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Original Article |

Haplotype-Based Localization of an Alcohol Dependence Gene to the 5q34 γ-Aminobutyric Acid Type A Gene Cluster FREE

Marta Radel, MD, PhD; Roger L. Vallejo, PhD; Nakao Iwata, MD, PhD; Richard Aragon, PhD; Jeffrey C. Long, PhD; Matti Virkkunen, MD; David Goldman, MD
[+] Author Affiliations

Author Affiliations: Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Md (Drs Radel, Vallejo, Aragon, Long, and Goldman); Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Japan (Dr Iwata); and Department of Psychiatry, University of Helsinki, Helsinki, Finland (Dr Virkkunen).


Arch Gen Psychiatry. 2005;62(1):47-55. doi:10.1001/archpsyc.62.1.47.
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Context  Pharmacobehavioral and pharmacogenetic evidence links γ-aminobutyric acid type A (GABAA) receptors and chromosomal regions containing GABAA receptor genes to ethanol-related responses. The GABAA gene cluster on chromosome 5q34 is of particular interest in the genetics of alcohol dependence because of the γ2 subunit requirement for ethanol’s modulatory action on GABAA receptors, previous linkage findings in mice and humans implicating both GABRA6 and GABRG2, and reported associations of GABRA6, GABRB2, and GABRG2 alleles with alcohol dependence.

Objective  To determine whether variation at the 5q34 GABAA gene cluster is implicated in differential susceptibility to alcohol dependence.

Methods  Two large psychiatrically interviewed samples, a Southwestern Native American population sample (N = 433) and a Finnish sample (N = 511) with alcohol-dependent subjects and unaffected individuals, were genotyped for 6 single nucleotide polymorphisms at the 5q34 GABAA gene cluster. In addition to sib-pair linkage and case-control association analyses, linkage disequilibrium mapping with haplotypes was used.

Results  Sib-pair linkage of GABRG2 to alcohol dependence was observed in Finns (P = .008). Association of the GABRB2 1412T allele with alcohol dependence was detected in both populations (Finns, P = .01; Southwestern Native Americans, P = .008), and the GABRA6 1519T allele was associated in both Finns (P = .01) and Southwestern Native Americans (P = .03). Linkage disequilibrium mapping with 3-locus haplotypes yielded evidence for an alcohol-dependence locus at the GABAA gene cluster region in both populations. The most highly significant signals were at 3-locus haplotypes that included 1 or more GABRA6 polymorphisms, with the peak signal at a GABRA6 3-locus haplotype (Finns, empirical P = .004; Southwestern Native Americans, empirical P = .02).

Conclusions  We detected sib-pair linkage of 5q34 GABAA receptor genes to alcohol dependence in Finns and found association both in Finns and in Southwestern Native Americans. In both populations, the haplotype localization implicates the region containing the Pro385Ser GABRA6 polymorphism and 2 other polymorphisms at GABRA6.

Figures in this Article

Alcohol dependence (“alcoholism”) is a common (10% in men, 4% in women),1 genetically influenced disorder with heritability estimates ranging from 40% to 60%.24 The vulnerability/protective alleles for alcoholism represent both the pharmacokinetic (detected and widely replicated) and the pharmacodynamic (detected but still putative) domains. A substantial part of the genetic component of variance in alcoholism vulnerability is substance specific, that is, not cross-inherited with vulnerability to other drugs.5 Such genetic epidemiological data implicate genes that are relatively substance specific. Although a few substance-specific polymorphisms have already been isolated in the pharmacokinetic genetic risk domain (ie, in genes coding for aldehyde dehydrogenase type 26 and for alcohol dehydrogenase type 27), they are common in East Asians but not in most other populations. Pharmacodynamic-specific gene targets for predisposition to alcohol dependence include the genes that encode receptors at which ethanol (alcohol) first acts in the brain and that may act as gatekeepers in alcohol response.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system8 and exerts most of its actions at GABA type A (GABAA) receptors, which are ligand-gated chloride-channel complexes. γ-Aminobutyric acid type A receptor complexes are generally heteropentamers composed of genetically distinct subunits; 16 related mammalian subunits have been reported.9,10 The human GABAA receptor subunit genes are grouped into 7 classes (α, β, γ, δ, ε, π, and θ).10 Within these classes, protein sequence identity is approximately 70% (ie, α1-α6, β1-β3, or γ1-γ3) and protein sequence identity is approximately 30% between classes. Most of the human GABAA receptor subunit genes have been assigned to chromosome regions, and subunit gene clusters have been identified on chromosomes 4, 5, 15, and X.10 Because of the number of subunits, the diversity of expressed GABAA receptor pentamers is large and would be larger except that these receptors are usually assembled from defined proportions of α, β, and γ subunits and except that there is considerable variation in the localization and developmental timing of expression of the different subunits.1114 The most abundant subunit combinations observed so far are α1β2γ2, α2β3γ2, and α3β3γ2, which make up about 80% of all GABAA receptors. The receptors containing a α1β2γ2 combination, believed to assemble following the coordinated expression of GABRA1, GABRB2, and GABRG2 genes on 5q34, constitute the major GABAA receptor subtype in the adult central nervous system (about 50%), have been identified in neurons at all levels of the neuraxis, and are believed to mediate the basic pharmacological spectrum of the classical, high-affinity BZ site ligands, except CL 218872.12,15 A variety of central nervous system–depressant drugs that show cross-tolerance, including ethanol, benzodiazepines, and barbiturates, as well as inhalant anesthetics and some endogenous neuroactive steroids, positively modulate the GABAA receptors.8,1618 Many behavioral effects of ethanol (eg, anxiolytic, ataxic, and sedative/hypnotic) may be explained by allosteric enhancement of GABAA receptor–mediated ionic influx and consequent hyperpolarization of the neuronal membrane.8 Agents that increase GABAA receptor activity in the central nervous system by acting as GABA positive modulators (ie, benzodiazepines, barbiturates, and depressant steroids) enhance acute sensitivity to ethanol and maintain ethanol preference, whereas drugs that act as GABA antagonists at GABAA receptors, such as picrotoxin, decrease many acute actions of ethanol and reduce ethanol preference.19 In addition, signs of ethanol withdrawal are diminished following treatments with those GABA agonists that increase GABAA receptor function, whereas GABA antagonists at GABAA receptors increase such signs.19 Effects of ethanol on GABAA receptor function and expression make the GABAA receptor subunit genes excellent candidates for vulnerability to alcohol dependence.20,21 In addition, genetic differences in the ethanol sensitivity of GABAA receptors were observed in short-sleep and long-sleep mice that differ in the sedative response to ethanol. Differential γ subunit function was observed between the 2 lines, which has been proposed as a critical determinant of individual differences in ethanol sensitivity.22 In BxD RI strains and other rodent genetic stocks, quantitative trait loci for sensitization of locomotor activation by ethanol and for predisposition to acute ethanol withdrawal map to the region containing the gene cluster that includes the γ2 and α6 subunit genes (gabrg2 and gabra6), and all of the mouse GABAA subunit gene clusters appear to have ethanol response–associated quantitative trait loci nearby.23 In the mouse, the Ala11Thr variant of the γ2 subunit correlates with acute ethanol withdrawal severity,24 and in the rat, an α6 Arg100Gln amino acid substitution that differentiates alcohol tolerant and nontolerant has been reported to alter response to benzodiazepines.25

