Key words: GENETIC STUDY DESIGN 213

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1 doi: /j x Genetics of alcohol dependencepcn_ Mitsuru Kimura, MD, PhD 1,2 * and Susumu Higuchi, MD, PhD 1 1 National Hospital Organization, Kurihama Alcoholism Center, Kanagawa, Japan, and 2 Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, Maryland, USA Twin, family, and adoption studies have consistently shown that genetic factors play an important role in the pathogenesis of alcohol dependence. Numerous studies have aimed to identify genes that contribute to susceptibility to alcohol dependence. Whole-genome linkage studies have identified several chromosomal regions that are linked with alcohol dependence. Association studies have also identified genes associated with alcohol dependence. Alcohol-metabolizing enzymes, such as alcohol dehydrogenase-1b and aldehyde dehydrogenase-2, are the most well-established genes that have polymorphisms associated with the risk for alcohol dependence. Polymorphisms in gammaaminobutyric acid receptor genes are also reported to be associated with alcohol dependence. The polymorphism of opioid receptor mu 1 gene is of interest because it alters the treatment effects of naltrexone. Several genes related to neural transmission have been reported to be associated with alcohol dependence, but results are inconsistent among studies. One reason for these inconsistent results is the great heterogeneity of alcohol dependence. Classifying alcohol dependence into homogeneous phenotypes is a good strategy to solve this problem. Recently, several genome-wide association studies have been reported. Genome-wide association studies enable hypothesis-free genome mapping of vulnerability-contributing genes and are expected to add data to identify genes associated with the susceptibility to alcohol dependence. Knowledge of the genetic basis of alcohol dependence is growing and leads to a better understanding of the biological mechanisms of addiction, which can help with strategies to prevent and treat this disease. Key words: alcoholism, genetic linkage, genetic polymorphism, genome-wide association study, single nucleotide polymorphisms. ALCOHOL DEPENDENCE IS a common disorder that causes physical, psychological, and social problems. The pathogenesis of alcohol dependence is multifactorial and includes shared genetic and environmental factors. Twin, family, and adoption studies have consistently shown that genetic factors play an important role in the development of alcohol dependence. Twin studies have estimated the heritability of alcohol dependence to be about 50 65% in both sexes. 1 3 Many studies have aimed to identify genes that contribute to the susceptibility to alcohol dependence. This article reviews the accumulated findings from genetic studies aimed at identifying genes associated with the risk for alcohol dependence. *Correspondence: Mitsuru Kimura, MD, PhD, National Hospital Organization, Kurihama Alcoholism Center, Nobi, Yokosuka, Kanagawa , Japan. kimura.mitsuru@gmail.com Accepted 15 December GENETIC STUDY DESIGN There are two strategies to identify genes related to the vulnerability for alcohol dependence: one is 213

2 214 M. Kimura and S. Higuchi Psychiatry and Clinical Neurosciences 2011; 65: genome-wide analysis including whole-genome linkage studies and genome-wide association studies (GWAS); and the other is candidate gene association studies. Whole-genome linkage studies use familybased samples and test genetic polymorphisms throughout the genome. Linkage studies can identify the region on the chromosome involved in the susceptibility to a disease or related traits. This method enables hypothesis-free scans of the entire genome, but the region of interest is usually broad and the resolution is low. Recent progress in genotyping allows for GWAS. These studies can use large case and control samples in the general population with increased genomic density. GWAS are a powerful tool in detecting relatively common polymorphisms that affect susceptibility to disease. Candidate gene association studies are conducted with a hypothesis based on the gene function that contributes to the pathogenesis of the disease or the pharmacological treatment process. The region covered by association studies is much smaller than in linkage studies and can be used for a subsequent analysis of wholegenome linkage studies. Association studies usually use case control designs and examine the association between the disease and gene variants like single nucleotide polymorphisms (SNP). A number of studies have identified gene polymorphisms associated with the risk for alcohol dependence. The quality of samples with regard to diagnosis and characterization is critical to achieve reliable results of genetic studies. One of the largest datasets that have provided successful results in identifying genes that influence the risk of alcohol dependence is the Collaborative Study on the Genetics of Alcoholism (COGA). 