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Original Article |

Systematic Analysis of Glutamatergic Neurotransmission Genes in Alcohol Dependence and Adolescent Risky Drinking Behavior FREE

Gunter Schumann, MD; Monika Johann, MD; Josef Frank, MA; Ulrich Preuss, MD; Norbert Dahmen, MD; Manfred Laucht, PhD; Marcella Rietschel, MD; Dan Rujescu, MD; Anbarasu Lourdusamy, PhD; Toni-Kim Clarke, MRes; Kristina Krause, MA; Anne Dyer, PhD; Martin Depner, MA; Stefan Wellek, PhD; Jens Treutlein, PhD; Armin Szegedi, MD; Ina Giegling, PhD; Sven Cichon, PhD; Dorothea Blomeyer, MA; Andreas Heinz, MD; Simon Heath, PhD; Mark Lathrop, PhD; Norbert Wodarz, MD; Michael Soyka, MD; Rainer Spanagel, PhD; Karl Mann, MD
[+] Author Affiliations

Author Affiliations: Central Institute of Mental Health, Mannheim, Germany (Drs Schumann, Laucht, Rietschel, Dyer, Wellek, Treutlein, Spanagel, and Mann, Mssrs Frank and Depner, and Ms Blomeyer); MRC, SGDP Center & Division of Psychological Medicine, Institute of Psychiatry at King's College, London, England (Drs Schumann and Lourdusamy and Mss Clarke and Krause); Department of Psychiatry and Psychotherapy, University of Regensburg Bezirksklinikum, Regensburg, Germany (Drs Johann and Wodarz); Department of Psychiatry and Psychotherapy, Martin-Luther-University, Halle, Germany (Dr Preuss); Department of Psychiatry and Psychotherapy, University of Mainz, Mainz, Germany (Dr Dahmen); Department of Psychology, University of Potsdam, Potsdam, Germany (Dr Laucht); Department of Psychiatry and Psychotherapy, University of Munich, Munich, Germany (Drs Rujescu, Giegling, and Soyka); Global Clinical Development, Organon, Roseland, New Jersey (Dr Szegedi); Department of Genomics, Life and Brain Centre, University of Bonn, Bonn, Germany (Dr Cichon); Department of Psychiatry and Psychotherapy; Charité-Universitätsmedizin, Berlin, Germany (Dr Heinz); and Centre National de Génotypage, Evry, France (Drs Heath and Lathrop).


Arch Gen Psychiatry. 2008;65(7):826-838. doi:10.1001/archpsyc.65.7.826.
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Context  Glutamatergic neurotransmission is implicated in alcohol-drinking behavior in animal models.

Objective  To investigate whether genetic variations in glutamatergic neurotransmission genes, which are known to alter alcohol effects in rodents, contribute to the genetic basis of alcoholism in humans.

Design  Association analysis of alcohol dependence and haplotype-tagging single nucleotide polymorphisms (SNPs) covering 10 glutamatergic genes. Resequencing of functional domains of these genes identified 204 SNPs. Haplotypes with a frequency of 5% or greater could be discriminated by 21 haplotype-tagging SNPs analyzed for association in 2 independent samples of alcohol-dependent adult patients and controls as well as adolescent trios.

Setting  Four university medical centers in the south of Germany.

Participants  One thousand three hundred thirty-seven patients and 1555 controls (study 1: 544 patients, 553 controls; study 2: 793 patients, 1002 controls). One hundred forty-four trios of 15-year-old adolescents assessed for risky drinking behavior.

Main Outcome Measures  Genotype profiles for GLAST; N-methyl-D-aspartate–receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB were analyzed for association with alcohol dependence using multivariate statistical analysis. Risky adolescent drinking was tested using the transmission disequilibrium test.

Results  Analysis of study 1 revealed that NR2A and MGLUR5 have the greatest relevance for human alcohol dependence among the genes selected with odds ratios of 2.35 and 1.69, respectively. Replication analysis in study 2 confirmed an association of alcohol dependence with NR2A (odds ratio, 2.01) but showed no association with MGLUR5. Combined analysis of study 1 and study 2 exhibited a more significant association on the Cochran-Mantel-Haenszel test (P < .001) for NR2A; NR2A was associated with positive family history, early onset of alcoholism, and maximum number of drinks in adults as well as risky drinking patterns in adolescents.

Conclusion  Genetic variations in NR2A have the greatest relevance for human alcohol dependence among the glutamatergic genes selected for their known alteration of alcohol effects in animal models.

Figures in this Article

Alcohol-related disorders cause 3.2% of deaths (1.8 million) and 4.0% of total disability-adjusted life-years.1 Alcohol dependence is a complex disorder with environmental as well as genetic components and does not have a main gene effect.2 As in other oligogenic neuropsychiatric disorders, multiple genes contribute to phenotypes of alcohol dependence. These genes involve different neurotransmitter systems, including glutamate, γ-aminobutyric acid, dopamine, opioids, serotonin, noradrenaline, and cannabinoids.3 Unlike many neuropsychiatric disorders, alcohol dependence offers a unique research potential to elucidate the contribution of each neurotransmitter system and apply translational approaches, as a wealth of behavioral animal models exist, which allow detailed assessment of drug-related behavior.4,5 To inform diagnostic and therapeutic developments in humans, the relevance of animal findings for human alcohol dependence and related phenotypes needs to be assessed in translational studies. Results of behavioral animal studies are often based on knockout models or pharmacological agents, which interfere to a much larger degree with signaling pathways than the usually more subtle consequences of functional genetic variations in frequent oligogenic disorders. For this reason and to explain a larger amount of genetic variance, we decided to analyze genes that encode functionally related proteins pertaining to one critical neurosignaling pathway, glutamatergic signaling.

Recently, a glutamatergic hypothesis has been developed to better understand the acute and chronic effects of alcohol on the brain. Alcohol affects the glutamatergic system on molecular, neurochemical, and cellular levels and this hypothesis proposes that alcohol consumption leads to enhanced glutamatergic activity in alcohol-dependent patients.6,7 This glutamate-induced hyperexcitability is uncovered during alcohol withdrawal. Furthermore, the hypothesis suggests that augmented glutamatergic activity can contribute to craving and relapse behavior, thus providing the rationale for using antiglutamatergic compounds, such as acamprosate, for relapse prevention.8 The role of glutamatergic neurotransmission in alcohol-drinking behavior has been analyzed using animal models and biochemical experimentation, and alterations have been identified at the presynaptic, synaptic, postsynaptic, or intracellular signaling level.7,9 It is suggested that increases in extracellular glutamate induced by ethanol exposure may be due in part to deficits in glutamate transport.10 Synaptic concentration of glutamate is partly regulated by glutamate aspartate transporter (GLAST). As shown in a recent animal study, decreased expression levels of GLAST result in increased synaptic glutamate concentration and increased amount of alcohol intake.11 Glutamate receptors are primary targets of alcohol action and alcohol inhibits the postsynaptic N-methyl-D-aspartate (NMDA)–receptor complex,12 thereby modulating alcohol sensitivity,13 self-administration, relapse behavior,7,14 and withdrawal responses.15 Chronic alcohol exposure results in compensatory upregulation of NMDA-receptor subunits, mainly NR1, NR2A, and NR2B,16 and can result in a hyperexcitatory state in periods of acute and conditioned alcohol withdrawal.6,7 Pharmacological inhibition experiments show that metabotropic glutamate receptor 5 (mGluR5) modulates alcohol self-administration17,18 and relapse behavior in rodents19 and is a potential target for acamprosate as well.20 Intracellularly, activation of NMDA receptors initiates a calcium-mediated signal transduction cascade activating calmodulin-dependent kinase IV and the transcription factor cyclic adenosine monophosphate–responsive element-binding protein 1 (CREB),21 which are implicated in alcohol withdrawal22,23 and self-administration in alcohol-preferring rats.24 Glutamate-induced activation of CREB also occurs through a parallel pathway,25 whereby mGluR5 and NMDA-receptor signaling converges on phosphatidyl inositol 3 kinase.26 Phosphatidylinositol 3-kinase then activates neuronal nitric oxide synthase and guanosine monophosphate–kinase II, all of which have been implicated in regulation of alcohol sensitivity and self-administration in knockout models.2730

This study is intended to address to what extent genetic variations in the glutamatergic neurotransmission genes that are shown to be correlated with alcohol drinking and relapse behavior in animal models contribute to the genetic basis of alcohol dependence in humans. In a broad resequencing analysis of each gene, we identified polymorphisms in regulatory domains, exons, and exon-intron boundaries, which constitute most functional glutamatergic variations. In a second step, all allelic information was contracted into multimarker-tagging single-nucleotide polymorphisms (SNPs), which concentrate the available functional allelic information into few predictive allelic markers.31 These haplotype-tagging SNPs (htSNPs) were then used to perform an association study with independent replication. The first sample consisted of 544 patients with alcohol dependence and 553 controls and was used to genotype the entire set of htSNPs. Genes associated with alcohol dependence were analyzed in a second sample of 793 alcohol-dependent patients and 1002 controls (Table 1) and were further assessed for their role in early risky drinking behavior in a sample of 144 trios of 15-year-old adolescents and their parents.