In the human, preliminary linkage findings have also indicated that variation in GABAA receptor subunit genes plays a role in differential vulnerability to alcohol dependence, and a specific polymorphism of the α6 subunit gene (ie, GABRA6) encoding an amino acid substitution (Pro385Ser) was implicated.26 As measured by slowing of saccadic eye movement velocity, sons of alcoholics appear to have diminished sensitivity to benzodiazepines as compared with young men who are at lower risk for alcohol dependence, leading to the proposal that differences in the expression or function of GABAA receptors alter vulnerability to alcohol dependence.27 In these same subjects, the GABAA α6 subunit Pro385Ser polymorphism was shown to predict benzodiazepine sensitivity.28

When ethanol itself is administered as a drug challenge, decreased intoxication, motor effects, and hormonal responses are observed in sons of alcoholics,29 which has been extensively replicated. In a 15-year follow-up study of 450 men, Schuckit30,31 showed that low level of response to alcohol in young adulthood is a predictor of later alcoholism. The risk attributable to the level of response is substantial (approximately 40% of the variance in vulnerability), largely independent of family history, and largely unshared with other drugs of abuse.31 By selective genotyping of a subsample of this same cohort, Schuckit et al26 found preliminary evidence that the GABAA α6 Pro385Ser amino acid substitution is associated both with alcohol sensitivity and with increased risk for alcohol dependence. Associations of markers at the 5q34 GABAA gene cluster have also been reported in Scottish,32 German,33 and Japanese34 samples.

Here we report both locus-based linkage analyses in sib pairs and allele-based localization by association using GABAA haplotypes in this Finnish population and in a second semi-isolated population of Southwestern Native Americans.

SAMPLES STUDIED

Subjects were selected from 2 semi-isolated populations: a Finnish population and a Southwestern Native American population.

FINNISH SAMPLE

A total of 511 psychiatrically interviewed Finns included 110 alcoholic offender probands, 277 relatives, and 124 unrelated controls.35 Participants were studied under a human research protocol approved by the institutional review board of the National Institutes of Health, Bethesda, Md; the National Institute of Mental Health, Bethesda; Office for Protection from Research Risks, Bethesda; the University of Helsinki Department of Psychiatry institutional review board, Helsinki, Finland; and the University of Helsinki Central Hospital institutional review board. All subjects were 17 years or older and provided written informed consent.

The probands were male criminal offenders undergoing forensic psychiatric evaluation as inpatients in the Department of Psychiatry, University of Helsinki. Therefore, the sample is enriched for the early-onset form of alcoholism associated with impulsivity and antisocial behaviors, so-called type II.36 Recently, it has become recognized that in addition to scoring high in novelty seeking (as hypothesized by Cloninger36), such alcoholics also tend to have higher harm avoidance and anxiety,37 and these particular alcoholics were above the norm for harm avoidance measured on the Tridimensional Personality Scale Questionnaire (data not shown).

A total of 110 women and 167 male relatives were ascertained through the index cases. There were 275 sib pairs among whom 82 sib pairs were concordant for alcoholism (DSM-III-R38 alcohol dependence), 64 were discordant, and 129 were unaffected. The 124 unaffected controls were unrelated healthy Finnish male volunteers recruited through local newspaper advertisements. Controls were also psychiatrically interviewed (Structured Clinical Interview for DSM-III-R38) and were paid for their participation. Controls were in a good state of general health as established by physical examination, erythrocyte and lymphocyte indices, liver enzyme and thyroid hormone levels, and serum electrolyte and creatinine concentrations.

SOUTHWESTERN NATIVE AMERICAN SAMPLE

The Southwestern Native American sample (N = 433) was collected without proband ascertainment bias for a family-based study on alcoholism and related psychiatric disorders.39 The human research protocol was approved by the tribal council and by the institutional review board of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health. All subjects provided written informed consent. Subjects were ascertained based on familial relationship; both the original selection of the families and the later recruitment of subjects were performed blind to diagnoses. Participants were members of the same genealogy, older than 21 years, in general good health, and eligible for tribal enrollment (ie, with at least one fourth tribal heritage).

The 433 psychiatrically interviewed Southwestern Native Americans included 192 individuals affected with alcoholism (DSM-III-R38 alcohol dependence) and 241 unaffected individuals. There were 419 sib pairs, which included 188 sib pairs concordant for alcoholism, 168 discordant, and 63 unaffected. Use of the tribal name and exact reservation location is avoided because these details are unnecessary for the analyses set out here.