4 COGA collected a large, family-based dataset from multiple sites; and the diagnosis of alcohol dependence was assessed with the Semi- Structured Assessment for the Genetics of Alcoholism (SSAGA). A notable feature of COGA is that it also collected detailed information including endophenotypes related to the risk for alcohol dependence. The diagnosis of alcohol dependence is highly heterogenic and it might mask the relatively small effect of genes. However, analyzing endophenotypes can reduce the heterogeneity of samples. For example, the pattern of event-related potentials (ERP) measured by electroencephalograms (EEG) differs between patients with alcohol dependence and controls, and the pattern is also seen in unaffected offspring of alcoholics. Such endophenotypes are considered more homogenous, reflecting the genetic basis of alcohol dependence. Therefore, comparisons between endophenotypes may be advantageous in the identification of genetic factors with strong statistical power. GENETIC LINKAGE STUDY Whole-genome linkage studies have identified several chromosomal regions that influence the vulnerability of alcohol dependence. The first reports were studies that used family subjects from the COGA study 5,6 and an American Indian population. 7 Several subsequent studies were reported using large family-based data of Mission Indians, 8 families in the Pittsburgh area, 9 Irish families, 10 and African- American families. 11 The most consistent findings of these linkage studies were detected on chromosome 4, that is, the chromosome 4q region that contains the alcohol dehydrogenase (ADH) gene cluster including ADH1B and ADH1C genes, 5,7,8,10 and the chromosome 4p region, which is close to the gene cluster of gamma-aminobutyric acid (GABA) receptors. 5,7 A linkage to chromosome 1 was also reported by multiple studies. 5,9 Several studies have shown evidence that some regions on genomes were linked with endophenotypes related to alcohol dependence, such as electrophysiological phenotypes, lowlevel response to alcohol, 16,17 and maximum number of drinks. 18 ASSOCIATION STUDIES Because the pathogenesis of alcohol dependence is so complex, many genes are anticipated to be involved and candidate genes have a wide range in function. Most of the fruitful works in association studies have been achieved by aiming to clarify the association in genes involved in alcohol metabolism and neural transmission. The genes of alcohol-metabolizing enzymes, aldehyde dehydrogenase-2 (ALDH2) and ADH1B, are the most well-established genes in which polymorphisms are associated with the risk for alcoholism. Many association studies have found polymorphic loci associated with the risk for alcohol dependence in the genes implicated in neural transmission (for example, dopamine, serotonin, and GABA receptors), but results were inconsistent among studies with different sample sets. Alcohol dehydrogenase Most alcohol taken into the body is eliminated in the liver; first, ethanol to acetaldehyde by ADH; second,

3 Psychiatry and Clinical Neurosciences 2011; 65: Genetics of alcohol dependence 215 acetaldehyde to acetic acid by ALDH. Variations in the activity of these enzymes alter drinking behavior, further affecting the vulnerability for alcohol dependence. Seven ADH genes have been identified in humans. These genes cluster on the chromosome 4q region on which several whole-genome studies suggested linkage with the risk for alcohol dependence. 