Table Graphic Jump LocationTable 1. Alcohol-Dependent Adult Patients and Controls and Adolescent Trio Sample
PARTICIPANTS AND PSYCHIATRIC ASSESSMENT
Alcohol-Dependent Adult Patients and Controls

For study 1, 544 patients were recruited from the psychiatry and addiction medicine departments at the university hospitals in Munich, Mainz, and Mannheim, Germany. Table 1 presents the characteristics of our sample. All patients were consecutively admitted for inpatient addiction treatment and fulfilled DSM-IV criteria for alcohol dependence. All participants were of German descent. In Munich and Mannheim, patients were assessed using the Semi-Structured Assessment for the Genetics of Alcoholism32 and the Structured Clinical Interview for DSM-III-R, respectively. In Mainz, the Composite International Diagnostic Interview33 and the Michigan Alcoholism Screening Test were used. Interviews were performed by trained staff members and rated independently. Genetically enriched phenotypes across study 1 were early onset, indicating onset of alcohol dependence before age 25 years. Positive family history for alcoholism included first- or second-degree relatives. One drink was defined as 17 g of alcohol.

Five hundred fifty-three controls were recruited in Munich, Mainz, and Mannheim (Table1). Munich controls were randomly selected from city registration. Controls from Mainz and Mannheim were from hospital personnel and board advertisement. A detailed medical and psychiatric history was performed and all Axis 1 psychiatric diagnoses were excluded.

For study 2, 793 patients were recruited from the Department of Psychiatry at Regensburg University (Table 1). All patients were admitted consecutively for inpatient treatment and met criteria of alcohol dependence according to DSM-IV. All participants were of German descent. Parents of the participants were living in Bavaria and participants themselves were born and raised in this area. Written informed consent was obtained from all participants before the investigation.

Diagnosis was assessed after alcohol withdrawal by the Composite International Diagnostic Interview33 performed by trained staff who rated participants independently. Patients with a lifetime history of schizophrenia or an addiction to drugs other than alcohol or tobacco were excluded. Genetically enriched phenotypes across sample 2 were defined similar to sample 1.

One thousand two individuals from Bonn were recruited from 2001 to 2003 within the German National Research Project to serve as controls for genetic studies in several neuropsychiatric phenotypes. Population-based recruitment was performed in collaboration with the local census bureau. Participants were screened for neurological and psychiatric disorders with self-report questionnaires adapted from the German version of the Inventory to Diagnose Depression34,35; smoking and drinking was screened with the Fagerström Tolerance Questionnaire and the Alcohol Use Disorders Identification Test, respectively.36,37 More than 96% of the participants were of German or Western European origin as ascertained by place of birth of their grandparents. For all patients and controls, written informed consent was obtained before study participation.

Adolescent Trio Sample

One hundred forty-two complete trios and 2 incomplete trios were recruited from the Mannheim Study of Risk Children, a cohort of first-born children (Table 1).38 Alcohol consumption in the last 6 months before assessment was evaluated at age 15 years using the Lifetime Drinking History interview.39 Lifetime prevalence of being drunk was defined as the participant never having been drunk vs having been drunk. High/low maximum amount of alcohol intake on 1 occasion was established using a median split at 2 or more standard alcoholic drinks. All participants were of Central European descent. Written informed consent was obtained from all individuals when they were in a state of full legal capacity. The study was approved by the ethics committees of the Landesärztekammer Rheinland-Pfalz and the universities of Regensburg, Heidelberg, and Munich.

MUTATION SCREENING AND IDENTIFICATION OF htSNPs

Identification of SNPs was performed by sequencing 32 DNA samples from white individuals. Sixteen DNA pools consisting of an equimolar mixture of 2 DNA samples were prepared and used as polymerase chain reaction templates. For each gene, primers were chosen to amplify the regulatory domains and the exon-containing DNA fragments, including exon-intron boundaries. Polymerase chain reaction was performed in a 15-μL reaction mixture containing 25 ng of DNA (the list of the primers for each gene is available at http://www.cng.fr). Sequencing reactions were performed using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, California). Alignment of experimental results and identification of SNPs were performed using the Genalys software developed by the Centre National de Génotypage. Haplotype-tagging SNPs were selected to discriminate haplotypes with a frequency of 5% or greater.

GENOTYPE ANALYSIS

Participants' DNA was prepared from whole blood with standard salting out methods. Single nucleotide polymorphisms were genotyped using the TaqMan system at the Centre National de Génotypage, University of Kiel Institute of Psychiatry. Probes and primers were from the Assay-by-Design system (Applied Biosystems). Polymerase chain reactions were performed in Biometra T1 thermocyclers (Biometra, Goettingen, Germany), and fluorescence results were determined by using an ABI Prism 7900HT sequence detector end-point read (Applied Biosystems). Process and genotyping data were exported into an internal laboratory information management system. In study 1, complete genotypes were obtained from 823 individuals, including 410 patients and 413 controls, which were included in the statistical analysis. In the follow-up study, 1795 individuals, including 793 patients and 1002 controls, were genotyped using 2 independent genotyping techniques, our in-house TaqMan and commercial genotyping (PreventionGenetics, Marshfield, Wisconsin) with 100% concordance.

STATISTICAL ANALYSIS

In study 1, multivariate statistical analysis for genotype × phenotype association was performed using a logistic regression approach. All htSNPs were in Hardy-Weinberg equilibrium (data not shown).40 Alternative haplotype analyses were done with COCAPHASE 2.404.41 In study 2, multilocus genotypes identified in study 1 were analyzed for replication. Trio analyses were performed using the transmission disequilibrium test. Transmission disequilibrium test was performed using tdtphase, version 2.404, which is part of the UNPHASED software package.42

GENEWISE LOGISTIC REGRESSION ANALYSIS

For logistic regression analysis, 4 htSNPs with a high correlation with other htSNPs (r > 0.7) were eliminated. The remaining htSNPs were coded into 10 categorical variables representing the 10 genes. Dummy variables were created, each representing 1 multilocus genotype within 1 gene. Rare genotypes (< 5% of the sample number) were grouped together. The most frequent genotype was taken as the reference group. These dummy variables were included as predictors in the regression model. Testing the relevance of these genes for prediction of alcohol dependence was performed by testing the null hypothesis that none of the analyzed genotypes within each gene differ from the reference group. The genes were ranked based on obtained P values (Table 2). Using regression coefficients, multilocus genotypes were identified for NR2A (OMIM *138253) and MGLUR5 (OMIM *604102), which predict a particular high (or low) risk for alcohol dependence compared with the reference group (Table 3). Taking sex into account led to the similar results for relevance of genes as well as for detecting risk groups within genes (data not shown). These extreme groups were compared to create an effect measure for the amount of the gene's influence on status and phenotypes.

Table Graphic Jump LocationTable 2. Genewise Multiple Logistic Regression of Alcohol Dependencea
Table Graphic Jump LocationTable 3. Genewise Identification of Risk vs Protective Multilocus Genotypesa

The extreme group analysis was carried out for the replication study. The Fisher exact test (2-sided) was used to evaluate the association between extreme groups for alcohol dependence. Evidence of replication, rather than multiple testing corrections, was used to evaluate the significance of associated genes. To comprehensively assess the reproducible results, we conducted the Cochran-Mantel-Haenszel test43 on the combined data set, which includes samples from original and replication case-control studies. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) and P values were obtained from Cochran-Mantel-Haenszel statistics (Figure 1).

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Figure 1.

Statistical modeling overview.