PSYCHIATRIC DIAGNOSES

The Finns were interviewed using the Structured Clinical Interview for DSM-III-R38 by 2 psychiatrists in the Department of Psychiatry, University of Helsinki. Diagnoses were made independently and blindly by the 2 psychiatrists under the supervision of a senior research psychiatrist and according to criteria from the DSM-III-R.38 The Southwestern Native Americans were interviewed using the Schedule for Affective Disorders and Schizophrenia–Lifetime Version by a psychologist experienced in psychiatric assessment with this and other Native American populations. Diagnoses for alcoholism and other psychiatric disorders according to criteria from the DSM-III-R38 were based on the interview data; medical, educational, court, and other records; and corroborative information from family members. Diagnoses were made by 2 blind raters: a clinical social worker and a clinical psychologist. Diagnostic differences were resolved in a consensus conference that included a senior psychiatrist experienced in diagnosis in Native American people.

In the Finnish sample, 1 patient was excluded because of chronic schizophrenia. No patient was excluded from the Southwestern Native American population sample.

GENOTYPING OF POLYMORPHISMS AT 5Q34 GABAA RECEPTOR GENE CLUSTER

Six polymorphisms at 3 GABAA receptor genes clustered on the long arm of human chromosome 5 were genotyped blind to diagnoses by polymerase chain reaction–restriction fragment length polymorphism analysis: GABRA6 1031G>C40; GABRA6 1236C>T (Pro385Ser)28; GABRA6 1519T>C33; GABRB2 1412C>T33; GABRG2 IVS9 + 99C>A41 (NCBI rs211014); and GABRG2 IVS10 + 3145A>G41 (NCBI rs211013). GenBank accession numbers for GABRA6, GABRB2, and GABRG2 are GABRA6, number S81944; GABRB2, number S67368; and GABRG2, number NM000816.

The primer pairs listed in Table 1 were used to amplify genomic DNA isolated from Epstein-Barr virus–immortalized lymphoblastoid cell lines. Each 25-μL polymerase chain reaction volume contained 50 to 100 ng of genomic DNA, 50 pmol of each primer, 0.125mM deoxyribonucleoside-5"-triphosphates (PerkinElmer, Fremont, Calif), and 1 unit of TaqGold (PerkinElmer) DNA polymerase. The final reaction volume also contained 10mM Tris base pH 9, 50mM potassium chloride (KCl), 1.5mM magnesium chloride (MgCl2), 0.1% Triton X-100 (PerkinElmer), and 0.01% gelatin. The reactions were performed using a hot-start procedure: TaqGold DNA polymerase was activated only after a first denaturation step of 12 minutes at 95°C. Amplifications were carried out using a modified step-down thermocycling procedure.42 A 12-μL volume of each amplicon was digested with the appropriate restriction enzyme (New England Biolabs, Beverly, Mass) (Table 1) according to the manufacturer’s specifications. The digested products were electrophoresed in 10% 1X TBE polyacrylamide gels, at 100 V for 1.5 hours at room temperature. Gels were stained with ethidium bromide and photographed under UV light.

Table Graphic Jump LocationTable 1 Primer and Enzymes for PCR-RFLP Assays*

Genotypes were read by 2 independent raters blind to diagnoses and were checked for mendelian segregation. Discrepant genotypes were regenotyped. The error rate was estimated to be less than 1%.

STATISTICAL ANALYSIS

Differences in allele and genotype frequencies and deviation of genotypes from Hardy-Weinberg equilibrium were evaluated using the χ2 test and the Fisher exact test for Hardy-Weinberg equilibrium, which is implemented in the computer program MENDEL version 5.0.43 Extreme cases of population admixture and/or stratification can be detected by marked departure from Hardy-Weinberg equilibrium. Evidence for population heterogeneity or admixture was further evaluated using the computer programs LINKMAP from the LINKAGE package version 5.244 and HOMOG.45 Population stratification was also evaluated using a variance-components approach that compares the between- and within-family components of association,46,47 which is implemented in the computer program QTDT version 2.2.1.47

Pair-wise linkage disequilibrium coefficients (D), normalized linkage disequilibrium (D′, D divided by the maximum possible value of D), and correlation coefficients (r2, the squared total linkage disequilibrium divided by the product of the allelic frequencies at both loci) were estimated with MLOCUS.48,49 Significance levels were estimated from the χ2 distribution.

The only phenotype analyzed was DSM-III-R38 alcohol dependence. Two-point linkage analysis was conducted using the nonparametric sib-pair regression method,50 as programmed in the Sibpal module of the S.A.G.E. package.51 Haplotype association and localization was performed with TRIMHAP, a family-based haplotype test for linkage disequilibrium.52 First, the 6 5q34 marker genotypes were used to estimate marker haplotypes for all pedigree members using GENEHUNTER.53 Afterwards, TRIMHAP was used to define a subset of markers that were feasible as ancestral haplotypes and to determine identity by descent within the pedigrees. For each 3-locus haplotype in the sample, a haplotype-sharing score was calculated. TRIMHAP determined the category of each haplotype, added it to the trimmed-haplotype table, and constructed the sum of haplotype-sharing scores over all categories. The empirical P values for the trimmed-haplotype statistic were estimated using 10 000 replicate samples generated by permutation bootstrapping. A feature of replicate samples constructed using a permutation scheme is that replicates are formed assuming the null hypothesis H0 of linkage but linkage disequilibrium is true.

The use of this method, which is a family-based test, also addresses the issue of association analysis of affected and unaffected individuals who share some degree of relationship, which is the case in the Southwestern Native American population sample.

Reflecting the semi-isolated nature of both populations, the Finns and the Southwestern Native Americans differed in allele frequencies, in 6-locus haplotype frequencies, and in overall patterns of linkage disequilibrium across the 5q34 GABAA cluster region (Figure 1). Within both populations, there were no significant deviations of genotypic distributions from Hardy-Weinberg equilibrium for all the polymorphisms in study. For both populations, there is no evidence of population heterogeneity or admixture.

Place holder to copy figure label and caption
Figure 1.

