Functional Genetic Variants That Increase Synaptic Serotonin and 5

Molecular Psychiatry (2011) 16, 1139–1146 & 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11 www.nature.com/mp ORIGINAL ARTICLE Functional genetic variants that increase synaptic serotonin and 5-HT3 receptor sensitivity predict alcohol and drug dependence M-A Enoch1, E Gorodetsky2, C Hodgkinson1, A Roy3 and D Goldman1 1Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA; 2Mood and Anxiety Program, National Institute of Mental Health, NIH, Bethesda, MD, USA and 3Department of Veterans Affairs, Psychiatry Service, New Jersey VA Health Care System, East Orange, NJ, USA

The 5-HT3 receptor is rapidly potentiated by ethanol and mediates fast excitatory serotonin (5-HT) transmission that modulates dopamine release in the reward circuitry. The 5-HT transporter regulates synaptic 5-HT availability. Functional polymorphisms in genes encoding the transporter and receptor may therefore influence addiction vulnerability. In this study, 360 treatment-seeking African American male patients with single and comorbid DSM-IV lifetime diagnoses of alcohol, cocaine and heroin dependence and 187 African American male controls were genotyped for the triallelic 5-HTTLPR functional polymorphism in the 5-HT transporter gene (SLC6A4) and 16 haplotype-tagging single-nucleotide polymorphisms (SNPs) across HTR3B (including the functional rs1176744 Tyr129Ser) and HTR3A, genes encoding 5-HT3 receptors. The HTR3B rs1176744 gain-of-function Ser129 allele predicted alcohol dependence (P = 0.002) and low 5-HTTLPR activity predicted cocaine/heroin dependence (P = 0.01). Both the HTR3B Ser129 allele (P = 0.014, odds ratio (OR) = 1.7 (1.1–2.6)) and low 5-HTTLPR activity (P = 0.011, OR = 2.5 (1.3–4.6)) were more common in men with alcohol þ drug dependence compared with controls. Moreover, the HTR3B Ser129 allele and low 5-HTTLPR activity had an additive (but not an interactive) effect on alcohol þ drug dependence (OR = 6.0 (2.1–16.6)) that accounted for 13% of the variance. One possible explanation of our findings is that increased synaptic 5-HT coupled with increased 5-HT3 receptor responsiveness may result in enhanced dopamine transmission in the reward pathway, a predictor of increased risk for addiction. Our results may have pharmacogenetic implications for 5-HT3 therapeutic antagonists such as ondansetron. Molecular Psychiatry (2011) 16, 1139–1146; doi:10.1038/mp.2010.94; published online 14 September 2010 Keywords: 5-HTTLPR; HTR3B; rs1176744; alcoholism; cocaine; heroin

