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Family-based and case-control study of DRD2, DAT, 5HTT, COMT genes polymorphisms in alcohol dependence
Neuroscience Letters 410 (2006) 1–5
Family-based and case-control study of DRD2, DAT, 5HTT, COMT genes polymorphisms in alcohol dependence Jerzy Samochowiec a,∗ , Jolanta Kucharska-Mazur a , Anna Grzywacz a , Marcin Jabło´nski a , Hans Rommelspacher b , Agnieszka Samochowiec a,c , Mariusz Sznabowicz a , Jan Horodnicki a , Leszek Sagan d , Justyna Pełka-Wysiecka a a
Department of Psychiatry, Pomeranian Medical University, Broniewskiego 26, 71-460 Szczecin, Poland Department of Clinical Neurobiology, Free University of Berlin, Eschenallee 3, 14050 Berlin, Germany c MSKP, University of Szczecin, Szwole˙zer´ ow 18a, 71-062 Szczecin, Poland d Department of Neurosurgery, Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland b
Received 10 March 2006; received in revised form 29 April 2006; accepted 2 May 2006
Abstract The paper focuses on such candidate gene polymorphisms that alter alcoholism-related intermediate phenotypes including: dopaminergic system polymorphic variants (DRD2 -141C Ins/Del in promoter region, exon 8 and DRD2 TaqI A and DAT 40bp VNTR genes polymorphisms) that cause predisposition to severe alcoholism (haplotype Ins/G/A2); COMT Val158Met gene polymorphism related to differences in executive cognitive function and 5-HTT gene promoter polymorphism, which alters stress response and affects anxiety and dysphoria. The transmission disequilibrium test (TDT) was used in the study. One hundred Polish families with alcohol dependence were recruited. The control subjects for the case-control study were 196 ethnically and gender matched healthy individuals. It was found that DRD2 TaqIA and DAT gene polymorphisms contained statistically significant differences in allele transmission. In the homogenous subgroups of patients with early onset and with withdrawal complications a statistically significant preferential A2 allele transmission was found in DRD2 TaqIA gene polymorphism. The alleles and genotypes distribution of the investigated polymorphisms did not differ significantly between the alcoholics and the controls in the case-control study. The results confirmed the fact that the candidate genes (DRD2 and DAT) are partially responsible for the development of alcohol dependence. The results are also in agreement with the hypothesis that there are various subtypes of alcohol dependence, which differ depending on their genetic background. Meanwhile, the currently available pharmacological therapies for alcoholism treatment are effective in some alcoholics but not for all of them. Some progress has been made in elucidating pharmacogenomic responses to drugs, particularly in the context of Clonninger and Lesch typology classification system for alcoholics. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol dependence; Genetics; TDT; Case-control study; Polymorphisms
It is now accepted that the etiology of alcohol dependence is attributable to genetic and environmental factors. The results from twin and adoption studies provide estimations of heritability, the results from chromosomal linkage studies identify regions of chromosomes that may contain relevant genes, and it finally leads to candidate genes studies. A thorough genetic mapping study was performed to identify predisposing genes for alcoholism dependence using the Collaborative Study on the Genetics of Alcoholism (COGA) data. By
∗
Corresponding author. Tel.: +4891 4541507; fax: +4891 4540733. E-mail address: [email protected] (J. Samochowiec).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.05.005
using different methods and markers, high linkage signals at 1, 2, 7 chromosomes were found [46]. Reck et al. reported linkage signals detected on chromosomes 3, 4, 7, 10, and 12 [34]. Previous genetic studies of alcoholism suggest the existence of inherited functional variants of genes that alter the neurobiology of reward, executive cognitive function, anxiety and dysphoria, and neuronal plasticity, playing a crucial role in the mechanism of alcohol dependence [19,32]. The investigated candidate genes polymorphisms that alter alcoholism-related intermediate phenotypes include: dopaminergic system polymorphic variants (DRD2: dopamine D2 receptor -141C Ins/Del in promoter region, exon 8 and TaqI A and DAT: dopamine transporter 40bp VNTR in 3 UTR genes polymorphisms) that
