between controls and patients with a history of delirium tremens and/or alcohol seizures (p = 0.043). A trend was also observed in the higher frequency of short alleles amongst individuals with an early age of onset of alcoholism (p = 0.063). CONCLUSION: The results of this study suggest that inherited short variants of DRD4 alleles may play role in pathogenesis of alcohol dependence.

Neurobiological and psychosocial processes associated with depressive and substance-related disorders in adolescents.

Adolescents are at heightened risk for the development of both depressive and substance-related disorders. These two disorders frequently co-occur in adolescents and are associated with significant morbidity and mortality. Given the substantial economic and psychosocial burden associated with the comorbid condition, the identification of causal mechanisms associated with their co-occurrence is of great public health importance. Although there is significant understanding of the environmental and neurobiological factors involved in depression and addictive disorders considered separately, the mechanisms underlying the comorbid illness have not been investigated carefully. The purpose of this review is to summarize the extant literature on genetic, environmental and neurobiological processes involved in the etiology of depressive and substance-related disorders in adolescents and adults. It is important to note that the data on common neurobiological systems that link addictive and depressive disorders are primarily from research with adult animals and humans. Given the ongoing maturation of these systems throughout adolescence and early adult life, it is not clear how these neurobiological processes influence the development and progression of both disorders. A better understanding of the pathophysiological mechanisms leading to the onset and course of these disorders during adolescence will be helpful in developing more effective preventive and treatment strategies not only for this population but also for adult patients with early-onset illness.

Genetic polymorphisms related to delirium tremens: a systematic review.

BACKGROUND: Delirium tremens (DT) is one of the more severe complications of alcohol withdrawal (AW), with a 5 to 10% lifetime risk for alcohol-dependent patients. The 2 most important neurosystems involved in AW are gamma-aminobutyric acid and glutamate. It is unknown whether these neurosystems are involved in the pathophysiology of DT as well. The candidate gene approach in DT could contribute to this knowledge and demonstrate a possible genetic predisposition for DT. The purpose of this study is to give an overview of all studied genetic polymorphisms in the diverse candidate genes related to DT and to summarize what these studies contribute to insights into the pathophysiology of DT. METHODS: The inclusion criteria for this literature study were articles in English analyzing the association between a genetic polymorphism and DT without other AW syndromes. Studies were identified until February 2006 in MEDLINE and EMBASE databases. RESULTS: We found 25 studies dealing with 30 polymorphisms, located in 19 different genes. Positive associations were found in 3 different candidate genes involved in the dopamine transmission, 1 gene involved in the glutamate pathway, 1 neuropeptide gene, and 1 cannabinoid gene. Two candidate genes involved in the dopamine transmission, dopamine receptor D3, and solute carrier family 6, were each associated with DT in 2 different study populations. The other 4 positive associations were not replicated in other studies. CONCLUSIONS: A total of 8 positive associations out of 30 polymorphisms makes a genetic base for DT plausible. Understanding the pathophysiological process of the development of DT has, indeed, been augmented by the reviewed genetic association studies. These studies suggest that the regulation of dopaminergic neurotransmission may play an important role.

Clinical and genetic characteristics of Korean male alcoholics with and without attention deficit hyperactivity disorder.