Introduction bound to 5-HT, produce fast activation of neurons, including in the mesolimbic reward circuitry.8,9 Variation in serotonin (5-HT) transmission in the 5-HT3 receptors share structural and functional central nervous system has been implicated in alcohol homology with GABA , nicotinic acetylcholine and and drug dependence. The 5-HT transporter regulates A glycine receptors. The members of this superfamily the availability of 5-HT in the synaptic cleft through re- are targets for the acute and chronic effects of uptake and is the target of selective serotonin re-uptake ethanol.10 Moreover, 5-HT3 receptors are the target inhibitors. A functional promoter polymorphism, 5- of ondansetron, which has been shown to diminish HTTLPR, in the 5-HT transporter gene (SLC6A4)has alcohol craving.8 The HTR3A and HTR3B genes both been associated with changes in neuronal circuitry lie in a 90 Kb region on chromosome 11q23.1, and implicated in negative affect.1 Results from studies encode the 5-HT3 and 5-HT3 receptor subunits, showing associations between 5-HTTLPR and alcohol, A B respectively. heroin or cocaine dependence are mixed.2–6 5-HT3 receptors can be homomeric (all 5-HT3 Unlike the other 12 5-HT receptors that are G A subunits) or heteromeric (5-HT3 and 5-HT3 sub- protein-coupled receptors,7 5-HT3 receptors are A B units). Although homomeric receptors are more pentameric, ligand-gated ion channels that, when widely distributed in the central nervous system than heteromeric receptors, the latter are expressed in the Correspondence: Dr M-A Enoch, Laboratory of Neurogenetics, amygdala, the caudate and the hippocampus, loca- National Institute on Alcohol Abuse and Alcoholism, NIH, 5625 tions implicated in addiction.11,12 A mis-sense poly- Fishers Lane, Room 3S32, MSC 9412, Bethesda, MD 20892, USA. morphism (Tyr129Ser, rs1176744) in HTR3B has E-mail: [email protected] Received 16 March 2010; revised 10 August 2010; accepted 11 been shown to alter the response of the heteromeric August 2010; published online 14 September 2010 receptor to 5-HT. The Ser129 substitution results in an Functional serotonin gene variants and addiction M-A Enoch et al 1140 increased maximum response to 5-HT, decreased The study was approved by the institutional review desensitization and deactivation kinetics (10–20-fold boards of the Veteran Affairs New Jersey Healthcare slower, respectively) and a sevenfold increase in System and University of Medicine and Dentistry, mean channel open time.13,14 As 5-HT3 receptors New Jersey. After a full description of the study was may have a role in reward by modulating dopamine provided, all participants gave written informed release in the mesolimbic pathway,15 we hypothe- consent. sized that this gain-of-function receptor might have implications for alcohol dependence (AD). Moreover, Genotyping we hypothesized that there might be an additive effect DNA was available for 547 participants (360 patients between the HTR3B Ser129 allele and low 5-HTTLPR and 187 controls). Missing DNA was random and activity that increases synaptic 5-HT and which has showed no selection bias. been associated with AD.2 We conducted our study in treatment-seeking HTR3B and HTR3A African American men who had both single and Sixteen haplotype-tagging SNPs were identified using comorbid lifetime DSM-IV diagnoses of alcohol, a previously described design pipeline17 to maximize heroin and cocaine dependence, and African Amer- haplotype capture for the region of chromosome 11 ican male controls. The primary association analyses extending from 5 kb upstream of HTR3B to 1 kb for AD, with and without comorbid drug dependence, downstream of HTR3A (NCBI Human Build 35.1) were conducted with the functional HTR3B single- (Figure 1).17 Nine HTR3B SNPs and seven HTR3A nucleotide polymorphism (SNP), rs1176744 Tyr129- SNPs were genotyped using the Illumina Golden Ser, and the functional triallelic 5-HTTLPR Gate platform.17 The SNPs listed in the direction polymorphism. In secondary analyses, we investi- of transcription (from left to right within Figure 1) gated whether further information could be derived with their minor allele frequencies in non-alcoholics from haplotype associations by genotyping an addi- are: HTR3B: rs3758987 (0.39); rs10502180 (0.04); tional 15 haplotype-tagging SNPs across the HTR3B rs11606194 (0.02); rs17116121 (0.08); rs1176744 (0.39) and HTR3A genes. Tyr129Ser; rs17116138 (0.08) Val183Ile; rs2276307 (0.10); rs3782025 (0.44); rs1176761 (0.17); HTR3A: Participants and methods rs897692 (0.39), rs1150226 (0.32), rs1176724 (0.34), rs2276302 (0.48), rs3737457 (0.15), rs897687 (0.33) Originally, 635 African American substance-depen- and rs1176713 (0.30). dent men were recruited: 590 from the substance abuse treatment program at the Department of Veteran Triallelic 5-HTTLPR polymorphism Affairs New Jersey Healthcare System, East Orange Genotyping was performed in two stages using size Campus and 45 men originally screened as controls discrimination for the S (103 bp) and L (146 bp) alleles