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by means of a structured interview, based on the substance abuse section of Composite International Diagnostic Interview (CIDI) [35] and also using a sociodemographic questionnaire. Alcoholdependence status of the parents was assessed using MAST, AUDIT and RDC (Research Diagnostic Criteria) [43]. Two overlapping subgroups were formed with respect to more homogeneous etiology of alcohol-dependent subjects according to the following criteria: (1) history of delirium tremens or seizures during withdrawal; (2) alcoholics characterized by early age at onset, i.e. under 26 years of age. The control subjects were 196 ethnically and gender matched, unrelated healthy individuals (171 males, 25 females), with excluded psychiatric disorder using Prime MD questionnaire (Primary Care Evaluation of Mental Disorders) [39], mean age 39 ± 17, recruited in West Pomeranian region. Written informed consent was obtained from all the participants. The study protocol was approved by the Ethical Committee of Pomeranian Medical University of Szczecin. Genomic DNA was extracted from uncoagulated venous blood samples using a salting out method [26]. PCR procedures for the examined gene polymorphisms were described in literature: dopamine transporter DAT [44], dopamine receptor D2 TaqIA [38], dopamine receptor D2 141C ins/del, promoter region [1], dopamine receptor D2 exon 8 [9], catechol-O-methyltransferase [20], serotonin transporter 44 bp del (5HTT LPR) [14]. The Statistical Package for the Social Sciences (SPSS) computer program (PC version for Windows, release 9 in English) was used for the statistical analysis of the data. The families’ data were calculated using transmission disequilibrium test [36]. Differences between the controls and the alcoholics were tested by the χ2 -test and considered significant if the type 1 error was less than 5% using SPSS. The Hardy–Weinberg equilibrium was calculated using the SAS computer program for Windows [40]. While performing our calculations we considered using Bonferroni correction. However, because our division of patients into homogenous subgroups seemed to have a strong biological background we eventually decided not to make use of this correction after all. The numbers of informative parents and transmitted alleles are shown in Table 1. Statistically significant differences in allele transmission for the whole group were found in the following genes poly-
cause predisposition to severe alcoholism (haplotype Ins/G/A2) [37]; catechol-O-methyltransferase (COMT) Val158Met gene polymorphism related to differences in executive cognitive function [25] and serotonin transporter 5-HTT gene-linked promoter region (5-HTT-LPR), which alters stress response and affects anxiety and dysphoria [32]. Several association studies which were published earlier support the hypothesis about the role of the above mentioned genes polymorphisms in alcohol dependence. The results of association studies of DRD2 gene polymorphisms with alcohol dependence turned out to be inconclusive. According to Foley et al. [10] and Noble et al. [30] DRD2TaqI A polymorphism may influence predisposition to alcoholism. Blomqvist et al. found no linkage disequilibrium of alcohol dependence with DRD2 polymorphic systems: TaqI A, and the functional -141C Ins/Del promoter systems [3]. Some association studies showed a possible role that DRD2 exon 8 gene polymorphism may play in alcohol dependence [24,37]. Because the association case-control studies of the above mentioned polymorphisms produced inconsistent results, it was necessary to use an alternative method. In our investigations we used a family-based association study called the transmission disequilibrium test (TDT). It detects an effect of the observed excess of investigated allele in transmission from a heterozygous parent to an affected offspring. If there is no association, then according to the Mendelian theory, the probability of a concrete allele transmission is 50%. A statistically significant rise of an allele’s transmission suggests the role of investigated gene polymorphism in the etiology of a given disorder. One hundred Polish families, Caucasians, with no history of psychiatric disorders of axis I of ICD- 10 other than alcohol or tobacco dependence were recruited in the Department of Psychiatry of the Pomeranian Medical University of Szczecin and Addiction Unit in Stanomino. Alcohol dependent offspring were: 88 males aged: 34 ± 9 and 12 females aged 40 ± 8. Mean alcohol consumption was: 224 ± 108 g per day, mean age at onset was: 25 ± 7 years; 64% of them had an early onset of alcoholism (<26 years), 51% of patients fulfilled the criteria of heavy drinkers, 33% had at least one alcoholic parent, 79% were type I alcoholics according to Clonninger. According to Lesch typology our subjects were: type I-60%, type II-20%, type III-17% and type IV-3% [22]. Alcohol and family history were assessed
Table 1 Family-based study: number of informative families, both parents, fathers, mothers and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Number of informative both parents Number of informative fathers Number of informative mothers Transmission of alleles
46 10 31 25 36 × A10 (63%), 21 × A9 (37%) 3.95 0.047
44 8 24 28 39 × A2 (75%), 13 × A1 (25%) 11.79 0.0001
57 8 32 33 31 × A (48%), 34 × G (52%) 0.14 0.71
13 1 8 6 8 × ins C (57%), 6 × del C (43%) 0.29 0.593
60 14 35 39 33 × Val (44%), 41 × Met (56%) 0.86 0.352
71 10 38 43 46 × L (57%), 35 × s (43%) 1.49 0.222
χ2 p-values
A9—nine-repeat allele; A10—ten repeat allele; L—long variant; s—short variant.