5 7 Class I ADH, encoded by ADH1A, ADH1B, and ADH1C, are considered to play a major role, and class II ADH, encoded by ADH4, plays some role in metabolizing ethanol in the liver. ADH1B, which encodes the beta subunit of class I ADH, has polymorphic loci: ADH1B*1, ADH1B*2, and ADH1B*3. The ADH1B*2 allele has an arginine to histidine substitution at the 48th position and the ADH1B*3 allele has an arginine to cysteine substitution at the 370th position. The ADH1B*2 allele is common in Asian populations; about 10% of Caucasians and 70% of Asians have the ADH1B*2 allele. ADH1B*3 is comparatively common in African ancestry populations. 19 The beta subunit encoded by ADH1B*2 and ADH1B*3 allele reveals greater activity of ethanol oxidization than the ADH1B*1 allele. 20 Association studies have consistently shown that the ADH1B*2 allele has a preventive effect against alcoholism. The allele frequencies of ADH1B*2 are lower in alcoholics than in controls regardless of sex and ethnicity. The mechanism for this effect of ADH1B is unclear. One possible explanation is that higher acetaldehyde production due to higher activity of ADH1B*2 causes adverse effects that prevent drinking behavior, similar to what occurs with inactive ALDH2. 21 A study using an animal model supports this hypothesis, although there has been no evidence of an accumulation of acetaldehyde after alcohol intake in humans with the ADH1B*2 allele. 22 The subunit encoded by the ADH1B*3 allele reveals higher activity of ethanol oxidization, and the ADH1B*3 allele has also been reported to have protective effects against the risk for alcohol dependence in the same manner as the ADH1B*2 allele. 23,24 The gamma subunit encoded by ADH1C also has a polymorphic locus at the 272nd and 350th amino acid position in high linkage equilibrium: ADH1C*1 with 272Arg-350Ile and ADH1C*2 with 272Gln- 350Val. Several studies have indicated that the ADH1C*2 allele, which codes the less active subunit, is associated with higher risk for alcohol dependence than the ADH1C*1 allele This association might be due to the linkage equilibrium between ADH1C*1 and ADH1B*2, 28 but the association is detected even in populations that rarely have the ADH1B*2 variant. 29 Compared with class I ADH, there are fewer studies about the association between variant class II ADH encoded by the ADH4 gene and alcohol dependence. Edenberg et al. analyzed SNP markers at the ADH gene cluster on chromosome 4q22 and found the strongest association with alcohol dependence was detected at the ADH4 gene region. 23 Some studies have supported that polymorphisms in the ADH4 gene are associated with the risk for alcohol dependence, but the particular locus that is responsible for the association and its function are still unclear. Aldehyde dehydrogenase Most acetaldehyde produced from ethanol by ADH is oxidized to acetic acid by ALDH in the liver. There are several ALDH family genes in humans. Mitochondrial ALDH2 is considered to play a major role in eliminating acetaldehyde, and cytosolic ALDH1A1 and mitochondrial ALDH1B1 are also involved in acetaldehyde metabolism. 33 The ALDH2 gene is located on chromosome12q24 and has 12 exons. There is a polymorphic locus on exon 12 with amino acid substitution from glutamic acid to lysine at the 504th position. The subunit encoded by 504Lys allele (ALDH2*2 allele) lacks any activity to catalyze acetaldehyde. The homozygote of ALDH2*2 allele has no activity to eliminate acetaldehyde, and even heterozygotes have much less activity. This polymorphism is found only in the East-Asian population. The significant impact on acetaldehyde elimination of inactive ALDH2 causes high blood acetaldehyde concentrations after drinking and results in a painful adverse effect called the flushing response. 34 Individuals with the ALDH2*2 allele experience facial flushing, nausea, headache, and rapid heartbeat after drinking. This response generally leads to the avoidance of excessive alcohol consumption. There is evidence that the ALDH2 polymorphism is associated with both the drinking behavior of healthy people and the risk for alcohol dependence. 35 Alcohol consumption in healthy people decreases with increasing gene doses. Our case control data among Japanese alcoholics and controls showed that the genotype frequencies of ALDH2*1/2*1, ALDH2*1/ 2*2, and ALDH2*2/2*2 in healthy controls were 55.4%, 38.8%, and 5.8%, respectively; however, the frequency of ALDH2*1/2*2 heterozygotes were much