Pair-wise normalized linkage disequilibrium (D′) and correlation (r2, in parentheses) coefficients between the 6 loci (boxed) in a Finnish sample (A) and in a Southwestern Native American population (B). Coefficients with P<.05 are in bold font. Cen indicates centromere; and tel, telomere.

Graphic Jump Location

Consistent with an autosomal linkage scan previously performed,54 sib-pair linkage of GABRG2 to alcohol dependence was observed in Finns (P = .008), although not in Southwestern Native Americans. Association of the GABRB2 1412T allele with alcohol dependence was detected in both Finns (P = .01) (Table 2) and Southwestern Native Americans (P = .008) (Table 3), and the GABRA6 1519T allele was also associated with alcohol dependence in both Finns (P = .01) (Table 2) and Southwestern Native Americans (P = .03) (Table 3).

Table Graphic Jump LocationTable 2 Genotype and Allele Associations of GABAA 5q Polymorphisms to Alcohol Dependence in Finns (n=234)*
Table Graphic Jump LocationTable 3 Genotype and Allele Associations of GABAA 5q Polymorphisms to Alcohol Dependence in Southwestern Native Americans (N=433)*

Linkage disequilibrium mapping with haplotypes yielded evidence for an alcohol dependence locus in the GABAA gene cluster region in both populations (Table 4 and Figure 2). For Finns, the most highly significant signals were at 3-locus haplotypes that included GABRA6 polymorphisms, with the peak signal at a 3-locus haplotype with the 3 GABRA6 polymorphisms (empirical P = .004). Results were also significant at haplotypes that included combinations of GABRA6 1236C>T, GABRA6 1519T>C, GABRG2 IVS9 + 99C>A, and GABRG2 IVS10 + 3415A>G polymorphisms (empirical P = .01 to .03). For Southwestern Native Americans, the most highly significant signal was also at a haplotype that included the 3 GABRA6 polymorphisms (empirical P = .02). Results were also significant at a haplotype that included the GABRB2 1412C>T, GABRA6 1031G>C, and GABRA6 1236C>T polymorphisms (empirical P = .01).

Table Graphic Jump LocationTable 4 Trimmed-Haplotype Test* for Linkage Disequilibrium of GABAA 5q Polymorphisms† to DSM-III-R Alcohol Dependence (AD) in Finns and Southwestern Native Americans
Place holder to copy figure label and caption
Figure 2.

(A) Chromosome 5q34 GABAA gene cluster fine structure and (B) haplotype-based localization of alcohol dependence to the 5q34 GABAA gene cluster: trimmed-haplotype test results. The P values correspond to the inter–single nucleotide polymorphism location for the putative alcohol-dependence susceptibility locus. Cen indicates centromere; SWNA, Southwestern Native Americans; and Tel, telomere.

Graphic Jump Location

Alcohol dependence has recently been the object of both positional cloning, or “reverse genetics,” and candidate gene, or “forward genetics,” approaches for gene mapping. Roles in alcoholism vulnerability for alcohol metabolic gene polymorphisms have been validated and reviewed,21,55 but these loci contribute a substantial portion of the variance in risk only in the Southeast Asian populations in which they are abundant. These findings, achieved by forward genetics, were made by relating functional alleles to alcohol pharmacokinetics to phenotype. On the other hand, positional cloning in humans56,57 and rodents5860 has yielded loci, several at least partially replicated, but not determinant alleles. Successes in other complex diseases, such as breast cancer61 and Alzheimer disease,62 clearly indicate that a combination of mapping methods can lead to the discovery of vulnerability alleles.

Both the positional cloning and the candidate gene paradigms that have been applied to the genetics of ethanol-related behaviors in humans20,21,55 and rodent genetic models5860 have produced convergent evidence implicating the region (5q34 in humans) containing a GABAA receptor gene cluster.20,21 These studies have focused further interest on genetic variation in GABAA receptors, which had already been implicated by pharmacobehavioral and receptor-pharmacology data as potential gatekeepers in ethanol response.9,10 Furthermore, locus-based linkage evidence in the Finnish sample studied here positionally implicated the chromosome 5q34 region in alcohol dependence.54 The allele-based associations we observed both in Finns and in Southwestern Native Americans narrow the region, so that it seems the linkage signals emanate from the GABAA gene cluster itself. Since the GABAA cluster spans more than 700 kilobases and includes 4 receptor subunit genes, we applied haplotype-based localization to further narrow the location of the risk gene, and we found the α6 Pro385Ser amino acid polymorphism positioned within the signal peak.

Semi-isolated populations with relatively smaller sizes, low ethnic diversity, and more homogeneous environments are invaluable in the genetic analysis of complex disorders because reduction in genetic and environmental heterogeneity facilitates the identification of specific factors that affect vulnerability.63 Since alcohol dependence is a complex disorder with diverse genetic and environmental contributions, we decided to study samples from 2 semi-isolated populations with well-defined genetic compositions and relatively homogeneous environments (ie, Finns and members of a Southwestern Native American population). In addition, because of founder effects and reduced population size, linkage disequilibrium in such populations is enhanced, leading to conservation of haplotypes containing disease alleles.64,65 The strategy was to increase the odds of finding out whether genetic changes in the 5q34 GABAA gene cluster have a role in the susceptibility to alcohol dependence. In addition to the testing for significant departure from Hardy-Weinberg equilibrium, which occurs in more extreme cases of population admixture and/or stratification and which was not detected in either population, the results from the tests that were used for detection of more subtle population heterogeneity or admixture and for population stratification confirmed the low genetic heterogeneity for both populations.