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Table 2 Family-based study: a homogenous subgroup of early onset patients (AOO <26 years old): number of informative families and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Transmission of alleles
34 23 × A10 (57.5%), 17 × A9 (42.5%) 0.9 0.343
35 12 × A1 (30%), 28 × A2 (70%) 6.4 0.011
36 20 × A (49%), 21 × G (51%) 0.024 0.876
9 4 × del (40%), 6 × ins (60%) 0.4 0.527
41 25 × Val (48%), 27 × Met (52%) 0.77 0.782
41 23 × L (49%), 24 × s (51%) 0.21 0.884
χ2 p-values
morphisms: DRD2 TaqIA (preferential A2 allele transmission; p = 0.0001), DAT (preferential A10 allele transmission; p = 0.047) (Table 1). In the homogenous subgroup of patients with early onset statistically significant differences in allele transmission were found in the following genes polymorphisms: DRD2 TaqIA (preferential A2 allele transmission; p = 0.011) (Table 2). In the homogenous subgroup of patients with withdrawal complicated by delirium and/or seizures significant differences in allele transmission was found only in: DRD2 TaqIA gene polymorphism (preferential A2 allele transmission; p = 0.005) (Table 3). The alleles and genotypes distribution of investigated polymorphisms did not differ significantly between the alcoholics and the controls. No significant differences of alleles and genotypes frequencies were found between the two above mentioned homogenous subgroups and controls (data not shown). The observed genotypes counts of all the investigated polymorphisms in the subjects did not differ significantly from those expected from the Hardy–Weinberg equilibrium (p > 0.05) calculated separately for patients and controls. The transmission disequilibrium test is a popular method of genetic investigations due to its simplicity and advantages over other methods of studies. Because population frequencies of these alleles vary considerably, conflicting results could be due to population stratification bias. It was possible to avoid the problem in the present study using the transmission disequilibrium test. The controversy over the association between DRD2 receptor density and alcoholism led some scholars to question the utility of population association studies in the genetic analysis of complex disorders. However, it is still worth examining relationships between DRD2 and alcoholism in carefully ascertained subjects in varied populations [4]. Our previous association study in Germans and some literature data showed a possible role of DRD2 exon 8 gene
polymorphism and promoter DRD2 -141C ins/del gene polymorphism in alcohol dependence [37]. The present case-control and TDT study on the Polish population revealed no significant differences in the genotypes distribution between alcoholics and controls and allele’s transmission of the investigated polymorphisms. The family-based study results are consistent with those from a recent study of alcohol dependence [3]. Together, these studies suggest that the conflicting findings from casecontrol studies of the association between alleles of DRD2 and substance dependence may be attributable to population stratification in some samples. A1 allele of DRD2 Taq IA gene polymorphism is known to be associated with severe alcoholism [5]. A meta-analysis of studies done in Caucasian populations showed a significantly higher DRD2 A1 allelic frequency and its prevalence in alcoholics as compared to controls [14], but the detailed results are controversial. As reviewed by Noble, perhaps the different results across studies are a function of differences among populations with respect to severity of alcoholism [31]. In Africa, the A2 allele frequencies range from 52 to 73% [16], in German population: 78% [42], Finnish population 80% [11]. Generally, the A2 allele frequency varies in range between 15 and 91% [2]. A haplotype analysis of DRD2 polymorphisms revealed that East Asia and New World populations have shown a single frequent A1-containing haplotype in contrast to the populations of Africa, Middle East and Europe. Consequently, there is considerable allelic diversity among haplotypes containing the A1 allele, indicating that association studies are ignoring potentially significant genetic variation [17]. Finckh study proved that in German population haplotype variants containing A2 allele are more common among alcoholics than haplotypes containing the A1 allele [8]. In our study A2 allele is preferably transmitted to affected offspring. This result remains stabile even in homogenous subgroups of early onset patients and in severe alcoholics (with-
Table 3 Family-based study: a homogenous subgroup of patients with severe alcoholism (with alcohol seizures and/or delirium tremens): number of informative families and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Transmission of alleles
17 11 × 10 (55%), 9 × A9 (45%) 0.2 0.655
15 15 × A2 (83%), 3 × A1 (17%) 8.0 0.005
20 12 × A (52%), 11 × G (48%) 0.43 0.835
5 3 × ins C (60%), 2 × del C (40%) 1 1
18 12 × Val (60%), 8 × Met (40%) 0.80 0.371
26 19 × L (61%), 12 × s (39%) 1.58 0.209
χ2 p-values