4 216 M. Kimura and S. Higuchi Psychiatry and Clinical Neurosciences 2011; 65: lower (12.0%) in alcoholic patients and no alcoholic patient was seen to have the ALDH2*2/2*2 homozygote genotype. 36 In addition, studies among Korean, Taiwanese, and Chinese populations consistently have indicated that the inactive type of ALDH2 allele (ALDH2*2) has a strong preventive effect against alcohol dependence, and the homozygote of this allele almost completely prevents individuals from becoming alcoholics, 37 although a few studies have reported rare cases who were homozygous for ALDH2* The ALDH2 polymorphism is also associated with alcohol-related organ damage. Alcoholics who have inactive ALDH2 have a high risk for upper gastrointestinal tract cancers, such as esophageal and oropharyngeal cancers. The repetitive exposure to acetaldehyde, which has a carcinogenic effect, increases the risk for upper gastrointestinal tract cancers in non-alcoholic moderate or heavy drinkers as well, interacting with both drinking and smoking habits. A few studies suggest that inactive ALDH2 increases the risk for polyneuropathy and central nervous system damage. 41,42 Alcoholics who have the inactive form of ALDH2 have interesting traits. Personality traits differ between alcoholics with active ALDH2 and those with inactive ALDH2. Alcoholics with inactive ALDH2 have higher novelty-seeking and lower harmavoidance scores on the tridimensional personality questionnaire. 43 In addition, alcoholics with inactive ALDH2 develop alcohol-related symptoms and problems more slowly than those with active ALDH2. 44 Hahn et al. studied alcohol expectancy scales in alcoholics with active and inactive ALDH2 and showed that negative alcohol expectancies scale scores were lower and positive alcohol expectancies scale scores were higher in alcoholics with inactive ALDH2 than in those with active ALDH2. 45 These distinguishing characteristics might suggest that alcoholics who have inactive ALDH2 possess other traits that make them keep drinking despite the painful response caused by deficiency of ALDH2 activity. Compared with the ALDH2 polymorphism, there are fewer studies about the association between the ALDH1A1 polymorphism and alcohol dependence. The deficiency of ALDH1A1 activity causes the same type of flushing seen in subjects with inactive ALDH2. 46 Some studies have indicated that polymorphisms in the ALDH1A1 gene are associated with the risk for alcohol dependence and drinking behavior GABA receptors GABA inhibitory neurotransmission is considered to mediate the pharmacological effects of alcohol in the brain. Several GABA receptor genes, including GABRG1, GABRA2, GABRA4, and GABRB1, cluster at chromosome 4p. This region is the locus where genetic linkage studies have shown the association of alcohol dependence with a microsatellite marker. 5,7 Edenberg et al. conducted an association study that focused on genetic markers in this region using COGA study samples, and reported that SNP in the GABRA2 gene, which encodes the GABA-A receptor alpha 2 subunit, were associated with increased risk for alcohol dependence. 51 Their results also suggested that SNP in GABRA2 were associated with increased power in the beta frequency band of the EEG, which is considered to reflect heritable vulnerability of alcohol dependence. Several subsequent studies in subjects from different populations have confirmed the association between GABRA2 and alcohol dependence, although not all studies replicated this association. 56,57 A haplotype analysis suggested that there is linkage disequilibrium between markers in GABRA2 and those in the adjacent GABRG1 gene encoding the GABA-A receptor gamma 1 subunit and that the association between GABRA2 and alcohol dependence might be due to the linkage disequilibrium with GABRG1. 58 In addition, some recent studies supported the association of GABRG1 with the risk for alcohol dependence 59 and drinking behavior. 60 The mechanism of the effect of GABRA2 or GABRG1 variants on the vulnerability for alcohol dependence is unclear because no functional polymorphism in these genes has been identified. Most of the loci associated with alcohol dependence in this region are non-coding SNP that can possibly change the gene expression of the GABA subunit. 61 There is another gene cluster of GABA receptors on chromosome 5q including GABRA1, GABRA6, GABRB2, and GABRG2. The association between SNP in these genes and alcohol dependence seems less evident than in genes on chromosome 4. Some studies suggest a positive association with alcohol dependence 62 or phenotype-like alcohol dependence with antisocial personality disorder (ASPD). 63,64 However, a comprehensive study that analyzed SNP in this region using COGA samples did not show any association with alcohol dependence and ASPD. 65