(see below) who were found to have a diagnosis of and for the rs25531 (LA (146 bp) and LG (61 bp)) lifetime substance dependence. Most of the patients alleles. This assay is also capable of detecting the SG recruited from the substance abuse treatment program and the super long L alleles, but they were not were inpatients on a 21-day residential treatment observed in these African Americans. The genotyping ward; however, some were recruited from the out- method is fully described in Supplementary Informa- patient clinic or from the methadone clinic. Criteria tion. Genotyping accuracy was determined empi- for inclusion in the study were that paticipants were rically by duplicate genotyping of 25% of the samples X18 years of age, met DSM-IV criteria for substance selected randomly. The error rate was < 0.005, and the dependence, self-identified as African American and completion rate was > 0.95. had been abstinent for at least 2 weeks. Exclusion Triallelic genotyping revealed the following

criteria included mental retardation, dementia and 5-HTTLPR allele frequencies: S = 0.25, LA = 0.51, acute psychosis. Patients were interviewed by a psy- LG = 0.24. Genotypes were grouped as low activity chiatrist (A.R.), with the substance abuse section of the (SS, SLG, LGLG) (0.23) medium activity (SLA,LALG) 16 Structured Clinical Interview for DSM-IV to determine (0.51) and high activity (LALA) (0.26). the lifetime substance dependence diagnoses. The mean (s.d.) age of the patients was 45.6 (7.8) years. Assessment of population stratification using Three hundred twenty African American male ancestry informative markers controls were recruited from churches and a blood A total of 186 ancestry informative markers17 were bank in Newark, NJ, USA (46%) and from among genotyped in the study sample and in the HGDP- insulin-dependent diabetic outpatients seen at an CEPH Human Genome Diversity Cell Line Panel ophthalmology clinic (54%) at the University of (1051 individuals from 51 worldwide populations) Medicine and Dentistry: New Jersey Medical School (http://www.cephb.fr/HGDP-CEPH-Panel). PHASE (Newark, NJ, USA). All controls had a semi-structured Structure 2.2 (http://pritch.bsd.uchicago.edu/software. psychiatric interview and were without a lifetime html) was run simultaneously using the ancestry history of any substance abuse or dependence or informative markers data from our sample and the 51 major Axis 1 psychiatric disorder. Their mean (s.d.) CEPH populations to identify population substruc- age was 34.0 (10.1) years. ture and compute individual ethnic factor scores.

Molecular Psychiatry Functional serotonin gene variants and addiction M-A Enoch et al 1141

Figure 1 Haplotype block structure across HTR3B and HTR3A showing the 16 genotyped single-nucleotide polymorphisms (SNPs). The numbers in the squares refer to pairwise linkage disequilibrium (LD) measured as D’. Haplotype blocks were defined using a setting of average pairwise D’ within-block of X0.80. The functional SNP rs1176744, Tyr129Ser, is outlined.

This ancestry assessment showed that the European variance, all analyses were conducted with haplo- factor score was on average 0.09 (median va- types X5% frequency. lue = 0.04). Both a Mid-East factor and an Asian factor had an average score of 0.06 (median 0.04). Results Statistical analyses Logistic regression analyses were undertaken using Primary analyses JMP 7 software. Backward stepwise regression was HTR3B SNP rs1176744 Tyr129Ser. The rs1176744 performed with variables (age, ethnic factor scores) 129Ser allele was more common in the total group of alcoholics compared with controls without being eliminated from the model in an iterative 2 process. Ethnic factor scores were included as addiction: 0. 47 vs 0.38, w = 6.3, 1 df, P = 0.012. As covariates in the final model if they had significant can be seen from Table 1, the strongest signal derived from the patients with only AD in whom the 129Ser effects (the European factor had a significant effect on 2 heroin addiction in most analyses but no effect on allele frequency was 0.51 (w = 6.4, 1 df, P = 0.012). cocaine or AD). Logistic regression models with There was no association between rs1176744 and nominal variables yielded likelihood ratio (L-R) w2 cocaine or heroin dependence (Table 1). results. The Ser129 allele exerted a dominant effect: the Although the mean (s.d.) age of the patients (45.6 frequencies of the Tyr129/Ser129 and the Ser129/ (7.8) years) was higher than the controls (34.0 (10.1) Ser129 genotypes were both increased in alcoholics, years), F = 211, P < 0.0001, there were no differences and indeed as shown in Table 1. the dominant model in age between carriers of the three HTR3B genotypes produced the strongest results. For this reason and (P = 0.7) or the three 5-HTTLPR genotype groups also for simplicity in analyses with 5-HTTLPR, two (P = 0.9). genotype groups were included in analyses: (1) Haplotype frequencies were estimated using a Tyr129/Tyr129 homozygotes and (2) Ser129 allele Bayesian approach implemented with PHASE.18 carriers. Haploview version 2.04 Software (Whitehead Insti- tute for Biomedical Research, USA) was used to 5-HTTLPR produce linkage disequilibrium matrices (Figure 1). Compared with non-addicted controls, low 5- As rare and uncommon haplotypes are subject to HTTLPR activity was significantly more abundant in estimation errors because of increased sampling the total group of patients with addiction (odds ratio