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IDENTIFICATION AND SELECTION OF htSNPS AND POWER CALCULATION

Based on behavioral animal experimentation and pharmacological data (Table 4), we selected the genes GLAST; NMDA-receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB (Figure 2). Identification of variations in the genes selected was performed by sequencing of regulatory domains, exons, and exon-intron boundaries (see “Methods” section) (Table 5). Two hundred four genetic variations were identified (http://www.cng.fr). Fifty-four polymorphisms had no reference SNP identifier at the time of discovery. To date, 4 additional SNPs have been given reference SNP identifiers (Table 5). Previously unknown polymorphisms added information to the haplotypes (frequency ≥ 5%) for the genes CAMK4, PIK3R1, PRKG2, and SLC1A3. Except for SNP SLC1A3-95103, which was genotyped in the present study, all other polymorphisms that contribute to haplotypes with a frequency of 5% or greater were tagged by multimarker combinations of other known variants (Table 5). The haplotypic structure (frequency ≥ 5%) of the analyzed genes was not diversified by the newly discovered variants (except in SLC1A3).

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Figure 2.

Glutamatergic neurotransmission (candidate genes selected for analysis are indicated by a bold frame). MAP, mitogen-activated protein; PSD-95, postsynaptic density-95.

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Table Graphic Jump LocationTable 4. Selection of Genes and htSNP Identification
Table Graphic Jump LocationTable 5. List of Polymorphisms Discovered by Resequencing

As a first step toward the identification of htSNPs, SNPs with a minor allele frequency of less than 5% were discarded. If groups of SNPs existed in which scored genotypes were identical for all samples, then a single SNP from the group was selected. The selected SNP was that with the least missing data or simply the first in the event of a tie. Haplotypes for the remaining SNPs were then estimated using the expectation-maximization algorithm, and a subset of htSNPs was selected so that all haplotypes with a frequency of 5% or greater could be distinguished. This process resulted in selection of 21 htSNPs (Table 4), which were genotyped in study 1. Genes shown to be associated with alcohol dependence were analyzed for replication in a second, independent sample. Disease severity, as defined by the mean of the number of DSM-IV symptoms in sample 1, was 5.74 compared with sample 2 with a mean of 6.07 (analysis of variance, F = 18.184; P < .001) (Table 6). Power calculation of the combined data set comprising 1555 controls and 1337 cases in an additive disease model revealed 98% power to detect a genotype relative risk of 1.25.

Table Graphic Jump LocationTable 6. Comparison of Severity of Alcohol Dependence in Study 1 vs Study 2a
IDENTIFICATION OF RISK GENOTYPES FOR ALCOHOL DEPENDENCE

Using a genewise, multiple regression approach, variations in 2 genes, NR2A and MGLUR5, were shown to be the most powerful predictors of alcohol dependence (Table 2). Detailed haplotype frequencies and haplotypic structures for GRIN2A and GRM5 are presented in Table 7. In these 2 genes, newly discovered variants added no additional information to haplotypes with a frequency of 5% or greater. Each of these genes was characterized by 2 htSNPs (the remaining 2 htSNPs of NR2A were eliminated because they were correlated [r > 0.7] to the htSNPs analyzed). In the case of NR2A, these variations were a C-to-A substitution in rs2072450 (intron 11) and a deletion/insertion in rs9924016 (intron 7). In the case of MGLUR5, these variations were the C-to-A exchange in rs3824927 (3′ flanking sequence) and the G-to-A exchange in rs3462 (exon 8) (Figure 3 and Figure 4). To assess the effect size of the genetic variations on alcohol dependence, protective and risk genotypes were identified based on their regression coefficients (Table 3). This analysis showed that carriers of the NR2A risk genotypes rs2072450 CC and rs9924016 Del/Del had an OR of 2.35 (95% CI, 1.37-4.03) of developing alcohol dependence compared with carriers of the protective genotypes rs2072450 AC and rs9924016 Del/Ins (Fisher exact test, P = .002) (Table 3). In the case of MGLUR5, carriers of the risk genotypes rs3824927 C/C and rs3462 G/G had an OR of 1.69 (95% CI, 1.06-2.69) of developing alcohol dependence compared with carriers of the protective genotypes rs3824927 CA and rs3462 GA (P = .03) (Table 8). In our samples, the protective genotype was heterozygous for all NR2A and MGLUR5 polymorphisms because no individuals were observed who carried a homozygous protective genotype. Haplotype analysis of NR2A and MGLUR5 genotypes did not yield an increase in OR compared with results of the regression analysis (data not shown). Correlation analysis revealed no evidence for confounding association between NR2A and MGLUR5 markers and age or sex (data not shown).

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Figure 3.

Schematic representation of the genomic organization of MGLUR5. Tagging single-nucleotide polymorphisms (SNPs) are indicated by bold frames. Introns are indicated by horizontal lines connecting the boxes. Localizations of the polymorphisms (arrows, not drawn to scale), nomenclature of the SNPs, nucleotide alleles, and base frequencies. Nomenclature according the Single-Nucleotide Polymorphism Database (reference SNP identification numbers). * Indicates SNP discovery numbers of the Centre National de Génotypage.

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Figure 4.

Schematic representation of the genomic organization of NR2A (exons 8 and 9 are fused into 1 rectangle). Tagging single-nucleotide polymorphisms (SNPs) are indicated by bold frames. Introns are indicated by horizontal lines connecting the boxes. Localizations of the polymorphisms (arrows, not drawn to scale), nomenclature of the SNPs, nucleotide alleles, and base frequencies. Nomenclature according to the Single-Nucleotide Polymorphism Database (reference SNP identification numbers). * Indicates SNP discovery numbers of the Centre National de Génotypage.

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Table Graphic Jump LocationTable 7. Haplotypes of GRIN2A and GRM5a
Table Graphic Jump LocationTable 8. Identification and Analysis of Extreme Genotypes for Alcohol Dependence in Study 1 and Study 2

In the replication, we performed a confirmatory independent analysis of the combined NR2A and MGLUR5 genotypes in a sample of 793 alcohol-dependent patients and 1002 controls. We found that carriers of the NR2A risk genotypes rs2072450 CC and rs9924016 Del/Del had an OR of 2.01 (95% CI, 1.15-3.50) of developing alcohol dependence compared with carriers of the protective genotypes rs2072450 AC and rs9924016 Del/Ins (P = .02) (Table 8). However, analysis of the MGLUR5 risk genotypes did not yield significant results (P = .62) (Table 8).

For joint analysis of studies 1 and 2, the contingency tables (2 × 2) with risk and protective genotypes were combined for NR2A and MGLUR5. This analysis revealed that for alcohol dependence, the NR2A risk genotypes rs2072450 CC and rs9924016 Del/Del had a pooled OR of 2.18 (95% CI, 1.48-3.21) compared with the protective genotypes rs2072450 AC and rs9924016 Del/Ins (P < .001) (Table 8). No evidence was found of an association of alcohol dependence with the MGLUR5 risk genotype vs protective genotype (P = .38) (Table 8). Using the Cochran-Armitage trend test, we obtained similar results for NR2A and MGLUR5 and their protective vs intermediate vs risk genotypes (data not shown). Correlation analysis revealed no evidence for a confounding association between the NR2A markers and age or sex (data not shown).

ASSOCIATION ANALYSIS OF NR2A GENOTYPES IN PHENOTYPES CARRYING A HIGH GENETIC LOAD

Patients with a positive family history and those with early onset of the disorder are thought to carry a high genetic load for alcohol dependence.3 Amount of alcohol intake measured as maximum number of drinks has been shown to be genetically influenced.46 We selected these phenotypes for further exploratory analysis of NR2A risk genotypes. We analyzed the association of these phenotypes and severity of the dependence as defined by the number of DSM-IV symptoms and found an association with amount of drinking in both samples (study 1: Spearman correlation coefficient, 0.167; P = .003; study 2: correlation coefficient, 0.255; P < .001) and positive family history in sample 2 (F = 5.789, P = .02, analysis of variance) but not in other phenotypes (data not shown). Positive family history was associated with NR2A risk vs protective genotype in study 1 (P = .001) and study 2 (OR, 2.37; 95% CI, 1.29-4.36; P = .007), with joint analysis of both samples revealing an OR of 2.68 (95% CI, 1.58-4.54; P < .001) (Table 9). Early onset of alcohol dependence was associated with NR2A risk vs protective genotypes in study 1 (OR, 3.54; 95% CI, 1.22-10.3; P = .01) and study 2 (OR, 2.67; 95% CI, 1.42-5.02; P = .002), with joint analysis revealing an OR of 3.69 (95% CI, 2.0-6.81; P < .001) (Table 9). While no association of maximum number of drinks with NR2A risk vs protective genotypes was observed in study 1 (P = .35), study 2 showed an association (OR, 3.21; 95% CI, 1.52-6.76; P = .002), with joint analysis revealing an OR of 2.70 (95% CI, 1.45-5.03; P = .001) (Table 9). The joint Cochran-Armitage trend test produced similar results for association between NR2A genotypes and the phenotypes in question (data not shown).