In this study, there were several instances where we found allele-based association in the absence of sib-pair linkage of the same phenotype to the same locus, and we detected linkage of GABRG2 to alcohol dependence in the Finnish sample but not in the Southwestern Native American sample. The explanation for this disparity need not be a biological mechanism whereby allele-based associations could occur in populations in the absence of locus linkage in families.66 Sib-pair linkage may fail to detect linkage to loci of minor effect,67 whereas allele effects at these loci may be readily detectable by association.68

The results we obtained are convergent with previously published data, despite the ethnic diversity of the populations represented. The GABRA6 1519T and the GABRB2 1412T alleles were associated with alcohol dependence in both Finns and Southwestern Native Americans. The GABRA6 1519T associations are consistent with previous studies in a Scottish sample32 and a German sample.33 The GABRB2 1412T associations in Finns and Southwestern Native Americans are consistent with the Scottish population findings but not the German sample findings. Furthermore, although we found sib-pair linkage to GABRG2 in 1 population (Finns) and neither population showed association of alcohol dependence with GABRG2 alleles, association between alcoholism and GABRG2 IVS10 + 3145 G has been reported in a Japanese sample.34

In addition to sib-pair linkage and association analyses, we also applied a family-based haplotype localization strategy, which exploits the power of linkage disequilibrium to identify ancestrally related chromosomes that carry an allele that influences disease.52 The trimmed-haplotype statistic allows detecting linkage disequilibrium due to ancestral haplotypes, and the algorithm is designed to be repeated with the disease-susceptibility locus located at a grid of positions covering a chromosomal region in study. This method addressed the need of a family-based association test for related individuals in the Southwestern Native American sample and allowed the localization of a putative alcohol-dependence susceptibility locus within the 5q34 GABAA cluster, with the lowest P values implicating GABRA6 polymorphisms in both Finnish and Southwestern Native American population samples (Figure 2).

Several disease-vulnerability alleles have been shown to reside on characteristic haplotype backgrounds so that their detection would have been possible through association to ancestrally related haplotypes.69,70 Linkage disequilibrium coefficient estimates between alleles at the 6 loci in the 5q34 GABAA gene cluster did not clearly vary as a function of distance in either Finns or Southwestern Native Americans, nor did the correlation coefficients (Figure 1). Decoupling between the extent of linkage disequilibrium and physical distance can reflect a variety of complexities in the histories (eg, mutation, selection, genetic drift, and migration) of populations64,65,71,72 and can vary even within the same population from one chromosome region to the other.64,65

Given that our findings in these 2 semi-isolated populations, in conjunction with data in the literature, strongly implicate the 5q34 GABAA cluster in alcohol dependence, the next critical steps in evaluating the genes in this region are likely to be single nucleotide polymorphism genotyping across the 5q34 GABAA gene cluster and the identification of functional alleles that may influence gene expression and of alleles that alter receptor structure. Among the 5q34 GABAA receptor genes, GABRA6, which encodes the α6 subunit, is peculiar in showing selective expression in the cerebellum. It is implicated in alcohol dependence both by haplotype localization and by association with Ser385, a relatively abundant nonconservative missense variant, which may alter phosphorylation of the receptor protein.40 However, functionality of Ser385 or any other human GABAA subunit variant has yet to be demonstrated. For GABAA receptors, this challenge is complicated by the heteropentameric nature of the receptor channel (creating a potential need to express subunit alleles in different molecular contexts) and the possibility that differences in cellular environment can affect the ability of an allele to alter expression or protein posttranslational modification. Isolation of the vulnerability allele(s) located within the chromosome 5q GABAA gene complex will enable us to better understand interindividual variation in vulnerability to alcohol dependence and would lead to improvements in detection and treatment of at least a subset of patients with this common, debilitating disorder.

Correspondence: Marta Radel, MD, PhD, Bioconsult, 17702 Calabar Dr, Gaithersburg, MD 20877 (martaradel@verizon.net).

Submitted for Publication: December 12, 2003; final revision received August 3, 2004; accepted August 19, 2004.

Funding/Support: This study was supported by grants from the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Md (Dr Goldman).

Acknowledgment: We would like to thank Longina Akhtar, MS, and Lisa Moore for their excellent technical assistance.