5 Psychiatry and Clinical Neurosciences 2011; 65: Genetics of alcohol dependence 217 Cholinergic muscarinic receptor-2 Cholinergic muscarinic receptor-2 (CHRM2) is located on chromosome 7q where earlier linkage studies showed linkage with alcohol dependence. 5,6 CHRM2 is the other gene on which polymorphisms in the region of interest have been replicated in association studies. An association study using COGA samples reported that SNP in the CHRM2 gene were associated with the risk for alcohol dependence as well as alcohol dependence with major depression. 66 A replication study supported the association between CHRM2 and alcohol dependence. 67 Further analysis suggested that the association was detected only in alcoholics with comorbid drug dependence but not in alcoholics without comorbid drug dependence. 68 Therefore, it is likely that the polymorphism of CHRM2 is associated with a personality trait or comorbid psychiatric disorder that leads to alcohol dependence, such as drug dependence, major depression, or ASPD. 69 Endogenous opioid and receptors The endogenous opioid system is considered to play an important role in mediating alcohol effects and developing drug addiction. There are several types of opioid receptors and opioid receptor mu 1 (OPRM1) is a particularly interesting candidate gene because there is a non-synonymous polymorphism with amino acid predisposition from asparagine to aspartic acid (Asn40Asp) that appears to be functional. 70 This polymorphism is reported to be associated with subjective responses to alcohol, such as craving and sensitivity Results of case control studies are mixed, but a meta-analysis reported that the Asn40Asp SNP in OPRM1 does not appear to affect the risk for alcohol dependence. 75 Naltrexone, an antagonist of opioid receptors, is used for the treatment of alcohol dependence. Interestingly, the OPRM1 polymorphism seems to influence the response to naltrexone treatment for alcohol dependence. Oslin et al. reported that naltrexonetreated patients who have at least one 40Asp allele of OPRM1 revealed significantly lower relapse rates than those who have the Asn40Asn genotype, although the sample size was relatively small. 76 Anton et al. also reported that good clinical outcome by naltrexone was detected only among patients with at least one 40Asp allele using COMBINE study subjects. 77 The interaction was replicated in a Korean population. 78 Another study failed to replicate the interaction, 79 although the difference might be due to the treatment dosage of naltrexone. Moreover, it was reported that the effect of naltrexone on the sensitivity to alcohol was different between non-treatment-seeking heavy drinkers with and without the 40Asp allele. 72 In addition to OPRM1, several studies have examined whether polymorphisms of endogenous opioid ligands and receptors are associated with the risk for alcohol dependence. Xuei et al. reported that SNP in opioid receptor kappa 1 (OPRK1) and its ligand prodynorphin (PDYN) were associated with alcohol dependence in a study using COGA samples. 80 Zhang et al. also showed a haplotype of OPRK1 SNP was associated with alcohol dependence, 81 although another study showed no association with OPRK1 polymorphisms. 82 Most studies have shown that opioid receptor delta 1 (OPRD1) is not likely to be associated with alcohol dependence, although one study did show a positive association. 81 There are few studies about nociceptin receptor (OPRL1), and results have been shown to be both positive 85 and negative. 84 There are also studies that indicate some effects of SNP in proopiomelanocortin (POMC) 86 and prepronociceptin (PNOC). 84 The relationship between SNP in these genes and alcohol dependence is still unclear, and further analysis is needed to determine whether polymorphisms of opioid-related genes have any effect on the vulnerability for alcohol dependence. Dopamine-related genes Dopamine is considered to mediate the reward system in mesolimbic neurons, and numerous studies have focused on genes related to dopamine function. The most frequently studied polymorphism in these genes was presumably TaqI polymorphism (rs ) in the dopamine receptor D2 (DRD2) gene. This polymorphism had been considered to exist in the DRD2 gene non-coding region, but was later found to be a missense mutation located in the coding region of the ankyrin repeat and kinase domain containing-1 (ANKK1) gene adjacent to DRD2. 87 The association between the ANKK1/DRD2 TaqI polymorphism and alcohol dependence was first reported in the early 1990s, 88 and a number of studies have tried to confirm the association, with mixed results A recent meta-analysis that overlooked 44 association studies reported a relatively small effect for the association of A1 allele with