Table Graphic Jump LocationTable 9. Association Analysis and Replication of NR2A With Genetically Enriched Phenotypes of Alcohol Dependence
RISKY DRINKING BEHAVIOR IN ADOLESCENTS

Since we found an association between NR2A genotypes and positive family history, early onset of alcohol dependence, and maximum number of drinks, we were interested to know if the genetic variations identified in alcohol-dependent adult patients may constitute a risk factor for risky drinking in adolescents. We used a transmission disequilibrium test in a sample of 144 trios composed of 15-year-old adolescents and their parents (see the “Methods” section). Our results show an overtransmission of the C allele of the rs2072450 polymorphism in NR2A in both lifetime prevalence of drunkenness (P = .04) and maximum amount of alcohol intake/occasion, a measure for heavy drinking (P = .02) (Table 10).

Table Graphic Jump LocationTable 10. Analysis of the Role of NR2A in Initiation and Risky Drinking in Adolescent Trios

We show that genetic variations in NR2A have the highest relevance for human alcohol dependence among the glutamatergic neurosignaling genes selected for their known alteration of alcohol effects in animal models. Our analysis is based on a broad resequencing approach of 10 genes, which led to the identification of 204 SNPs, 50 of which are not listed in the National Center for Biotechnology Information (NCBI) Reference Sequence database. Haplotype analysis revealed that the most frequent haplotypes (> 5%) could be discriminated by 21 htSNPs, which serve to decrease redundancy of SNP information and represent the most parsimonious set to describe the sum of genetic variations of functionally relevant domains of our candidate genes. Because the DSM-IV diagnosis of alcohol dependence contains a number of genetically heterogenous phenotypes resulting in limited effect sizes of each individual gene, we were interested in maximizing sensitivity of our analysis by using a statistical model comparing combined risk with protective genotypes. To minimize the danger of false-positive associations, we performed an independent replication of positive results in a combined sample, which carried 98% power to detect even small genetic effects (genotype risk ratio, 1.25).

Our approach establishes the relevance of NR2A for alcohol dependence, as indicated by significant associations and comparable ORs of risk vs a protective genotype of 2.35 and 1.83 in the first and second samples, respectively, which result in a joint OR of 2.18. Analysis of phenotypes enriched for genetic loading increased the OR in the case of positive family history to 2.68, in the case of early onset to 3.69, and in the case of maximum number of drinks to 2.70. Our finding of an association of NR2A genotypes with phenotypes of risky drinking, including prevalence of drunkenness and a measure for heavy drinking patterns in 144 adolescent trios, suggests a role for NR2A in early stages of alcohol use and indicates its relevance for developmental trajectories of alcohol use disorders. A number of prospective and retrospective studies have shown that early-adolescent alcohol use is significantly associated with alcohol-related problems and disorders in adulthood.47,48

Genetic variants in public databases are not an exhaustive collection of existing human variation. Resequencing of regions with particular biological importance can complete this collection. However, despite the fact that resequencing the major functional portions of the glutamatergic genes identified 50 SNPs that are not listed in the NCBI Reference Sequence database, this exercise contributed only limited information compared with the frequent haplotypic structures revealed solely by NCBI Reference SNP polymorphisms.

Alcohol has been shown to regulate expression of NR2A in brain regions relevant for addiction-related neurobiological processes, including amygdala and hippocampus.14,49,50 The NR2A subunits are among the NMDA-receptor subunits that are most sensitive to the inhibitory effects of ethanol.51 A recent study in healthy individuals with a familial vulnerability to alcoholism showed an attenuated response to ketamine, which acts partially as an NR2A antagonist52 to perceptual alterations and dysphoric mood in those individuals with a positive family history of alcohol dependence, suggesting a contribution of NR2A to subjective responses to alcohol.53 Animal experimentation indicates 2 behavioral mechanisms through which NR2A may contribute to alcohol-drinking behavior and alcohol dependence; Nr2a knockout mice failed to show evidence for conditioned place preference, suggesting an impairment in learned reward-related responses to ethanol.49 An alternative hypothesis rests on the finding that Nr2a knockout mice exhibit decreased anxiety-like behavior across multiple tests,50 suggesting stress-induced gene × environment interactions resulting in increased alcohol drinking. While our samples were not suited for confirmatory gene × environment analyses, exploratory findings in the adolescent trios suggest that association of an NR2A genotype with amount of drinking is only present in those adolescents with high psychosocial stress but not in those with low psychosocial stress (data not shown). This indication clearly requires formal analysis in a larger sample set.

While this study is the first to find evidence of an association of NR2A with alcohol dependence and alcohol-drinking behavior in humans, previous studies have investigated the role of additional NMDA-receptor subtypes and observed an association of genetic variations of NR1 with alcohol dependence and NR2B with Cloninger type 2 alcoholism.54 This finding could be replicated in part in a study that observed an association of NR1 only in patients with a history of withdrawal-induced seizures.55 Given the hypothesis of glutamatergic hyperexcitability in alcohol withdrawal, the observed discrepancy may be a result of genetic heterogeneity.56 In this example, selection of withdrawal-related phenotypes may have enriched for a neurobiological mechanism that is particularly sensitive to alterations in glutamatergic function and thus identified an association, which in this sample was not detectable on analysis of the more heterogenous phenotype of alcohol dependence.

European studies—including our current work—did not find additional evidence of an association between alcohol dependence and NR2B in rs1806201.57,58 However, a recent study from South Korea reports an association of the same genetic variation with alcohol dependence.59 Allelic distributions of rs1806201 in the HapMap database are 0.22 in Europeans and 0.54 in Asians, suggesting significant ethnic differences, which may contribute to the observed discrepant results.

This phenomenon may also have contributed to conflicting results of a genetic variation of the protein tyrosine kinase FYN gene, which was found to be associated with alcohol dependence in a German60 but not a Japanese61 sample. Further analysis of this gene is relevant, as Fyn knockout mice show increased alcohol sensitivity and lack of tolerance to the effects of ethanol62 owing to a reduction of fyn-dependent phosphorylation of NR2A.63

While in the first step of our analysis, both NR2A and MGLUR5 were shown to be associated with alcohol dependence; our replication analysis did not support an association of MGLUR5 with alcohol dependence. This may have been a consequence of a limitation of the study: controls in study 2 were selected on the basis of a questionnaire as opposed to controls in study 1, who were selected on the basis of a diagnostic interview, which may have led to the erroneous inclusion of alcohol-dependent participants in the control sample and resulted in decreased power in the second study to detect differences between cases and controls. An absence of association between MGLUR5 and alcohol dependence stands in contrast to results from animal studies, which clearly identify a role of mGluR5 in alcohol-related behavior. In pharmacological studies using the mGluR5 antagonist 2-methyl-6-phenylethynylpyridine, the relevance of this gene for alcohol self-administration and relapse behavior in rats17,19 and mice18 has been established. This discrepancy may suggest that the role of mGluR5 is limited to specific (intermediate) phenotypes, which the current study was unable to identify, or it may indicate an involvement of a gene farther downstream in the mGluR5-signaling cascade, which so far has not been implicated in the behavioral effects observed in animal studies.

Interestingly, we observed only an association with alcohol dependence in genes encoding transmembrane proteins but not in intracellular signaling genes. This is somewhat unexpected, as various animal models have shown pronounced effects on alcohol-drinking behavior mediated by genes encoding intracellular proteins, such as neuronal nitric oxide synthase27 and cyclic guanosine monophosphate–kinase II.28 While this may be in part explained by a greater complexity of the diagnosis of alcohol dependence compared with behavioral animal paradigms of alcohol-drinking behavior, it may also indicate a lack of specificity of knockout models, whereby deletion of a single gene may lead to inactivation of an entire signaling cascade.

The limited analogy of knockout models and analysis of human genetic variations clearly constitutes one of the limitations of the translational claim of studies such as the one presented. Other limitations of our study result from the parsimonious selection of genetic variations. While designed to cover the most frequent haplotypes selected for functionally relevant genetic variations, this analysis does not consider potentially functional SNPs in intronic enhancer sequences nor does it take into account a variable number of tandem repeats in the genes analyzed.64 Based on the robust results generated from our large data set, further genotype-specific molecular characterizations of NR2A may result in its application for pharmacogenetic studies of response to the anticraving drug acamprosate as well as providing a potential target for pharmacological interventions in human alcohol dependence.