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Korpi  ERKleingoor  CKettenmann  HSeeburg  PH Benzodiazepine-induced motor impairment linked to point mutation at cerebellar GABAA receptor. Nature 1993;361356- 359
PubMed
Schuckit  MASmith  TLRadel  MIwata  NGoldman  D Selective genotyping for the role of 5-HT2A, 5-HT2C, and the GABAA α6 receptors and the serotonin transporter in the level of response to alcohol: a pilot study. Biol Psychiatry 1999;45647- 651
PubMed
Cowley  DSRoy-Byrne  PPRadant  AHommer  DWGreenblatt  DJVitaliano  PPGodon  C Eye movement effects of diazepam in sons of alcoholic fathers and male control subjects. Alcohol Clin Exp Res 1994;18324- 332
PubMed
Iwata  NCowley  DRadel  MRoy-Byrne  PGoldman  DA Pro385Ser substitution in the GABAA receptor α6 subunit gene associates with benzodiazepine sensitivity. Am J Psychiatry 1999;1561447- 1449
PubMed
Schuckit  MA Ethanol-induced changes in body sway in men at high alcoholism risk. Arch Gen Psychiatry 1985;42375- 379
PubMed
Schuckit  MA Low level of response to alcohol as a predictor of future alcoholism. Am J Psychiatry 1994;151184- 193
PubMed
Schuckit  MA Alcohol-related disorders. Kaplan  HISadok  BJeds.Comprehensive Textbook of Psychiatry 6th ed. New York, NY Plennum Press1995;775- 791
Loh  EWSmith  IMurray  RMcLaughlin  MMcNulty  SBall  D Association between variants at the GABAAbeta2, GABAAalpha6 and GABAAgamma2 gene cluster and alcohol dependence in a Scottish population. Mol Psychiatry 1999;4539- 544
PubMed
Sander  TBall  DMurray  RPatel  JSamochowiec  JWinterer  GRommelspacher  HSchmidt  LGLoh  EW Association analysis of sequence variants of the GABAA alpha6, beta2, and gamma2 gene cluster and alcohol dependence. Alcohol Clin Exp Res 1999;23427- 431
PubMed
Loh  EWHiguchi  SMatsushita  SMurray  RChen  CKBall  D Association analysis of the GABA(A) receptor subunit genes cluster on 5q33-34 and alcohol dependence in a Japanese population. Mol Psychiatry 2000;5301- 307
PubMed
Lappalainen  JLong  JCEggert  MOzaki  NRobin  RWBrown  GLNaukkarinen  HVirkkunen  MLinnoila  MGoldman  D Linkage of antisocial alcoholism to the serotonin 5-HT1B receptor gene in 2 populations. Arch Gen Psychiatry 1998;55989- 994
PubMed
Cloninger  CR Neurogenetic adaptive mechanisms in alcoholism. Science 1987;236410- 416
PubMed
Babor  TFHofmann  MDelBoca  FKHesselbrock  VMeyer  REDolinsky  ZSRounsaville  B Types of alcoholics, I: evidence for an empirically derived typology based on indicators of vulnerability and severity. Arch Gen Psychiatry 1992;49599- 608
PubMed
American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition.   Washington, DC American Psychiatric Press1987;
Robin  RWLong  JCRasmussen  JKAlbaugh  BGoldman  D Relationship of binge drinking to alcohol dependence, other psychiatric disorders, and behavioral problems in an American Indian tribe. Alcohol Clin Exp Res 1998;22518- 523
PubMed
Iwata  NVirkkunen  MGoldman  D Identification of a naturally occurring Pro385-Ser385 substitution in the GABA(A) receptor alpha6 subunit gene in alcoholics and healthy volunteers. Mol Psychiatry 2000;5316- 319
PubMed
Cheng  ATLoh  EWCheng  CYWang  YCHsu  YP Polymorphisms and intron sequences flanking the alternatively spliced 8-amino-acid exon of gamma2 subunit gene for GABAA receptors. Biochem Biophys Res Commun 1997;238683- 685
PubMed
Cotton  RGH Mutation detection. E.E.A.F.S.,ed.The Practical Approach Series New York, NY Oxford University Press1998;241
Lange  KCantor  RHorvath  SPerola  MSabatti  CSinsheimer  JSobel  E Mendel version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets. Am J Hum Genet 2001;69 ((suppl)) A1886
Lathrop  GMLalouel  JMJulier  COtt  J Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A 1984;813443- 3446
PubMed
Ott  J Analysis of Human Genetic Linkage. Rev ed. Baltimore, Md Johns Hopkins University Press1991;
Fulker  DWCherny  SSSham  PCHewitt  JK Combined linkage and association sib-pair analysis for quantitative traits. Am J Hum Genet 1999;64259- 267
PubMed
Abecasis  GRCardon  LRCookson  WOC A general test of association for quantitative traits in nuclear families. Am J Hum Genet 2000;66279- 292
PubMed
Long  JC Multiple Locus Haplotype Analysis. Version 2.0. Bethesda, Md National Institutes of Health1999;
Long  JCWilliams  RCUrbanek  M An E-M algorithm and testing strategy for multiple locus haplotypes. Am J Hum Genet 1995;56799- 810
PubMed
Haseman  JKElston  RC The investigation of linkage between a quantitative trait and a marker locus. Behav Genet 1972;23- 19