AIMS: To examine the clinical and genetic characteristics of Korean male alcoholics with and without attention deficit hyperactivity disorder (ADHD). METHODS: The present study included 85 male alcoholics who were diagnosed as having DSM-IV alcohol dependence. A total of 28 (32.9%) alcoholics were diagnosed as having DSM-IV ADHD with ongoing symptoms in adulthood. For the evaluation of their psychiatric conditions, the alcohol dependence scale (ADS), Beck depression inventory (BDI), Beck anxiety inventory (BAI), Barratt impulsiveness scale (BIS), brief anger-aggression questionnaire (BAQ), overt aggression scale (OAS), codependence test, and obsessive compulsive drinking scale (OCDS) were administered. The genotype frequencies of the dopamine type 2 receptor gene (DRD2), aldehyde dehydrogenase type 2 gene (ALDH2), functional polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR), and catechol-O-methyltransferase gene (COMT) polymorphisms were examined. RESULTS: Compared with alcoholics without ADHD, the mean ages for the onset of pathological drinking and alcohol withdrawal hallucinations were significantly earlier in alcoholics with ADHD. There was also a significant difference in the history of antisocial behaviour between the two groups. Compared with alcoholics without ADHD, the mean scores of the ADS, BDI, BAI, OAS, and OCDS were significantly higher in alcoholics with ADHD. With regard to the codependence test results, the mean scores of the interpersonal problem, low self-esteem and anxiety/fear subscales, and the mean total score of the codependence test were significantly higher in alcoholics with ADHD when compared with those without ADHD. There were no significant differences in the genotype frequencies of the DRD2, ALDH2, 5-HTTLPR, and COMT polymorphisms between alcoholics with and without ADHD. CONCLUSIONS: The results of this study suggest that the comorbidity of alcohol dependence and ADHD in this Korean sample forms a distinct clinical phenotype that shows an increased severity of alcohol-related symptoms and behavioural/emotional problems and that ADHD is associated with an increased risk for the early onset of alcohol dependence in Korean male alcoholics.

Impact of sex: determination of alcohol neuroadaptation and reinforcement.

This article represents the proceedings of a symposium at the Research Society on Alcoholism meeting in Santa Barbara, California. The organizers/chairs were Kristine M. Wiren and Deborah A. Finn. Following a brief introduction by Deborah Finn, the presentations were (1) The Importance of Gender in Determining Expression Differences in Mouse Lines Selected for Chronic Ethanol Withdrawal Severity, by Kristine M. Wiren and Joel G. Hashimoto; (2) Sex Differences in Ethanol Withdrawal Involve GABAergic and Stress Systems, by Paul E. Alele and Leslie L. Devaud; (3) The Influence of Sex on Ethanol Consumption and Reward in C57BL/6 Mice, by Kimber L. Price and Lawrence D. Middaugh; and (4) Sex Differences in Alcohol Self-administration in Cynomolgus Monkeys, by Kathleen A. Grant.

DBH*444G/A polymorphism of the dopamine-beta-hydroxylase gene is associated with alcoholism but not with severe alcohol withdrawal symptoms.

As the enzyme dopamine-beta-hydroxylase (DbetaH) converts dopamine to norepinephrine and both transmitters seem to be involved in the pathology of alcoholism and severe alcohol withdrawal symptoms, the gene encoding DbetaH (DBH) was applied to explore the genetic background of alcoholism and severe withdrawal symptoms. 102 healthy control subjects and 208 alcoholics, including 97 patients with a history of mild withdrawal symptoms, 57 with a history of alcohol withdrawal seizure (AWS) and 82 with a history of delirium tremens (DT) were genotyped for the DBH*444G/A polymorphism revealing a significantly elevated frequency of genotypes carrying the A-allele (p = 0.02; after Bonferroni adjustment for multiple tests) in alcoholics compared to healthy controls. Frequencies of alleles and genotypes of individuals with mild withdrawal symptoms did not differ significantly from those of patients with DT or AWS.

Ionotropic glutamate receptor gene GRIK3 SER310ALA functional polymorphism is related to delirium tremens in alcoholics.