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drawal complications with seizures and/or delirium). In the case-control study no association was found. One of the possible reasons of the opposite findings can be the fact, that in the studies the amount of alcohol intake was not taken into account. Hallikainen et al. reported that alcohol consumption in non-alcoholic group was significantly lower in A1/A1 and A1/A2 subjects than in A2/A2 subjects [13]. Munafo et al. replicated these findings in two independent samples and proved that DRD2 A1 allele was closely associated with reduced alcohol consumption in men [28]. Therefore, our hypothesis is that DRD2 Taq IA polymorphism may be linked not with alcoholism per se but rather with the quantity of alcohol consumption. Another possible explanation could be that A1 allele is linked with stimulation seeking (due to less potent dopamine binding). The A2 allele would be linked with severe alcoholism (with complications of alcohol withdrawal like delirium and/or seizures) [15]. This result should be further investigated in subgroups according, for example, to Lesch typology which qualitatively differentiates patients into different treatment response subgroups since those patients may show negative response to neuroleptics in alcoholism relapse prevention [22,45]. DAT 40 bp VNTR gene polymorphism A9 allele seems to be associated with severe alcoholism (withdrawal complicated with delirium and/or seizures) [23,39,41]. In the present study a preferential transmission of A10 allele to the affected offspring was found (p = 0.047). After a division into homogenous subgroups this effect disappeared. In the case-control study no association was found. The case-control study results for the whole group of alcoholics are in good agreement with the literature despite the fact that the significance of A9 allele in severe alcoholism was not confirmed. TDT study results suggest the role of A10 allele in genetic background of alcoholism, although the role of A9 allele was postulated by several previously cited authors of association studies. We did not find any literature of TDT studies with this gene polymorphism and alcoholism. Laine et al. suggested that TaqI A RFLP is in linkage disequilibrium with a gene variant modifying DAT density in alcoholics [21]. It is possible, that A2 allele of DRD2 TaqIA and A10 allele of DAT are inherited together more often than it would have been expected by a mere chance and this, in fact, could explain the results of the present study. COMT Val158Met and 5-HTT-LPR genes polymorphisms revealed adequate transmissions of alleles to the affected offspring. The results of this study are in good agreement with literature data [6,18]. The 5-HTT-LPR polymorphism is associated rather with anxiety-related personality traits [27], and therefore may influence the amount of alcohol consumption in neurotic subjects [29]. The data provided by Ploski et al. [33] revealed no variation among populations of different regions of Poland but showed their distinctiveness from other parts of Europe which should also be taken into consideration while discussing genetic results of Polish alcoholic families. Further studies are undoubtedly necessary in order to replicate this study’s findings and analyze the data by creating haplo-
types, and this is going to be done shortly. However, the results obtained in the present study confirmed a partial role of candidate genes (especially DRD2 and DAT) in the development of alcohol dependence and they are in agreement with the hypothesis that there are some subtypes of alcohol dependence, specially severe alcoholism complicated by alcohol seizures/delirium, which differ from other types of the disease depending on the genetic background. This may play a crucial role in the process of individuation of alcoholism therapy. Genetic studies, including case-control association studies and genome wide linkage studies have identified associations between alcoholism and common functional polymorphisms in several candidate genes. Meanwhile, the currently available pharmacological therapies for alcoholism are effective in some alcoholics but not for all of them. Some progress has been made in elucidating pharmacogenomic responses to drugs, particularly in the context of the Clonninger and Lesch typology classification system for alcoholics [7,12]. Acknowledgments This work was supported by KBN: 3 PO5D14622 and BMBF, POL 01/063. References [1] T. Arinami, M. Gao, H. Hamaguchi, M. Toru, A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia, Hum. Mol. Genet. 6 (1997) 577–582. [2] C. Barr, K. Kidd, Population frequencies of the A1 allele at the dopamine D2 receptor locus, Biol. Psychiatry 34 (1993) 204–209. [3] O. Blomqvist, J. Gelernter, H. Kranzler, Family-based study of DRD2 alleles in alcohol and drug dependence, Am. J. Med. Genet. 96 (2000) 659– 664. [4] A. Bowirrat, M. Oscar-Berman, Relationship between dopaminergic neurotransmission, alcoholism, and Reward Deficiency syndrome, Am. J. Med. Genet. B Neuropsychiatr. Genet. 132 (2005) 29–37. [5] J.P. Connor, R.M. Young, B.R. Lawford, T.L. Ritchie, E.P. Noble, D(2) dopamine receptor (DRD2) polymorphism is associated with severity of alcohol dependence, Eur. Psychiatry 17 (2002) 17–23. [6] P.R. Dodd, P.F. Foley, S.T. Buckley, A.L. Eckert, D.J. Innes, Genes and gene expression in the brain of the alcoholic, Addict. Behav. 