6 218 M. Kimura and S. Higuchi Psychiatry and Clinical Neurosciences 2011; 65: alcohol dependence, 92 but this polymorphism is not likely to have an important effect on the pathology of alcohol dependence when combined with the idea that the TaqI polymorphism does not affect the function of DRD2. Some studies suggest that the polymorphism of the dopamine D4 receptor (DRD4) gene is associated with drinking behavior. DRD4 has a polymorphic 48-base pair variable number of tandem repeats (VNTR) sequence in exon 3, and the seven repeat alleles blunt intracellular response to dopamine. 93 The DRD4 VNTR polymorphism does not seem to be associated with alcohol dependence directly, but might be associated with cravings for alcohol 94 and binge drinking. 95 Laucht et al. reported that the effects of DRD4 VNTR polymorphism on drinking behavior are mediated by novelty seeking, which is a known personality trait implicated in alcohol abuse. 96 In terms of genes related to metabolism and elimination of dopamine in the neural system, the effects of catechol-o-methyltransferase (COMT) and dopamine transporter (SLC6A3/DAT1) gene polymorphism have been studied. COMT contains a functional missense mutation (Val158Met), and several studies showed that the low activity allele (Met158) was associated with the risk of late-onset male alcohol dependence, 97 early-onset alcohol dependence, 98 and the comorbidity of alcohol dependence and smoking in women. 99 However, an analysis using larger samples failed to confirm the association between COMT polymorphism and alcohol dependence. 100 DAT1 has a VNTR polymorphic locus in the 3 untranslated region. This VNTR polymorphism does not appear to be associated with alcohol dependence in the general case control design, 101 but some studies suggest that nine repeat alleles of DAT1 are associated with severe symptoms of alcohol withdrawal or alcoholics with a paternal history of alcohol dependence. 106 Serotonin-related genes Serotonin (5-hydroxytryptamine, 5-HT) is believed to modulate alcohol craving and drinking behavior. The most well-studied gene polymorphism related to the serotonin system is the insertion/deletion variant (5-HTTLPR) in the promoter region of the serotonin transporter (SLC6A4/5-HTT) gene. The short (S) allele has lower transcription activity of the 5-HTT gene than the long (L) allele, 107 and the polymorphism has been reported to be associated with several psychiatric disorders and personality traits, such as major depression and suicide. 108,109 Results of association studies that examined the relation between this polymorphism and alcohol dependence are inconsistent, but a meta-analysis of 17 primary studies showed that the frequency of the S allele was significantly associated with the risk for alcohol dependence (odds ratio, 1.18) and greater association with the S allele was detected among individuals with a comorbid psychopathology, early-onset, or more severe alcohol dependence. 110 Some studies indicate that polymorphisms in serotonin 2A receptor (HTR2A) and serotonin 1B receptor (HTR1B) are associated with some subtype of alcohol dependence, but these polymorphisms do not seem to be associated with the general risk of alcohol dependence. 36,111 Enoch et al. recently reported that the 5-HT3 receptor gene polymorphism was associated with the risk of alcohol dependence. 