Correspondence: Gunter Schumann, MD, Interdisciplinary Research Group Addiction, MRC–SGDP Center, Institute of Psychiatry at King's College, POB 080, London SE5 8AF, England (g.schumann@iop.kcl.ac.uk).

Submitted for Publication: October 30, 2007; final revision received February 21, 2008; accepted March 17, 2008.

Financial Disclosure: None reported.

Funding/Support: This study was supported in part by grant PL037286 from the European Commission FP-6 Integrated Project IMAGEN (Dr Schumann), by the United Kingdom Department of Health National Institute for Health Research–Biomedical Research Centre Mental Health (Dr Schumann), and by grants FKZ 01GS0117/NGFN and FKZ EB 01011300 from the Bundesministerium für Bildung und Forschung (Drs Schumann, Spanagel, and Mann).

Additional Contributions: We thank Marina Füg and Christine Hohmeyer for their expert technical assistance. We also thank Ivo Gut, PhD, Centre National de Génotypage, Evry, France, for his helpful discussions.

World Health Organization, The World Health Report 2002: Reducing Risks, Promoting Healthy Life.  Geneva, Switzerland World Health Organization2002;
Reich  THinrichs  ACulverhouse  RBierut  L Genetic studies of alcoholism and substance dependence. Am J Hum Genet 1999;65 (3) 599- 605
PubMed
Gorwood  PSchumann  GTreutlein  JAdes  J Pharmacogenetics of alcohol dependence. Gorwood  PHamon  MPsychopharmacogenetics. Heidelberg, Germany Springer2006;177- 202
Schumann  GSpanagel  RMann  K Candidate genes for alcohol dependence: animal studies. Alcohol Clin Exp Res 2003;27 (5) 880- 888
PubMed
Sanchis-Segura  CSpanagel  R Behavioural assessment of drug reinforcement and addictive features in rodents: an overview. Addict Biol 2006;11 (1) 2- 38
PubMed
Tsai  GCoyle  JT The role of glutamatergic neurotransmission in the pathophysiology of alcoholism. Annu Rev Med 1998;49173- 184
PubMed
Gass  JTOlive  MF Glutamatergic substrates of drug addiction and alcoholism [published online ahead of print June 30, 2007]. Biochem Pharmacol 2008;75 (1) 218- 265
PubMed10.1016/j.bcp.2007.06.039
Spanagel  RKiefer  F Drugs for relapse prevention of alcoholism: ten years of progress [published online ahead of print February 11, 2008]. Trends Phramcol Sci 2008;29 (3) 109- 115
PubMed10.1016/j.tips.2007.12.005
Vengeliene  VBilbao  AMolander  ASpanagel  R Neuropharmacology of alcohol addiction [published online ahead of print March 3, 2008]. Brit J Pharmacol
PubMed10.1038/bjp.2008.30
Melendez  RIHicks  MPCagle  SSKalivas  PW Ethanol exposure decreases glutamate uptake in the nucleus accumbens. Alcohol Clin Exp Res 2005;29 (3) 326- 333
PubMed
Spanagel  RPendyala  GAbarca  CZghoul  TSanchis-Segura  CMagnone  MCLascorz  JDepner  MHolzberg  DSoyka  MSchreiber  SMatsuda  FLathrop  MSchumann  GAlbrecht  U The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med 2005;11 (1) 35- 42
PubMed
Lovinger  DMWhite  GWeight  FF Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 1989;243 (4899) 1721- 1724
PubMed
Gordey  MMekmanee  LMody  I Altered effects of ethanol in NR2A (DeltaC/DeltaC) mice expressing C-terminally truncated NR2A subunit of NMDA receptor. Neuroscience 2001;105 (4) 987- 997
PubMed
Vengeliene  VBachteler  DDanysz  WSpanagel  R The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect. Neuropharmacology 2005;48 (6) 822- 829
PubMed
Narita  MSoma  MMizoguchi  HTseng  LFSuzuki  T Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence. Eur J Pharmacol 2000;401 (2) 191- 195
PubMed
Gulya  KGrant  KAValverius  PHoffman  PLTabakoff  B Brain regional specificity and time-course of changes in the NMDA receptor-ionophore complex during ethanol withdrawal. Brain Res 1991;547 (1) 129- 134
PubMed
Cowen  MSDjouma  ELawrence  AJ The metabotropic glutamate 5 receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine reduces ethanol self-administration in multiple strains of alcohol-preferring rats and regulates olfactory glutamatergic systems. J Pharmacol Exp Ther 2005;315 (2) 590- 600
PubMed
Hodge  CWMiles  MFSharko  ACStevenson  RAHillmann  JRLepoutre  VBesheer  JSchroeder  JP The mGluR5 antagonist MPEP selectively inhibits the onset and maintenance of ethanol self-administration in C57BL/6J mice. Psychopharmacology (Berl) 2006;183 (4) 429- 438
PubMed
Backstrom  PBachteler  DKoch  SHyytia  PSpanagel  R mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior. Neuropsychopharmacology 2004;29 (5) 921- 928
PubMed
Harris  BRPrendergast  MAGibson  DARogers  DTBlanchard  JAHolley  RCFu  MCHart  SRPedigo  NWLittleton  JM Acamprosate inhibits the binding and neurotoxic effects of trans-ACPD, suggesting a novel site of action at metabotropic glutamate receptors. Alcohol Clin Exp Res 2002;26 (12) 1779- 1793
PubMed
Ron  D Signaling cascades regulating NMDA receptor sensitivity to ethanol. Neuroscientist 2004;10 (4) 325- 336
PubMed
Pandey  SCRoy  AMittal  N Effects of chronic ethanol intake and its withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat cortex. J Pharmacol Exp Ther 2001;296 (3) 857- 868
PubMed
Pandey  SCRoy  AZhang  H The decreased phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in the central amygdala acts as a molecular substrate for anxiety related to ethanol withdrawal in rats. Alcohol Clin Exp Res 2003;27 (3) 396- 409
PubMed
Pandey  SCZhang  HRoy  AXu  T Deficits in amygdaloid cAMP-responsive element-binding protein signaling play a role in genetic predisposition to anxiety and alcoholism. J Clin Invest 2005;115 (10) 2762- 2773
PubMed
Paul  IASkolnick  P Glutamate and depression: clinical and preclinical studies. Ann N Y Acad Sci 2003;1003250- 272
PubMed
Daw  MIBortolotto  AZSaulle  EZaman  SCollingridge  GLIsaac  JT Phosphatidylinositol 3 kinase regulates synapse specificity of hippocampal long-term depression. Nat Neurosci 2002;5 (9) 835- 836
PubMed
Spanagel  RSiegmund  SCowen  MSchroff  KSchumann  GFiserova  MSillaber  IHolzinger  JWellek  SSinger  MPutke  J The neuronal nitric oxide synthase gene is critically involved in neurobehavioral effects of alcohol. J Neurosci 2002;22 (19) 8676- 8683
PubMed
Werner  CRaivich  GCowen  MStrekalova  TSillaber  IButers  JTSpanagel  RHofmann  F Importance of NO/cGMP signalling via cGMP-dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol. Eur J Neurosci 2004;20 (12) 3498- 3506
PubMed
Corl  ABRodan  ARHeberlein  U Insulin signaling in the nervous system regulates ethanol intoxication in Drosophila melanogaster. Nat Neurosci 2005;8 (1) 18- 19
PubMed
Crabbe  JCPhillips  TJHarris  RAArends  MAKoob  GF Alcohol-related genes: contributions from studies with genetically engineered mice. Addict Biol 2006;11 (3-4) 195- 269
PubMed
Crawford  DCNickerson  DA Definition and clinical importance of haplotypes. Annu Rev Med 2005;56303- 320