29 (2004) 1295–1309. [7] M. Enoch, Pharmacogenomics of alcohol response and addiction, Am. J. Pharmacogenomics 4 (2003) 3217–3232. [8] U. Finckh, In: D. Agarwal, H. Seitz (Eds.), Alcohol in health and disease, Copyright by Marcel Dekker, Inc, New York-Basel, (2001) s.163. [9] U. Finckh, H. Rommelspacher, S. Kuhn, P. Dufeu, G. Otto, A. Heinz, M. Dettling, M. Giraldo-Velasquez, J. Pelz, K.J. Graf, H. Harms, T. Sander, L.G. Schmidt, A. Rolfs, Influence of the dopamine D2 receptor (DRD2) genotype on neuroadaptive effects of alcohol and the clinical outcome of alcoholism, Pharmacogenetics 7 (1997) 271–281. [10] P.F. Foley, E.W. Loh, D.J. Innes, S.M. Williams, A.E. Tannenberg, C.G. Harper, P.R. Dodd, Association studies of neurotransmitter gene polymorphisms in alcoholic Caucasians, Ann. N.Y. Acad. Sci. 1025 (2004) 39–46. [11] T. Geijer, J. Neiman, U. Rydberg, L. Terenius, Dopamine D2-receptor gene polymorphisms in Scandinavian chronic alcoholics, Eur. Arch. Psychiatry Clin. Neurosci. 244 (1994) 26–32. [12] D. Goldman, G. Oroszi, S. O’Malley, R. Anton, COMBINE genetics study: the pharmacogenetics of alcoholism treatment response: genes and mechanisms, J. Stud. Alcohol. Suppl. 15 (2005) 56–64. [13] T. Hallikainen, J. Hietala, J. Kauhanen, T. Pohjalainen, E. Syvalahti, J.T. Salonen, J. Tiihonen, Ethanol consumption and DRD2 gene TaqI a poly-
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Family-based and case-control study of DRD2, DAT, 5HTT, COMT genes polymorphisms in alcohol dependence Jerzy Samochowiec a,∗ , Jolanta Kucharska-Mazur a , Anna Grzywacz a , Marcin Jabło´nski a , Hans Rommelspacher b , Agnieszka Samochowiec a,c , Mariusz Sznabowicz a , Jan Horodnicki a , Leszek Sagan d , Justyna Pełka-Wysiecka a a
Department of Psychiatry, Pomeranian Medical University, Broniewskiego 26, 71-460 Szczecin, Poland Department of Clinical Neurobiology, Free University of Berlin, Eschenallee 3, 14050 Berlin, Germany c MSKP, University of Szczecin, Szwole˙zer´ ow 18a, 71-062 Szczecin, Poland d Department of Neurosurgery, Pomeranian Medical University, Unii Lubelskiej 1, 71-252 Szczecin, Poland b
Received 10 March 2006; received in revised form 29 April 2006; accepted 2 May 2006
Abstract The paper focuses on such candidate gene polymorphisms that alter alcoholism-related intermediate phenotypes including: dopaminergic system polymorphic variants (DRD2 -141C Ins/Del in promoter region, exon 8 and DRD2 TaqI A and DAT 40bp VNTR genes polymorphisms) that cause predisposition to severe alcoholism (haplotype Ins/G/A2); COMT Val158Met gene polymorphism related to differences in executive cognitive function and 5-HTT gene promoter polymorphism, which alters stress response and affects anxiety and dysphoria. The transmission disequilibrium test (TDT) was used in the study. One hundred Polish families with alcohol dependence were recruited. The control subjects for the case-control study were 196 ethnically and gender matched healthy individuals. It was found that DRD2 TaqIA and DAT gene polymorphisms contained statistically significant differences in allele transmission. In the homogenous subgroups of patients with early onset and with withdrawal complications a statistically significant preferential A2 allele transmission was found in DRD2 TaqIA gene polymorphism. The alleles and genotypes distribution of the investigated polymorphisms did not differ significantly between the alcoholics and the controls in the case-control study. The results confirmed the fact that the candidate genes (DRD2 and DAT) are partially responsible for the development of alcohol dependence. The results are also in agreement with the hypothesis that there are various subtypes of alcohol dependence, which differ depending on their genetic background. Meanwhile, the currently available pharmacological therapies for alcoholism treatment are effective in some alcoholics but not for all of them. Some progress has been made in elucidating pharmacogenomic responses to drugs, particularly in the context of Clonninger and Lesch typology classification system for alcoholics. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol dependence; Genetics; TDT; Case-control study; Polymorphisms
It is now accepted that the etiology of alcohol dependence is attributable to genetic and environmental factors. The results from twin and adoption studies provide estimations of heritability, the results from chromosomal linkage studies identify regions of chromosomes that may contain relevant genes, and it finally leads to candidate genes studies. A thorough genetic mapping study was performed to identify predisposing genes for alcoholism dependence using the Collaborative Study on the Genetics of Alcoholism (COGA) data. By
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using different methods and markers, high linkage signals at 1, 2, 7 chromosomes were found [46]. Reck et al. reported linkage signals detected on chromosomes 3, 4, 7, 10, and 12 [34]. Previous genetic studies of alcoholism suggest the existence of inherited functional variants of genes that alter the neurobiology of reward, executive cognitive function, anxiety and dysphoria, and neuronal plasticity, playing a crucial role in the mechanism of alcohol dependence [19,32]. The investigated candidate genes polymorphisms that alter alcoholism-related intermediate phenotypes include: dopaminergic system polymorphic variants (DRD2: dopamine D2 receptor -141C Ins/Del in promoter region, exon 8 and TaqI A and DAT: dopamine transporter 40bp VNTR in 3 UTR genes polymorphisms) that