112 The 5-HT3 receptor can be heteromeric with 5-HT3A (HTR3A) and 5-HT3B (HTR3B) subunits in the amygdala, the caudate, and the hippocampus. There is a missense SNP (Tyr129Ser), and the Ser129 allele causes increased function of 5-HT3 receptors. A case control study in an African- American population showed that the Ser129 allele was associated with an increased risk for alcohol dependence. Another report suggested a different SNP in the HTR3B gene was associated with alcohol dependence with ASPD. 113 GENOME-WIDE ASSOCIATION STUDIES GWAS are a powerful and promising method to clarify the genes underlying complex genetic traits. The completion of the HapMap project and advances in genotyping tools enable the comprehensive genome mapping of SNP and hypothesis-free identification of genes that affect the risk of polygenic diseases. Several GWAS that focused on alcohol dependence were published in the last few years (Table 1) The first trial of a genome-wide association scan for alcohol dependence was reported in 2006 by Johnson and collegues. 114 They examined SNP of pooled samples of 120 alcohol dependence cases and 160 controls from the COGA study. Results showed that 188 SNP clustering in 51 regions satisfied their criteria of significance, including genes

7 Psychiatry and Clinical Neurosciences 2011; 65: Genetics of alcohol dependence 219 Table 1. GWAS of alcohol dependence Sample size Source Analyzed SNP Replication sample Findings Johnson et al. 114 (2006) Treutlein et al. 115 (2009) Bierut et al. 116 (2010) Edenberg et al. 117 (2010) 120 EA cases 160 EA controls 487 cases, male inpatients, early-onset 1358 controls 1235 EA cases 662 AA cases 1433 EA controls 499 AA controls 847 EA cases 345 AA cases 552 EA controls 140 AA controls COGA None 188 SNP in 51 regions satisfied their criteria (t value >3, clustering) German population SAGE (COGA, FSCD, COGEND) COGA male inpatients 996 controls families 487 German cases 1358 German controls (EA) (AA) 2 SNP in PECR gene showed genome-wide significance None of the SNP met genome-wide significance 15 SNP with P < 10-5 in GWAS analysis 262 families None of the SNP met genome-wide significance 11 SNP with P < 10-5 in GWAS analysis A region on chromosome 11 with SNP with low P-value AA, African-American; COGA, Collaborative Study on the Genetics of Alcoholism; COGEND, Collaborative Genetic Study of Nicotine Dependence; EA, European-American; FSCD, Family Study of Cocaine Dependence; GWAS, genome-wide association studies; PECR, peroxisomal trans-2-enol-coa reductase; SAGE, Study of Addiction: Genetics and Environment; SNP, single nucleotide polymorphism. whose products are implicated in cellular signaling, gene regulation, development, and cell adhesion, although the samples size and the number of SNP markers were relatively small. Treutlein et al. reported GWAS results using German cases and controls. 115 They initially tested SNP of 487 male inpatients with an onset of alcohol dependence at younger than 28 years and 1358 controls. Results showed that 121 SNP were with nominal P < 10-4 from the GWAS, and 15 of them were confirmed in the follow-up sample set. The genome-wide significance across the GWAS and follow-up samples was detected in two SNP on the 3 -flanking region of the gene encoding peroxisomal trans-2-enol-coa reductase (PECR) located on the chromosome region 2q35. In 2010, two reports on GWAS of alcohol dependence were added. Bierut et al. performed GWAS testing SNP among 1897 cases and 1932 controls from the Study of Addiction: Genetics and Environment (SAGE) that contains COGA, Family Study of Cocaine Dependence (FSCD), and the Collaborative Genetic Study of Nicotine Dependence (COGEND) datasets. 116 This large study indentified 15 SNP with P < 10-5 in the GWAS analysis, but none of them revealed a significant association in the replication stage. Edenberg et al. reported GWAS of COGA samples that included 847 European- American cases and 552 European-American controls along with 345 African-American cases and 140 African-American controls. 117 None of the SNP met conventional criteria for genome-wide significance, but the strongest association was found in the region of chromosome 11. These studies did not find a particular gene that consistently showed a significant association across studies. One of the possible reasons for this lack of findings is that there were ethnic and sex differences in subjects, and the phenotype of alcohol dependence might have differed among the studies. Moreover, GWAS analysis needs a low threshold for P-value significance because of the correction for multiple comparisons of large numbers of SNP. A careful meta-analysis would provide more power to identify SNP associated with alcohol dependence from multiple GWAS results.