PubMed
Bucholz  KKCadoret  RCloninger  CRDinwiddie  SHHesselbrock  VMNurnberger  JI  JrReich  TSchmidt  ISchuckit  MA A new, semi-structured psychiatric interview for use in genetic linkage studies: a report on the reliability of the SSAGA. J Stud Alcohol 1994;55 (2) 149- 158
PubMed
Wittchen  HU Reliability and validity studies of the WHO–Composite International Diagnostic Interview (CIDI): a critical review. J Psychiatr Res 1994;28 (1) 57- 84
PubMed
Kühner  C Fragebogen zur Depressionsdiagnostik nach DSM-IV (FDD-DSM-IV).  Göttingen, Germany Hogrefe1997;
Zimmerman  MCoryell  WCorenthal  CWilson  S A self-report scale to diagnose major depressive disorder. Arch Gen Psychiatry 1986;43 (11) 1076- 1081
PubMed
Fagerström  K-O Measuring degree of physical dependence to tobacco smoking with reference to individualization of treatment. Addict Behav 1978;3 (3-4) 235- 241
PubMed
Babor  TFHiggins-Biddle  JCSaunders  JBMonteiro  MG The Alcohol Use Disorders Identification Test: Guidelines for Use in Primary Care. 2nd ed. Geneva, Switzerland World Health Organization2001;
Laucht  MEsser  GBaving  LGerhold  MHoesch  IIhle  WSteigleider  PStock  BStoehr  RMWeindrich  DSchmidt  MH Behavioral sequelae of perinatal insults and early family adversity at 8 years of age. J Am Acad Child Adolesc Psychiatry 2000;39 (10) 1229- 1237
PubMed
Skinner  HASheu  WJ Reliability of alcohol use indices: the Lifetime Drinking History and the MAST. J Stud Alcohol 1982;43 (11) 1157- 1170
PubMed
Balding  DJ A tutorial on statistical methods for population association studies. Nat Rev Genet 2006;7 (10) 781- 791
PubMed
Dudbridge  F Pedigree disequilibrium tests for multilocus haplotypes. Genet Epidemiol 2003;25 (2) 115- 121
PubMed
Dudbridge  F A survey of current software for linkage analysis. Hum Genomics 2003;1 (1) 63- 65
PubMed
Mantel  NHaenszel  W Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22 (4) 719- 748
PubMed
Ko  SWJia  YXu  HYim  SJJang  DHLee  YSZhao  MGToyoda  HWu  LJChatila  TKaang  BKZhuo  M Evidence for a role of CaMKIV in the development of opioid analgesic tolerance. Eur J Neurosci 2006;23 (8) 2158- 2168
PubMed
Izzo  EMartin-Fardon  RKoob  GFWeiss  FSanna  PP Neural plasticity and addiction: PI3-kinase and cocaine behavioral sensitization. Nat Neurosci 2002;5 (12) 1263- 1264
PubMed
Saccone  NLKwon  JMCorbett  JGoate  ARochberg  NEdenberg  HJForoud  TLi  TKBegleiter  HReich  TRice  JP A genome screen of maximum number of drinks as an alcoholism phenotype. Am J Med Genet 2000;96 (5) 632- 637
PubMed
Grant  BFDawson  DA Age at onset of alcohol use and its association with DSM-IV alcohol abuse and dependence: results from the National Longitudinal Alcohol Epidemiologic Survey. J Subst Abuse 1997;9103- 110
PubMed
Grant  BFStinson  FSHarford  TC Age at onset of alcohol use and DSM-IV alcohol abuse and dependence: a 12-year follow-up. J Subst Abuse 2001;13 (4) 493- 504
PubMed
Boyce-Rustay  JMHolmes  A Ethanol-related behaviors in mice lacking the NMDA receptor NR2A subunit. Psychopharmacology (Berl) 2006;187 (4) 455- 466
PubMed
Boyce-Rustay  JMHolmes  A Genetic inactivation of the NMDA receptor NR2A subunit has anxiolytic- and antidepressant-like effects in mice. Neuropsychopharmacology 2006;31 (11) 2405- 2414
PubMed
Mirshahi  TWoodward  JJ Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2+)-insensitive mutants. Neuropharmacology 1995;34 (3) 347- 355
PubMed
Petrenko  ABYamakura  TFujiwara  NAskalany  ARBaba  HSakimura  K Reduced sensitivity to ketamine and pentobarbital in mice lacking the N-methyl-D-aspartate receptor GluRepsilon1 subunit. Anesth Analg 2004;99 (4) 1136- 1140
PubMed
Petrakis  ILLimoncelli  DGueorguieva  RJatlow  PBoutros  NNTrevisan  LGelernter  JKrystal  JH Altered NMDA glutamate receptor antagonist response in individuals with a family vulnerability to alcoholism. Am J Psychiatry 2004;161 (10) 1776- 1782
PubMed
Wernicke  CSamochowiec  JSchmidt  LGWinterer  GSmolka  MKucharska-Mazur  JHorodnicki  JGallinat  JRommelspacher  H Polymorphisms in the N-methyl-D-aspartate receptor 1 and 2B subunits are associated with alcoholism-related traits. Biol Psychiatry 2003;54 (9) 922- 928
PubMed
Rujescu  DSoyka  MDahmen  NPreuss  UHartmann  AMGiegling  IKoller  GBondy  BMöller  HJSzegedi  A GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal. Am J Med Genet B Neuropsychiatr Genet 2005;133 (1) 85- 87
PubMed
Goldman  DOroszi  GDucci  F The genetics of addictions: uncovering the genes. Nat Rev Genet 2005;6 (7) 521- 532
PubMed
Schumann  GRujescu  DSzegedi  ASinger  PWiemann  SWellek  SGiegling  IKlawe  CAnghelescu  IHeinz  ASpanagel  RMann  KHenn  FADahmen  N No association of alcohol dependence with a NMDA-receptor 2B gene variant. Mol Psychiatry 2003;8 (1) 11- 12
PubMed
Tadic  ADahmen  NSzegedi  ARujescu  DGiegling  IKoller  GAnghelescu  IFehr  CKlawe  CPreuss  UWSander  TToliat  MRSinger  PBondy  BSoyka  M Polymorphisms in the NMDA subunit 2B are not associated with alcohol dependence and alcohol withdrawal-induced seizures and delirium tremens. Eur Arch Psychiatry Clin Neurosci 2005;255 (2) 129- 135
PubMed
Kim  JHPark  MYang  SYJeong  BSYoo  HJKim  JWChung  JHKim  SA Association study of polymorphisms in N-methyl-D-aspartate receptor 2B subunits (GRIN2B) gene with Korean alcoholism. Neurosci Res 2006;56 (2) 220- 223
PubMed
Schumann  GRujescu  DKissling  CSoyka  MDahmen  NPreuss  UWWieman  SDepner  MWellek  SLascorz  JBondy  BGiegling  IAnghelescu  ICowen  MSPoustka  ASpanagel  RMann  KHenn  FASzegedi  A Analysis of genetic variations of protein tyrosine kinase fyn and their association with alcohol dependence in two independent cohorts. Biol Psychiatry 2003;54 (12) 1422- 1426
PubMed
Ishiguro  HSaito  TShibuya  HToru  MArinami  T Mutation and association analysis of the Fyn kinase gene with alcoholism and schizophrenia. Am J Med Genet 2000;96 (6) 716- 720
PubMed
Miyakawa  TYagi  TKitazawa  HYasuda  MKawai  NTsuboi  KNiki  H Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function. Science 1997;278 (5338) 698- 701
PubMed
Tezuka  TUmemori  HAkiyama  TNakanishi  SYamamoto  T PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A. Proc Natl Acad Sci U S A 1999;96 (2) 435- 440
PubMed
Itokawa  MYamada  KYoshitsugu  KToyota  TSuga  TOhba  HWatanabe  AHattori  EShimizu  HKumakura  TEbihara  MMeerabux  JMToru  MYoshikawa  T A microsatellite repeat in the promoter of the N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene suppresses transcriptional activity and correlates with chronic outcome in schizophrenia. Pharmacogenetics 2003;13 (5) 271- 278
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Statistical modeling overview.

Graphic Jump Location
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Figure 2.