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by means of a structured interview, based on the substance abuse section of Composite International Diagnostic Interview (CIDI) [35] and also using a sociodemographic questionnaire. Alcoholdependence status of the parents was assessed using MAST, AUDIT and RDC (Research Diagnostic Criteria) [43]. Two overlapping subgroups were formed with respect to more homogeneous etiology of alcohol-dependent subjects according to the following criteria: (1) history of delirium tremens or seizures during withdrawal; (2) alcoholics characterized by early age at onset, i.e. under 26 years of age. The control subjects were 196 ethnically and gender matched, unrelated healthy individuals (171 males, 25 females), with excluded psychiatric disorder using Prime MD questionnaire (Primary Care Evaluation of Mental Disorders) [39], mean age 39 ± 17, recruited in West Pomeranian region. Written informed consent was obtained from all the participants. The study protocol was approved by the Ethical Committee of Pomeranian Medical University of Szczecin. Genomic DNA was extracted from uncoagulated venous blood samples using a salting out method [26]. PCR procedures for the examined gene polymorphisms were described in literature: dopamine transporter DAT [44], dopamine receptor D2 TaqIA [38], dopamine receptor D2 141C ins/del, promoter region [1], dopamine receptor D2 exon 8 [9], catechol-O-methyltransferase [20], serotonin transporter 44 bp del (5HTT LPR) [14]. The Statistical Package for the Social Sciences (SPSS) computer program (PC version for Windows, release 9 in English) was used for the statistical analysis of the data. The families’ data were calculated using transmission disequilibrium test [36]. Differences between the controls and the alcoholics were tested by the χ2 -test and considered significant if the type 1 error was less than 5% using SPSS. The Hardy–Weinberg equilibrium was calculated using the SAS computer program for Windows [40]. While performing our calculations we considered using Bonferroni correction. However, because our division of patients into homogenous subgroups seemed to have a strong biological background we eventually decided not to make use of this correction after all. The numbers of informative parents and transmitted alleles are shown in Table 1. Statistically significant differences in allele transmission for the whole group were found in the following genes poly-
cause predisposition to severe alcoholism (haplotype Ins/G/A2) [37]; catechol-O-methyltransferase (COMT) Val158Met gene polymorphism related to differences in executive cognitive function [25] and serotonin transporter 5-HTT gene-linked promoter region (5-HTT-LPR), which alters stress response and affects anxiety and dysphoria [32]. Several association studies which were published earlier support the hypothesis about the role of the above mentioned genes polymorphisms in alcohol dependence. The results of association studies of DRD2 gene polymorphisms with alcohol dependence turned out to be inconclusive. According to Foley et al. [10] and Noble et al. [30] DRD2TaqI A polymorphism may influence predisposition to alcoholism. Blomqvist et al. found no linkage disequilibrium of alcohol dependence with DRD2 polymorphic systems: TaqI A, and the functional -141C Ins/Del promoter systems [3]. Some association studies showed a possible role that DRD2 exon 8 gene polymorphism may play in alcohol dependence [24,37]. Because the association case-control studies of the above mentioned polymorphisms produced inconsistent results, it was necessary to use an alternative method. In our investigations we used a family-based association study called the transmission disequilibrium test (TDT). It detects an effect of the observed excess of investigated allele in transmission from a heterozygous parent to an affected offspring. If there is no association, then according to the Mendelian theory, the probability of a concrete allele transmission is 50%. A statistically significant rise of an allele’s transmission suggests the role of investigated gene polymorphism in the etiology of a given disorder. One hundred Polish families, Caucasians, with no history of psychiatric disorders of axis I of ICD- 10 other than alcohol or tobacco dependence were recruited in the Department of Psychiatry of the Pomeranian Medical University of Szczecin and Addiction Unit in Stanomino. Alcohol dependent offspring were: 88 males aged: 34 ± 9 and 12 females aged 40 ± 8. Mean alcohol consumption was: 224 ± 108 g per day, mean age at onset was: 25 ± 7 years; 64% of them had an early onset of alcoholism (<26 years), 51% of patients fulfilled the criteria of heavy drinkers, 33% had at least one alcoholic parent, 79% were type I alcoholics according to Clonninger. According to Lesch typology our subjects were: type I-60%, type II-20%, type III-17% and type IV-3% [22]. Alcohol and family history were assessed
Table 1 Family-based study: number of informative families, both parents, fathers, mothers and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Number of informative both parents Number of informative fathers Number of informative mothers Transmission of alleles
46 10 31 25 36 × A10 (63%), 21 × A9 (37%) 3.95 0.047
44 8 24 28 39 × A2 (75%), 13 × A1 (25%) 11.79 0.0001
57 8 32 33 31 × A (48%), 34 × G (52%) 0.14 0.71
13 1 8 6 8 × ins C (57%), 6 × del C (43%) 0.29 0.593
60 14 35 39 33 × Val (44%), 41 × Met (56%) 0.86 0.352
71 10 38 43 46 × L (57%), 35 × s (43%) 1.49 0.222
χ2 p-values
A9—nine-repeat allele; A10—ten repeat allele; L—long variant; s—short variant.