Upregulation of glutamatergic neurotransmission resulting from chronic ethanol intoxication may cause a hyperexcitable state during alcohol withdrawal, which may lead to seizures and delirium tremens. The aim of our study was to evaluate the association between a history of alcohol withdrawal-induced seizures and delirium tremens, and a functional polymorphism (Ser310Ala) of the GRIK3 gene coding for the glutamatergic kainate receptor subunit GlurR7 in a sample of well-characterized alcoholics compared to controls. In total, 233 patients meeting DSM-IV alcohol dependence criteria and 309 controls, all of German descent, were investigated. GRIK3 functional polymorphism was determined using PCR (polymerase chain reaction) of lymphocyte DNA. History of alcohol withdrawal-induced delirium tremens and seizures were obtained using the SSAGA (Semi-Structured Assessment for the Genetics of Alcoholism). Data were cross-checked with in-patients' clinical files. While a significant relationship between history of delirium tremens and the Ser310 allele was detected, no significant results were obtained for alcohol withdrawal-related seizures. Although this result is suggestive for a significant role of this polymorphism in the pathogenesis of delirium tremens in alcohol-dependent individuals, further investigation and confirmation are warranted.

Alcohol consumption and the body's biological clock.

This review summarizes new findings on the bidirectional interactions between alcohol and the clock genes, underlying the generation of circadian rhythmicity. At the behavioral level, both adult and perinatal ethanol treatments after the free-running period and light response of the circadian clock in rodents; genetic ethanol preference in alcohol-preferring rat lines is also associated with alterations in circadian pacemaker function. At the neuronal level, it has been shown that ethanol consumption alters the circadian expression patterns of period (per) genes in various brain regions, including the suprachiasmatic nucleus. Notably, circadian functions of beta-endorphin-containing neurons that participate in the control of alcohol reinforcement become disturbed after chronic alcohol intake. In turn, per2 gene activity regulates alcohol intake through its effects on the glutamatergic system through glutamate reuptake mechanisms and thereby may affect a variety of physiological processes that are governed by our internal clock. In summary, a new pathologic chain has been identified that contributes to the negative health consequences of chronic alcohol intake. Thus, chronic alcohol intake alters the expression of per genes, and as a consequence, a variety of neurochemical and neuroendocrine functions become disturbed. Further steps in this pathologic chain are alterations in physiological and immune functions that are under circadian control, and, as a final consequence, addictive behavior might be triggered or sustained by this cascade.

Alcohol withdrawal severity in inbred mouse (Mus musculus) strains.

Male mice (Mus musculus) from 15 standard inbred strains were exposed to a nearly constant concentration of ethanol (EtOH) vapor for 72 hr, averaging 1.59 +/- 0.03 mg EtOH/mL blood at withdrawal. EtOH- and air-exposed groups were tested hourly for handling-induced convulsions for 10 hr and at Hours 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of control values), and by design these differences were independent of strain differences in EtOH metabolism. Correlation of strain mean withdrawal severity with other responses to EtOH supported previously reported genetic relationships of high EtOH withdrawal with low drinking, high conditioned taste aversion, low tolerance to EtOH-induced hypothermia, and high stimulated activity after low-dose EtOH. Also supported were the positive genetic correlations among EtOH, barbiturate, and benzodiazepine withdrawal. Sensitivity of naive mice to several chemical convulsant-induced seizures was also correlated with EtOH withdrawal.

Association of the dopamine transporter gene with alcoholism.

AIMS: It was investigated whether the allele A9 of the dopamine transporter gene (DAT1; SLC6A3) is associated with alcoholism, delirium tremens (DT), alcohol withdrawal seizures (AWS), or the daily alcohol intake. METHODS: A group of 102 healthy subjects and 216 alcoholics, including 97 patients with a history of mild withdrawal symptoms, 65 with a history of AWS and 83 with a history of DT were genotyped and personal data were achieved for statistical evaluation in a case-control design. RESULTS: The frequency of individuals carrying the allele A9 [f(A9+)] was significantly higher (P = 0.01) in the group of alcoholics [f(A9+) = 0.48] compared with healthy controls [f(A9+) = 0.32]. There was no significant association of the allele A9 with severe withdrawal symptoms or the daily amount of alcohol consumed. CONCLUSIONS: Our results reveal that the allele A9 is strongly associated with alcoholism but not with withdrawal symptoms or daily alcohol intake.