PubMed
 S.A.G.E.: Statistical Analysis for Genetic Epidemiology. Release 2.2. New Orleans, La Department of Biometry and Genetics, LSU Medical Center1996;
MacLean  CJMartin  RBSham  PCWang  HStraub  REKendler  KS The trimmed-haplotype test for linkage disequilibrium. Am J Hum Genet 2000;661062- 1075
PubMed
Kruglyak  LDaly  MJReeve-Daly  MPLander  ES Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;581347- 1363
PubMed
Vallejo  RLLong  JCHoopes  DVirkkunen  MGoldman  D Autosomal genome scan for loci linked to alcohol abuse and related psychiatric disorders in Finnish pedigrees. Alcohol Clin Exp Res 1998;1197A
Li  TK Pharmacogenetics of responses to alcohol and genes that influence alcohol drinking. J Stud Alcohol 2000;615- 12
PubMed
Reich  TEdenberg  HJGoate  AWilliams  JTRice  JPVan Eerdewegh  PForoud  THesselbrock  VSchuckit  MABucholz  KPorjesz  BLi  TKConneally  PMNurnberger  JI  JrTischfield  JACrowe  RRCloninger  CRWu  WShears  SCarr  KCrose  CWillig  CBegleiter  H Genome-wide search for genes affecting the risk for alcohol dependence. Am J Med Genet 1998;81207- 215
PubMed
Long  JCKnowler  WCHanson  RLRobin  RWUrbanek  MMoore  EBennett  PHGoldman  D Evidence for genetic linkage to alcohol dependence on chromosomes 4 and 11 from autosome-wide scan in an American Indian population. Am J Med Genet 1998;81216- 221
PubMed
Crabbe  JCBelknap  JKMitchell  SRCrawshaw  LI Quantitative trait loci mapping of genes that influence the sensitivity and tolerance to ethanol-induced hypothermia in BXD recombinant inbred mice. J Pharmacol Exp Ther 1994;269184- 188
PubMed
Johnson  TEDeFries  JCMarkel  PD Mapping quantitative trait loci for behavioral traits in the mouse. Behav Genet 1992;22635- 653
PubMed
Carr  LGForoud  TBice  PGobbett  TIvashina  JEdenberg  HLumeng  LLi  TK A quantitative trait locus for alcohol consumption in selectively bred rat lines. Alcohol Clin Exp Res 1998;22884- 887
PubMed
Hall  JMLee  MKNewman  BMorrow  JEAnderson  LAHuey  BKing  MC Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990;2501684- 1689
PubMed
Corder  EHSaunders  AMStrittmatter  WJSchmechel  DEGaskell  PCSmall  GWRoses  ADHaines  JLPericak-Vance  MA Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993;261921- 923
PubMed
Lander  EKruglyak  L Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995;11241- 247
PubMed
Taillon-Miller  PBauer-Sardina  ISaccone  NLPutzel  JLaitinen  TCao  AKere  JPilia  GRice  JPKwok  PY Juxtaposed regions of extensive and minimal linkage disequilibrium in human Xq25 and Xq28. Nat Genet 2000;25324- 328
PubMed
Eaves  IAMerriman  TRBarber  RANutland  STuomilehto-Wolf  ETuomilehto  JCucca  FTodd  JA The genetically isolated populations of Finland and Sardinia may not be a panacea for linkage disequilibrium mapping of common disease genes. Nat Genet 2000;25320- 323
PubMed
Hodge  SE What association analysis can and cannot tell us about the genetics of complex disease. Am J Med Genet 1994;54318- 323
PubMed
Lander  ESSchork  NJ Genetic dissection of complex traits. Science 1994;2652037- 2048
PubMed
Risch  NMerikangas  K The future of genetic studies of complex human diseases. Science 1996;2731516- 1517
PubMed
Tishkoff  SAGoldman  ACalafell  FSpeed  WCDeinard  ASBonne-Tamir  BKidd  JRPakstis  AJJenkins  TKidd  KK A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations. Am J Hum Genet 1998;621389- 1402
PubMed
Osier  MVPakstis  AJSoodyall  HComas  DGoldman  DOdunsi  AOkonofua  FParnas  JSchulz  LOBertranpetit  JBonne-Tamir  BLu  RBKidd  JRKidd  KK A global perspective on genetic variation at the ADH genes reveals unusual patterns of linkage disequilibrium and diversity. Am J Hum Genet 2002;7184- 99
PubMed
Kidd  KKMorar  BCastiglione  CMZhao  HPakstis  AJSpeed  WCBonne-Tamir  BLu  RBGoldman  DLee  CNam  YSGrandy  DKJenkins  TKidd  JR A global survey of haplotype frequencies and linkage disequilibrium at the DRD2 locus. Hum Genet 1998;103211- 227
PubMed
Peterson  RJGoldman  DLong  JC Nucleotide sequence diversity in non-coding regions of ALDH2 as revealed by restriction enzyme and SSCP analysis. Hum Genet 1999;104177- 187
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Pair-wise normalized linkage disequilibrium (D′) and correlation (r2, in parentheses) coefficients between the 6 loci (boxed) in a Finnish sample (A) and in a Southwestern Native American population (B). Coefficients with P<.05 are in bold font. Cen indicates centromere; and tel, telomere.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