Glutamatergic neurotransmission (candidate genes selected for analysis are indicated by a bold frame). MAP, mitogen-activated protein; PSD-95, postsynaptic density-95.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Schematic representation of the genomic organization of MGLUR5. Tagging single-nucleotide polymorphisms (SNPs) are indicated by bold frames. Introns are indicated by horizontal lines connecting the boxes. Localizations of the polymorphisms (arrows, not drawn to scale), nomenclature of the SNPs, nucleotide alleles, and base frequencies. Nomenclature according the Single-Nucleotide Polymorphism Database (reference SNP identification numbers). * Indicates SNP discovery numbers of the Centre National de Génotypage.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Schematic representation of the genomic organization of NR2A (exons 8 and 9 are fused into 1 rectangle). Tagging single-nucleotide polymorphisms (SNPs) are indicated by bold frames. Introns are indicated by horizontal lines connecting the boxes. Localizations of the polymorphisms (arrows, not drawn to scale), nomenclature of the SNPs, nucleotide alleles, and base frequencies. Nomenclature according to the Single-Nucleotide Polymorphism Database (reference SNP identification numbers). * Indicates SNP discovery numbers of the Centre National de Génotypage.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Alcohol-Dependent Adult Patients and Controls and Adolescent Trio Sample
Table Graphic Jump LocationTable 2. Genewise Multiple Logistic Regression of Alcohol Dependencea
Table Graphic Jump LocationTable 3. Genewise Identification of Risk vs Protective Multilocus Genotypesa
Table Graphic Jump LocationTable 4. Selection of Genes and htSNP Identification
Table Graphic Jump LocationTable 5. List of Polymorphisms Discovered by Resequencing
Table Graphic Jump LocationTable 6. Comparison of Severity of Alcohol Dependence in Study 1 vs Study 2a
Table Graphic Jump LocationTable 7. Haplotypes of GRIN2A and GRM5a
Table Graphic Jump LocationTable 8. Identification and Analysis of Extreme Genotypes for Alcohol Dependence in Study 1 and Study 2
Table Graphic Jump LocationTable 9. Association Analysis and Replication of NR2A With Genetically Enriched Phenotypes of Alcohol Dependence
Table Graphic Jump LocationTable 10. Analysis of the Role of NR2A in Initiation and Risky Drinking in Adolescent Trios