8 220 M. Kimura and S. Higuchi Psychiatry and Clinical Neurosciences 2011; 65: CONCLUSION Evidence that several genes are associated with the development of alcohol dependence has been accumulating for 2 decades. One of the most successful findings is that polymorphisms in ALDH2 and ADH1B influence the risk for alcohol dependence. Association studies have consistently supported these associations. It is interesting that individuals who develop alcohol dependence despite possessing the protective genotype have distinguishing traits, because studying them might allow differentiation of the pathological mechanism of the disease from alcohol metabolism. In addition, GABRA2 is a promising gene. The association between the polymorphism of GABRA2 and alcohol dependence has been replicated in multiple association studies as well as the whole-genome study, which supports the linkage to the chromosome region of GABRA2. GABA receptor is a primary target for the behavioral effects of alcohol; therefore, functional alternation of the GABA receptor probably plays a major role in developing alcohol addiction. Findings of associations between other genes and alcohol dependence are less consistent among studies, and further collection of data seems necessary to identify the effect of the gene polymorphisms. One of the major reasons for the inconsistent results among association studies is that alcohol dependence is a heterogeneous disease. The variety of subtypes associated with different genetic risk factors might mask the differences of a particular genetic factor in case control designs. Classifying alcohol dependence into a more homogeneous phenotype is a good way to solve this problem. Several typological concepts have been proposed, such as type I/type II alcoholics 118 and alcoholics with or without ASPD. 119 Studying biological endophenotypes associated with the genetic risk of alcohol dependence is also a solution; for example, P300 amplitude of ERP 120 or brain oscillations 121,122 in EEG are also powerful tools that can be used to reduce the heterogeneity of alcoholism. A drawback of such a classification system is that each phenotype covers a limited population; however, this strategy provides additional power to detect small to moderate genetic factors. In summary, knowledge of the genetic basis of alcohol dependence is growing. Genetic studies have shown increased evidence that polymorphisms in some genes are associated with vulnerability for alcohol dependence. Identification of the genes that affect the risk of alcohol dependence leads to a better understanding of the biological mechanism of addiction, which can help prevent and treat this disease. The recent revolution in molecular technology will accelerate advances in the genetic study of alcohol dependence. REFERENCES 1. Kendler KS, Heath AC, Neale MC, Kessler RC, Eaves LJ. A population-based twin study of alcoholism in women. JAMA 1992; 268: Kendler KS, Prescott CA, Neale MC, Pedersen NL. Temperance board registration for alcohol abuse in a national sample of Swedish male twins, born 1902 to Arch. Gen. Psychiatry 1997; 54: Heath AC, Bucholz KK, Madden PA et al. Genetic and environmental contributions to alcohol dependence risk in a national twin sample: consistency of findings in women and men. Psychol. Med. 1997; 27: Edenberg HJ, Foroud T. The genetics of alcoholism: identifying specific genes through family studies. Addict. Biol. 2006; 11: Reich T, Edenberg HJ, Goate A et al. Genome-wide search for genes affecting the risk for alcohol dependence. Am. J. Med. Genet. 1998; 81: Foroud T, Edenberg HJ, Goate A et al. Alcoholism susceptibility loci: confirmation studies in a replicate sample and further mapping. Alcohol. Clin. Exp. Res. 2000; 24: Long JC, Knowler WC, Hanson RL et al. Evidence for genetic linkage to alcohol dependence on chromosomes 4 and 11 from an autosome-wide scan in an American Indian population. Am. J. Med. Genet. 1998; 81: Ehlers CL, Gilder DA, Wall TL, Phillips E, Feiler H, Wilhelmsen KC. Genomic screen for loci associated with alcohol dependence in Mission Indians. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2004; 129B: Hill SY, Shen S, Zezza N, Hoffman EK, Perlin M, Allan W. A genome wide search for alcoholism susceptibility genes. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2004; 128B: Prescott CA, Sullivan PF, Kuo PH et al. Genomewide linkage study in the Irish affected sib pair study of alcohol dependence: evidence for a susceptibility region for symptoms of alcohol dependence on chromosome 4. Mol. Psychiatry 2006; 11: Gelernter J, Kranzler HR, Panhuysen C et al. Dense genomewide linkage scan for alcohol dependence in African Americans: significant linkage on chromosome 10. Biol. Psychiatry 2009; 65:

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