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Table 2 Family-based study: a homogenous subgroup of early onset patients (AOO <26 years old): number of informative families and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Transmission of alleles
34 23 × A10 (57.5%), 17 × A9 (42.5%) 0.9 0.343
35 12 × A1 (30%), 28 × A2 (70%) 6.4 0.011
36 20 × A (49%), 21 × G (51%) 0.024 0.876
9 4 × del (40%), 6 × ins (60%) 0.4 0.527
41 25 × Val (48%), 27 × Met (52%) 0.77 0.782
41 23 × L (49%), 24 × s (51%) 0.21 0.884
χ2 p-values
morphisms: DRD2 TaqIA (preferential A2 allele transmission; p = 0.0001), DAT (preferential A10 allele transmission; p = 0.047) (Table 1). In the homogenous subgroup of patients with early onset statistically significant differences in allele transmission were found in the following genes polymorphisms: DRD2 TaqIA (preferential A2 allele transmission; p = 0.011) (Table 2). In the homogenous subgroup of patients with withdrawal complicated by delirium and/or seizures significant differences in allele transmission was found only in: DRD2 TaqIA gene polymorphism (preferential A2 allele transmission; p = 0.005) (Table 3). The alleles and genotypes distribution of investigated polymorphisms did not differ significantly between the alcoholics and the controls. No significant differences of alleles and genotypes frequencies were found between the two above mentioned homogenous subgroups and controls (data not shown). The observed genotypes counts of all the investigated polymorphisms in the subjects did not differ significantly from those expected from the Hardy–Weinberg equilibrium (p > 0.05) calculated separately for patients and controls. The transmission disequilibrium test is a popular method of genetic investigations due to its simplicity and advantages over other methods of studies. Because population frequencies of these alleles vary considerably, conflicting results could be due to population stratification bias. It was possible to avoid the problem in the present study using the transmission disequilibrium test. The controversy over the association between DRD2 receptor density and alcoholism led some scholars to question the utility of population association studies in the genetic analysis of complex disorders. However, it is still worth examining relationships between DRD2 and alcoholism in carefully ascertained subjects in varied populations [4]. Our previous association study in Germans and some literature data showed a possible role of DRD2 exon 8 gene
polymorphism and promoter DRD2 -141C ins/del gene polymorphism in alcohol dependence [37]. The present case-control and TDT study on the Polish population revealed no significant differences in the genotypes distribution between alcoholics and controls and allele’s transmission of the investigated polymorphisms. The family-based study results are consistent with those from a recent study of alcohol dependence [3]. Together, these studies suggest that the conflicting findings from casecontrol studies of the association between alleles of DRD2 and substance dependence may be attributable to population stratification in some samples. A1 allele of DRD2 Taq IA gene polymorphism is known to be associated with severe alcoholism [5]. A meta-analysis of studies done in Caucasian populations showed a significantly higher DRD2 A1 allelic frequency and its prevalence in alcoholics as compared to controls [14], but the detailed results are controversial. As reviewed by Noble, perhaps the different results across studies are a function of differences among populations with respect to severity of alcoholism [31]. In Africa, the A2 allele frequencies range from 52 to 73% [16], in German population: 78% [42], Finnish population 80% [11]. Generally, the A2 allele frequency varies in range between 15 and 91% [2]. A haplotype analysis of DRD2 polymorphisms revealed that East Asia and New World populations have shown a single frequent A1-containing haplotype in contrast to the populations of Africa, Middle East and Europe. Consequently, there is considerable allelic diversity among haplotypes containing the A1 allele, indicating that association studies are ignoring potentially significant genetic variation [17]. Finckh study proved that in German population haplotype variants containing A2 allele are more common among alcoholics than haplotypes containing the A1 allele [8]. In our study A2 allele is preferably transmitted to affected offspring. This result remains stabile even in homogenous subgroups of early onset patients and in severe alcoholics (with-
Table 3 Family-based study: a homogenous subgroup of patients with severe alcoholism (with alcohol seizures and/or delirium tremens): number of informative families and the transmission of alleles to the affected offspring Genes polymorphisms
DAT
DRD2 TaqIA
DRD2 Ex 8
DRD2 -141C ins/del
COMT
5HTT
Number of informative families Transmission of alleles
17 11 × 10 (55%), 9 × A9 (45%) 0.2 0.655
15 15 × A2 (83%), 3 × A1 (17%) 8.0 0.005
20 12 × A (52%), 11 × G (48%) 0.43 0.835
5 3 × ins C (60%), 2 × del C (40%) 1 1
18 12 × Val (60%), 8 × Met (40%) 0.80 0.371
26 19 × L (61%), 12 × s (39%) 1.58 0.209
χ2 p-values