References

World Health Organization, The World Health Report 2002: Reducing Risks, Promoting Healthy Life.  Geneva, Switzerland World Health Organization2002;
Reich  THinrichs  ACulverhouse  RBierut  L Genetic studies of alcoholism and substance dependence. Am J Hum Genet 1999;65 (3) 599- 605
PubMed
Gorwood  PSchumann  GTreutlein  JAdes  J Pharmacogenetics of alcohol dependence. Gorwood  PHamon  MPsychopharmacogenetics. Heidelberg, Germany Springer2006;177- 202
Schumann  GSpanagel  RMann  K Candidate genes for alcohol dependence: animal studies. Alcohol Clin Exp Res 2003;27 (5) 880- 888
PubMed
Sanchis-Segura  CSpanagel  R Behavioural assessment of drug reinforcement and addictive features in rodents: an overview. Addict Biol 2006;11 (1) 2- 38
PubMed
Tsai  GCoyle  JT The role of glutamatergic neurotransmission in the pathophysiology of alcoholism. Annu Rev Med 1998;49173- 184
PubMed
Gass  JTOlive  MF Glutamatergic substrates of drug addiction and alcoholism [published online ahead of print June 30, 2007]. Biochem Pharmacol 2008;75 (1) 218- 265
PubMed10.1016/j.bcp.2007.06.039
Spanagel  RKiefer  F Drugs for relapse prevention of alcoholism: ten years of progress [published online ahead of print February 11, 2008]. Trends Phramcol Sci 2008;29 (3) 109- 115
PubMed10.1016/j.tips.2007.12.005
Vengeliene  VBilbao  AMolander  ASpanagel  R Neuropharmacology of alcohol addiction [published online ahead of print March 3, 2008]. Brit J Pharmacol
PubMed10.1038/bjp.2008.30
Melendez  RIHicks  MPCagle  SSKalivas  PW Ethanol exposure decreases glutamate uptake in the nucleus accumbens. Alcohol Clin Exp Res 2005;29 (3) 326- 333
PubMed
Spanagel  RPendyala  GAbarca  CZghoul  TSanchis-Segura  CMagnone  MCLascorz  JDepner  MHolzberg  DSoyka  MSchreiber  SMatsuda  FLathrop  MSchumann  GAlbrecht  U The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med 2005;11 (1) 35- 42
PubMed
Lovinger  DMWhite  GWeight  FF Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 1989;243 (4899) 1721- 1724
PubMed
Gordey  MMekmanee  LMody  I Altered effects of ethanol in NR2A (DeltaC/DeltaC) mice expressing C-terminally truncated NR2A subunit of NMDA receptor. Neuroscience 2001;105 (4) 987- 997
PubMed
Vengeliene  VBachteler  DDanysz  WSpanagel  R The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect. Neuropharmacology 2005;48 (6) 822- 829
PubMed
Narita  MSoma  MMizoguchi  HTseng  LFSuzuki  T Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence. Eur J Pharmacol 2000;401 (2) 191- 195
PubMed
Gulya  KGrant  KAValverius  PHoffman  PLTabakoff  B Brain regional specificity and time-course of changes in the NMDA receptor-ionophore complex during ethanol withdrawal. Brain Res 1991;547 (1) 129- 134
PubMed
Cowen  MSDjouma  ELawrence  AJ The metabotropic glutamate 5 receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine reduces ethanol self-administration in multiple strains of alcohol-preferring rats and regulates olfactory glutamatergic systems. J Pharmacol Exp Ther 2005;315 (2) 590- 600
PubMed
Hodge  CWMiles  MFSharko  ACStevenson  RAHillmann  JRLepoutre  VBesheer  JSchroeder  JP The mGluR5 antagonist MPEP selectively inhibits the onset and maintenance of ethanol self-administration in C57BL/6J mice. Psychopharmacology (Berl) 2006;183 (4) 429- 438
PubMed
Backstrom  PBachteler  DKoch  SHyytia  PSpanagel  R mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior. Neuropsychopharmacology 2004;29 (5) 921- 928
PubMed
Harris  BRPrendergast  MAGibson  DARogers  DTBlanchard  JAHolley  RCFu  MCHart  SRPedigo  NWLittleton  JM Acamprosate inhibits the binding and neurotoxic effects of trans-ACPD, suggesting a novel site of action at metabotropic glutamate receptors. Alcohol Clin Exp Res 2002;26 (12) 1779- 1793
PubMed
Ron  D Signaling cascades regulating NMDA receptor sensitivity to ethanol. Neuroscientist 2004;10 (4) 325- 336
PubMed
Pandey  SCRoy  AMittal  N Effects of chronic ethanol intake and its withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat cortex. J Pharmacol Exp Ther 2001;296 (3) 857- 868
PubMed
Pandey  SCRoy  AZhang  H The decreased phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein in the central amygdala acts as a molecular substrate for anxiety related to ethanol withdrawal in rats. Alcohol Clin Exp Res 2003;27 (3) 396- 409
PubMed
Pandey  SCZhang  HRoy  AXu  T Deficits in amygdaloid cAMP-responsive element-binding protein signaling play a role in genetic predisposition to anxiety and alcoholism. J Clin Invest 2005;115 (10) 2762- 2773
PubMed
Paul  IASkolnick  P Glutamate and depression: clinical and preclinical studies. Ann N Y Acad Sci 2003;1003250- 272
PubMed
Daw  MIBortolotto  AZSaulle  EZaman  SCollingridge  GLIsaac  JT Phosphatidylinositol 3 kinase regulates synapse specificity of hippocampal long-term depression. Nat Neurosci 2002;5 (9) 835- 836
PubMed
Spanagel  RSiegmund  SCowen  MSchroff  KSchumann  GFiserova  MSillaber  IHolzinger  JWellek  SSinger  MPutke  J The neuronal nitric oxide synthase gene is critically involved in neurobehavioral effects of alcohol. J Neurosci 2002;22 (19) 8676- 8683
PubMed
Werner  CRaivich  GCowen  MStrekalova  TSillaber  IButers  JTSpanagel  RHofmann  F Importance of NO/cGMP signalling via cGMP-dependent protein kinase II for controlling emotionality and neurobehavioural effects of alcohol. Eur J Neurosci 2004;20 (12) 3498- 3506
PubMed
Corl  ABRodan  ARHeberlein  U Insulin signaling in the nervous system regulates ethanol intoxication in Drosophila melanogaster. Nat Neurosci 2005;8 (1) 18- 19
PubMed
Crabbe  JCPhillips  TJHarris  RAArends  MAKoob  GF Alcohol-related genes: contributions from studies with genetically engineered mice. Addict Biol 2006;11 (3-4) 195- 269
PubMed
Crawford  DCNickerson  DA Definition and clinical importance of haplotypes. Annu Rev Med 2005;56303- 320
PubMed
Bucholz  KKCadoret  RCloninger  CRDinwiddie  SHHesselbrock  VMNurnberger  JI  JrReich  TSchmidt  ISchuckit  MA A new, semi-structured psychiatric interview for use in genetic linkage studies: a report on the reliability of the SSAGA. J Stud Alcohol 1994;55 (2) 149- 158
PubMed
Wittchen  HU Reliability and validity studies of the WHO–Composite International Diagnostic Interview (CIDI): a critical review. J Psychiatr Res 1994;28 (1) 57- 84
PubMed
Kühner  C Fragebogen zur Depressionsdiagnostik nach DSM-IV (FDD-DSM-IV).  Göttingen, Germany Hogrefe1997;
Zimmerman  MCoryell  WCorenthal  CWilson  S A self-report scale to diagnose major depressive disorder. Arch Gen Psychiatry 1986;43 (11) 1076- 1081
PubMed
Fagerström  K-O Measuring degree of physical dependence to tobacco smoking with reference to individualization of treatment. Addict Behav 1978;3 (3-4) 235- 241
PubMed
Babor  TFHiggins-Biddle  JCSaunders  JBMonteiro  MG The Alcohol Use Disorders Identification Test: Guidelines for Use in Primary Care. 2nd ed. Geneva, Switzerland World Health Organization2001;
Laucht  MEsser  GBaving  LGerhold  MHoesch  IIhle  WSteigleider  PStock  BStoehr  RMWeindrich  DSchmidt  MH Behavioral sequelae of perinatal insults and early family adversity at 8 years of age. J Am Acad Child Adolesc Psychiatry 2000;39 (10) 1229- 1237
PubMed
Skinner  HASheu  WJ Reliability of alcohol use indices: the Lifetime Drinking History and the MAST. J Stud Alcohol 1982;43 (11) 1157- 1170
PubMed
Balding  DJ A tutorial on statistical methods for population association studies. Nat Rev Genet 2006;7 (10) 781- 791
PubMed
Dudbridge  F Pedigree disequilibrium tests for multilocus haplotypes. Genet Epidemiol 2003;25 (2) 115- 121
PubMed
Dudbridge  F A survey of current software for linkage analysis. Hum Genomics 2003;1 (1) 63- 65
PubMed
Mantel  NHaenszel  W Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22 (4) 719- 748
PubMed
Ko  SWJia  YXu  HYim  SJJang  DHLee  YSZhao  MGToyoda  HWu  LJChatila  TKaang  BKZhuo  M Evidence for a role of CaMKIV in the development of opioid analgesic tolerance. Eur J Neurosci 2006;23 (8) 2158- 2168
PubMed
Izzo  EMartin-Fardon  RKoob  GFWeiss  FSanna  PP Neural plasticity and addiction: PI3-kinase and cocaine behavioral sensitization. Nat Neurosci 2002;5 (12) 1263- 1264
PubMed
Saccone  NLKwon  JMCorbett  JGoate  ARochberg  NEdenberg  HJForoud  TLi  TKBegleiter  HReich  TRice  JP A genome screen of maximum number of drinks as an alcoholism phenotype. Am J Med Genet 2000;96 (5) 632- 637
PubMed
Grant  BFDawson  DA Age at onset of alcohol use and its association with DSM-IV alcohol abuse and dependence: results from the National Longitudinal Alcohol Epidemiologic Survey. J Subst Abuse 1997;9103- 110
PubMed
Grant  BFStinson  FSHarford  TC Age at onset of alcohol use and DSM-IV alcohol abuse and dependence: a 12-year follow-up. J Subst Abuse 2001;13 (4) 493- 504
PubMed
Boyce-Rustay  JMHolmes  A Ethanol-related behaviors in mice lacking the NMDA receptor NR2A subunit. Psychopharmacology (Berl) 2006;187 (4) 455- 466
PubMed
Boyce-Rustay  JMHolmes  A Genetic inactivation of the NMDA receptor NR2A subunit has anxiolytic- and antidepressant-like effects in mice. Neuropsychopharmacology 2006;31 (11) 2405- 2414
PubMed
Mirshahi  TWoodward  JJ Ethanol sensitivity of heteromeric NMDA receptors: effects of subunit assembly, glycine and NMDAR1 Mg(2+)-insensitive mutants. Neuropharmacology 1995;34 (3) 347- 355
PubMed
Petrenko  ABYamakura  TFujiwara  NAskalany  ARBaba  HSakimura  K Reduced sensitivity to ketamine and pentobarbital in mice lacking the N-methyl-D-aspartate receptor GluRepsilon1 subunit. Anesth Analg 2004;99 (4) 1136- 1140
PubMed
Petrakis  ILLimoncelli  DGueorguieva  RJatlow  PBoutros  NNTrevisan  LGelernter  JKrystal  JH Altered NMDA glutamate receptor antagonist response in individuals with a family vulnerability to alcoholism. Am J Psychiatry 2004;161 (10) 1776- 1782
PubMed
Wernicke  CSamochowiec  JSchmidt  LGWinterer  GSmolka  MKucharska-Mazur  JHorodnicki  JGallinat  JRommelspacher  H Polymorphisms in the N-methyl-D-aspartate receptor 1 and 2B subunits are associated with alcoholism-related traits. Biol Psychiatry 2003;54 (9) 922- 928
PubMed
Rujescu  DSoyka  MDahmen  NPreuss  UHartmann  AMGiegling  IKoller  GBondy  BMöller  HJSzegedi  A GRIN1 locus may modify the susceptibility to seizures during alcohol withdrawal. Am J Med Genet B Neuropsychiatr Genet 2005;133 (1) 85- 87
PubMed
Goldman  DOroszi  GDucci  F The genetics of addictions: uncovering the genes. Nat Rev Genet 2005;6 (7) 521- 532
PubMed
Schumann  GRujescu  DSzegedi  ASinger  PWiemann  SWellek  SGiegling  IKlawe  CAnghelescu  IHeinz  ASpanagel  RMann  KHenn  FADahmen  N No association of alcohol dependence with a NMDA-receptor 2B gene variant. Mol Psychiatry 2003;8 (1) 11- 12
PubMed
Tadic  ADahmen  NSzegedi  ARujescu  DGiegling  IKoller  GAnghelescu  IFehr  CKlawe  CPreuss  UWSander  TToliat  MRSinger  PBondy  BSoyka  M Polymorphisms in the NMDA subunit 2B are not associated with alcohol dependence and alcohol withdrawal-induced seizures and delirium tremens. Eur Arch Psychiatry Clin Neurosci 2005;255 (2) 129- 135
PubMed
Kim  JHPark  MYang  SYJeong  BSYoo  HJKim  JWChung  JHKim  SA Association study of polymorphisms in N-methyl-D-aspartate receptor 2B subunits (GRIN2B) gene with Korean alcoholism. Neurosci Res 2006;56 (2) 220- 223
PubMed
Schumann  GRujescu  DKissling  CSoyka  MDahmen  NPreuss  UWWieman  SDepner  MWellek  SLascorz  JBondy  BGiegling  IAnghelescu  ICowen  MSPoustka  ASpanagel  RMann  KHenn  FASzegedi  A Analysis of genetic variations of protein tyrosine kinase fyn and their association with alcohol dependence in two independent cohorts. Biol Psychiatry 2003;54 (12) 1422- 1426
PubMed
Ishiguro  HSaito  TShibuya  HToru  MArinami  T Mutation and association analysis of the Fyn kinase gene with alcoholism and schizophrenia. Am J Med Genet 2000;96 (6) 716- 720
PubMed
Miyakawa  TYagi  TKitazawa  HYasuda  MKawai  NTsuboi  KNiki  H Fyn-kinase as a determinant of ethanol sensitivity: relation to NMDA-receptor function. Science 1997;278 (5338) 698- 701
PubMed
Tezuka  TUmemori  HAkiyama  TNakanishi  SYamamoto  T PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-D-aspartate receptor subunit NR2A. Proc Natl Acad Sci U S A 1999;96 (2) 435- 440
PubMed
Itokawa  MYamada  KYoshitsugu  KToyota  TSuga  TOhba  HWatanabe  AHattori  EShimizu  HKumakura  TEbihara  MMeerabux  JMToru  MYoshikawa  T A microsatellite repeat in the promoter of the N-methyl-D-aspartate receptor 2A subunit (GRIN2A) gene suppresses transcriptional activity and correlates with chronic outcome in schizophrenia. Pharmacogenetics 2003;13 (5) 271- 278
PubMed

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