(A) Chromosome 5q34 GABAA gene cluster fine structure and (B) haplotype-based localization of alcohol dependence to the 5q34 GABAA gene cluster: trimmed-haplotype test results. The P values correspond to the inter–single nucleotide polymorphism location for the putative alcohol-dependence susceptibility locus. Cen indicates centromere; SWNA, Southwestern Native Americans; and Tel, telomere.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1 Primer and Enzymes for PCR-RFLP Assays*
Table Graphic Jump LocationTable 2 Genotype and Allele Associations of GABAA 5q Polymorphisms to Alcohol Dependence in Finns (n=234)*
Table Graphic Jump LocationTable 3 Genotype and Allele Associations of GABAA 5q Polymorphisms to Alcohol Dependence in Southwestern Native Americans (N=433)*
Table Graphic Jump LocationTable 4 Trimmed-Haplotype Test* for Linkage Disequilibrium of GABAA 5q Polymorphisms† to DSM-III-R Alcohol Dependence (AD) in Finns and Southwestern Native Americans

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Schuckit  MASmith  TLRadel  MIwata  NGoldman  D Selective genotyping for the role of 5-HT2A, 5-HT2C, and the GABAA α6 receptors and the serotonin transporter in the level of response to alcohol: a pilot study. Biol Psychiatry 1999;45647- 651
PubMed
Cowley  DSRoy-Byrne  PPRadant  AHommer  DWGreenblatt  DJVitaliano  PPGodon  C Eye movement effects of diazepam in sons of alcoholic fathers and male control subjects. Alcohol Clin Exp Res 1994;18324- 332
PubMed
Iwata  NCowley  DRadel  MRoy-Byrne  PGoldman  DA Pro385Ser substitution in the GABAA receptor α6 subunit gene associates with benzodiazepine sensitivity. Am J Psychiatry 1999;1561447- 1449
PubMed
Schuckit  MA Ethanol-induced changes in body sway in men at high alcoholism risk. Arch Gen Psychiatry 1985;42375- 379
PubMed
Schuckit  MA Low level of response to alcohol as a predictor of future alcoholism. Am J Psychiatry 1994;151184- 193
PubMed
Schuckit  MA Alcohol-related disorders. Kaplan  HISadok  BJeds.Comprehensive Textbook of Psychiatry 6th ed. New York, NY Plennum Press1995;775- 791
Loh  EWSmith  IMurray  RMcLaughlin  MMcNulty  SBall  D Association between variants at the GABAAbeta2, GABAAalpha6 and GABAAgamma2 gene cluster and alcohol dependence in a Scottish population. Mol Psychiatry 1999;4539- 544
PubMed
Sander  TBall  DMurray  RPatel  JSamochowiec  JWinterer  GRommelspacher  HSchmidt  LGLoh  EW Association analysis of sequence variants of the GABAA alpha6, beta2, and gamma2 gene cluster and alcohol dependence. Alcohol Clin Exp Res 1999;23427- 431
PubMed
Loh  EWHiguchi  SMatsushita  SMurray  RChen  CKBall  D Association analysis of the GABA(A) receptor subunit genes cluster on 5q33-34 and alcohol dependence in a Japanese population. Mol Psychiatry 2000;5301- 307
PubMed
Lappalainen  JLong  JCEggert  MOzaki  NRobin  RWBrown  GLNaukkarinen  HVirkkunen  MLinnoila  MGoldman  D Linkage of antisocial alcoholism to the serotonin 5-HT1B receptor gene in 2 populations. Arch Gen Psychiatry 1998;55989- 994
PubMed
Cloninger  CR Neurogenetic adaptive mechanisms in alcoholism. Science 1987;236410- 416
PubMed
Babor  TFHofmann  MDelBoca  FKHesselbrock  VMeyer  REDolinsky  ZSRounsaville  B Types of alcoholics, I: evidence for an empirically derived typology based on indicators of vulnerability and severity. Arch Gen Psychiatry 1992;49599- 608
PubMed
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PubMed
Iwata  NVirkkunen  MGoldman  D Identification of a naturally occurring Pro385-Ser385 substitution in the GABA(A) receptor alpha6 subunit gene in alcoholics and healthy volunteers. Mol Psychiatry 2000;5316- 319
PubMed
Cheng  ATLoh  EWCheng  CYWang  YCHsu  YP Polymorphisms and intron sequences flanking the alternatively spliced 8-amino-acid exon of gamma2 subunit gene for GABAA receptors. Biochem Biophys Res Commun 1997;238683- 685
PubMed
Cotton  RGH Mutation detection. E.E.A.F.S.,ed.The Practical Approach Series New York, NY Oxford University Press1998;241
Lange  KCantor  RHorvath  SPerola  MSabatti  CSinsheimer  JSobel  E Mendel version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets. Am J Hum Genet 2001;69 ((suppl)) A1886
Lathrop  GMLalouel  JMJulier  COtt  J Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A 1984;813443- 3446
PubMed
Ott  J Analysis of Human Genetic Linkage. Rev ed. Baltimore, Md Johns Hopkins University Press1991;
Fulker  DWCherny  SSSham  PCHewitt  JK Combined linkage and association sib-pair analysis for quantitative traits. Am J Hum Genet 1999;64259- 267
PubMed
Abecasis  GRCardon  LRCookson  WOC A general test of association for quantitative traits in nuclear families. Am J Hum Genet 2000;66279- 292
PubMed
Long  JC Multiple Locus Haplotype Analysis. Version 2.0. Bethesda, Md National Institutes of Health1999;
Long  JCWilliams  RCUrbanek  M An E-M algorithm and testing strategy for multiple locus haplotypes. Am J Hum Genet 1995;56799- 810
PubMed
Haseman  JKElston  RC The investigation of linkage between a quantitative trait and a marker locus. Behav Genet 1972;23- 19
PubMed
 S.A.G.E.: Statistical Analysis for Genetic Epidemiology. Release 2.2. New Orleans, La Department of Biometry and Genetics, LSU Medical Center1996;
MacLean  CJMartin  RBSham  PCWang  HStraub  REKendler  KS The trimmed-haplotype test for linkage disequilibrium. Am J Hum Genet 2000;661062- 1075
PubMed
Kruglyak  LDaly  MJReeve-Daly  MPLander  ES Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;581347- 1363
PubMed
Vallejo  RLLong  JCHoopes  DVirkkunen  MGoldman  D Autosomal genome scan for loci linked to alcohol abuse and related psychiatric disorders in Finnish pedigrees. Alcohol Clin Exp Res 1998;1197A
Li  TK Pharmacogenetics of responses to alcohol and genes that influence alcohol drinking. J Stud Alcohol 2000;615- 12
PubMed
Reich  TEdenberg  HJGoate  AWilliams  JTRice  JPVan Eerdewegh  PForoud  THesselbrock  VSchuckit  MABucholz  KPorjesz  BLi  TKConneally  PMNurnberger  JI  JrTischfield  JACrowe  RRCloninger  CRWu  WShears  SCarr  KCrose  CWillig  CBegleiter  H Genome-wide search for genes affecting the risk for alcohol dependence. Am J Med Genet 1998;81207- 215
PubMed
Long  JCKnowler  WCHanson  RLRobin  RWUrbanek  MMoore  EBennett  PHGoldman  D Evidence for genetic linkage to alcohol dependence on chromosomes 4 and 11 from autosome-wide scan in an American Indian population. Am J Med Genet 1998;81216- 221
PubMed
Crabbe  JCBelknap  JKMitchell  SRCrawshaw  LI Quantitative trait loci mapping of genes that influence the sensitivity and tolerance to ethanol-induced hypothermia in BXD recombinant inbred mice. J Pharmacol Exp Ther 1994;269184- 188
PubMed
Johnson  TEDeFries  JCMarkel  PD Mapping quantitative trait loci for behavioral traits in the mouse. Behav Genet 1992;22635- 653
PubMed
Carr  LGForoud  TBice  PGobbett  TIvashina  JEdenberg  HLumeng  LLi  TK A quantitative trait locus for alcohol consumption in selectively bred rat lines. Alcohol Clin Exp Res 1998;22884- 887
PubMed
Hall  JMLee  MKNewman  BMorrow  JEAnderson  LAHuey  BKing  MC Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990;2501684- 1689
PubMed
Corder  EHSaunders  AMStrittmatter  WJSchmechel  DEGaskell  PCSmall  GWRoses  ADHaines  JLPericak-Vance  MA Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993;261921- 923
PubMed
Lander  EKruglyak  L Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995;11241- 247
PubMed
Taillon-Miller  PBauer-Sardina  ISaccone  NLPutzel  JLaitinen  TCao  AKere  JPilia  GRice  JPKwok  PY Juxtaposed regions of extensive and minimal linkage disequilibrium in human Xq25 and Xq28. Nat Genet 2000;25324- 328
PubMed
Eaves  IAMerriman  TRBarber  RANutland  STuomilehto-Wolf  ETuomilehto  JCucca  FTodd  JA The genetically isolated populations of Finland and Sardinia may not be a panacea for linkage disequilibrium mapping of common disease genes. Nat Genet 2000;25320- 323
PubMed
Hodge  SE What association analysis can and cannot tell us about the genetics of complex disease. Am J Med Genet 1994;54318- 323
PubMed
Lander  ESSchork  NJ Genetic dissection of complex traits. Science 1994;2652037- 2048
PubMed
Risch  NMerikangas  K The future of genetic studies of complex human diseases. Science 1996;2731516- 1517
PubMed
Tishkoff  SAGoldman  ACalafell  FSpeed  WCDeinard  ASBonne-Tamir  BKidd  JRPakstis  AJJenkins  TKidd  KK A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations. Am J Hum Genet 1998;621389- 1402
PubMed
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