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drawal complications with seizures and/or delirium). In the case-control study no association was found. One of the possible reasons of the opposite findings can be the fact, that in the studies the amount of alcohol intake was not taken into account. Hallikainen et al. reported that alcohol consumption in non-alcoholic group was significantly lower in A1/A1 and A1/A2 subjects than in A2/A2 subjects [13]. Munafo et al. replicated these findings in two independent samples and proved that DRD2 A1 allele was closely associated with reduced alcohol consumption in men [28]. Therefore, our hypothesis is that DRD2 Taq IA polymorphism may be linked not with alcoholism per se but rather with the quantity of alcohol consumption. Another possible explanation could be that A1 allele is linked with stimulation seeking (due to less potent dopamine binding). The A2 allele would be linked with severe alcoholism (with complications of alcohol withdrawal like delirium and/or seizures) [15]. This result should be further investigated in subgroups according, for example, to Lesch typology which qualitatively differentiates patients into different treatment response subgroups since those patients may show negative response to neuroleptics in alcoholism relapse prevention [22,45]. DAT 40 bp VNTR gene polymorphism A9 allele seems to be associated with severe alcoholism (withdrawal complicated with delirium and/or seizures) [23,39,41]. In the present study a preferential transmission of A10 allele to the affected offspring was found (p = 0.047). After a division into homogenous subgroups this effect disappeared. In the case-control study no association was found. The case-control study results for the whole group of alcoholics are in good agreement with the literature despite the fact that the significance of A9 allele in severe alcoholism was not confirmed. TDT study results suggest the role of A10 allele in genetic background of alcoholism, although the role of A9 allele was postulated by several previously cited authors of association studies. We did not find any literature of TDT studies with this gene polymorphism and alcoholism. Laine et al. suggested that TaqI A RFLP is in linkage disequilibrium with a gene variant modifying DAT density in alcoholics [21]. It is possible, that A2 allele of DRD2 TaqIA and A10 allele of DAT are inherited together more often than it would have been expected by a mere chance and this, in fact, could explain the results of the present study. COMT Val158Met and 5-HTT-LPR genes polymorphisms revealed adequate transmissions of alleles to the affected offspring. The results of this study are in good agreement with literature data [6,18]. The 5-HTT-LPR polymorphism is associated rather with anxiety-related personality traits [27], and therefore may influence the amount of alcohol consumption in neurotic subjects [29]. The data provided by Ploski et al. [33] revealed no variation among populations of different regions of Poland but showed their distinctiveness from other parts of Europe which should also be taken into consideration while discussing genetic results of Polish alcoholic families. Further studies are undoubtedly necessary in order to replicate this study’s findings and analyze the data by creating haplo-
types, and this is going to be done shortly. However, the results obtained in the present study confirmed a partial role of candidate genes (especially DRD2 and DAT) in the development of alcohol dependence and they are in agreement with the hypothesis that there are some subtypes of alcohol dependence, specially severe alcoholism complicated by alcohol seizures/delirium, which differ from other types of the disease depending on the genetic background. This may play a crucial role in the process of individuation of alcoholism therapy. Genetic studies, including case-control association studies and genome wide linkage studies have identified associations between alcoholism and common functional polymorphisms in several candidate genes. Meanwhile, the currently available pharmacological therapies for alcoholism are effective in some alcoholics but not for all of them. Some progress has been made in elucidating pharmacogenomic responses to drugs, particularly in the context of the Clonninger and Lesch typology classification system for alcoholics [7,12]. Acknowledgments This work was supported by KBN: 3 PO5D14622 and BMBF, POL 01/063. References [1] T. Arinami, M. Gao, H. Hamaguchi, M. Toru, A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia, Hum. Mol. Genet. 6 (1997) 577–582. [2] C. Barr, K. Kidd, Population frequencies of the A1 allele at the dopamine D2 receptor locus, Biol. Psychiatry 34 (1993) 204–209. [3] O. Blomqvist, J. Gelernter, H. Kranzler, Family-based study of DRD2 alleles in alcohol and drug dependence, Am. J. Med. Genet. 96 (2000) 659– 664. [4] A. Bowirrat, M. Oscar-Berman, Relationship between dopaminergic neurotransmission, alcoholism, and Reward Deficiency syndrome, Am. J. Med. Genet. B Neuropsychiatr. Genet. 132 (2005) 29–37. [5] J.P. Connor, R.M. Young, B.R. Lawford, T.L. Ritchie, E.P. Noble, D(2) dopamine receptor (DRD2) polymorphism is associated with severity of alcohol dependence, Eur. Psychiatry 17 (2002) 17–23. [6] P.R. Dodd, P.F. Foley, S.T. Buckley, A.L. Eckert, D.J. Innes, Genes and gene expression in the brain of the alcoholic, Addict. Behav. 29 (2004) 1295–1309. [7] M. Enoch, Pharmacogenomics of alcohol response and addiction, Am. J. Pharmacogenomics 4 (2003) 3217–3232. [8] U. Finckh, In: D. Agarwal, H. Seitz (Eds.), Alcohol in health and disease, Copyright by Marcel Dekker, Inc, New York-Basel, (2001) s.163. [9] U. Finckh, H. Rommelspacher, S. Kuhn, P. Dufeu, G. Otto, A. Heinz, M. Dettling, M. Giraldo-Velasquez, J. Pelz, K.J. Graf, H. Harms, T. Sander, L.G. Schmidt, A. Rolfs, Influence of the dopamine D2 receptor (DRD2) genotype on neuroadaptive effects of alcohol and the clinical outcome of alcoholism, Pharmacogenetics 7 (1997) 271–281. [10] P.F. Foley, E.W. Loh, D.J. Innes, S.M. Williams, A.E. Tannenberg, C.G. Harper, P.R. Dodd, Association studies of neurotransmitter gene polymorphisms in alcoholic Caucasians, Ann. N.Y. Acad. Sci. 1025 (2004) 39–46. [11] T. Geijer, J. Neiman, U. Rydberg, L. Terenius, Dopamine D2-receptor gene polymorphisms in Scandinavian chronic alcoholics, Eur. Arch. Psychiatry Clin. Neurosci. 244 (1994) 26–32. [12] D. Goldman, G. Oroszi, S. O’Malley, R. Anton, COMBINE genetics study: the pharmacogenetics of alcoholism treatment response: genes and mechanisms, J. Stud. Alcohol. Suppl. 15 (2005) 56–64. [13] T. Hallikainen, J. Hietala, J. Kauhanen, T. Pohjalainen, E. Syvalahti, J.T. Salonen, J. Tiihonen, Ethanol consumption and DRD2 gene TaqI a poly-
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