Molecular Psychiatry Functional serotonin gene variants and addiction M-A Enoch et al 1142 Table 1 Influence of the functional HTR3B Rs1176744 Tyr129Ser on addiction diagnoses

Diagnosis N Genotype Tyr/Tyr vs Ser Allele frequency Genotype Tyr/Ser þ Ser/Ser allele freq Tyr/Tyr Tyr/Ser Ser/Ser w2 P-valuea w2 P-valueb w2 P-valueb

All patients with addiction 357 0.29 0.53 0.18 5.1 0.077 5.0 0.025 0.45 4.3 0.037 (1) Heroin/cocaine dependence only 131 0.34 0.50 0.16 0.6 0.742 0.6 0.44 0.41 0.6 0.453 (2) All alcoholics 226 0.26 0.55 0.19 7.7 0.021 7.5 0.006 0.47 6.3 0.012 Alcohol only 62 0.21 0.56 0.23 7.2 0.028 6.6 0.010 0.51 6.4 0.012 Alcohol þ drug dependence 164 0.27 0.55 0.18 4.7 0.094 4.4 0.035 0.45 3.7 0.054 No addiction 177 0.38 0.48 0.14 0.38

a2 df. b1 df. All groups were compared with controls without addiction. The total group of patients are divided into subsets (1) and (2). P-values in bold indicate significant effects.

(OR) = 2.49, 95% confidence interval (CI) = 1.45–4.26) the normal function HTR3B Tyr129/Tyr129 genotype (Table 2). This signal derived from the patients with (N = 31): OR = 5.95, 95% CI = 2.12–16.56. Within these AD and comorbid drug dependence (OR = 2.45, 95% 74 individuals, the additive effect of variation in these CI = 1.30–4.62) and the non-alcoholic patients with two genes accounted for 13% of the variance in cocaine and/or heroin dependence (OR = 2.81, 95% alcohol and drug dependence. There was no statis- CI = 1.43–5.48) but (in contrast to HTR3B Ser129) not tical gene–gene interaction (P = 0.49). Thus, 5- from the patients with only AD (Table 2). HTTLPR and HTR3B Tyr129Ser appear to have additive effects on the risk for alcohol þ drug depen- Additive effects of 5-HTTLPR þ HTR3B rs1176744 dence. Tyr129Ser Low, medium and high 5-HTTLPR activity and the Secondary analyses HTR3B Tyr129/Tyr129 and Ser129 carrier genotypes Secondary analyses were undertaken to determine were included together as independent variables in whether HTR3B and HTR3A haplotype analyses logistic regression models, with diagnosis as the could confer further information about genetic risk dependent variable. Table 2 shows that the results for alcohol and drug dependence. for the whole model tests for each diagnosis were all significant (P = 0.01–0.0007). When the total group of HTR3B and HTR3A haplotype blocks patients with addiction was divided into three HTR3B and HTR3A are closely adjacent genes but are component parts: (1) AD only; (2) alcohol þ drug in different haplotype blocks although there is dependence; and (3) heroin and/or cocaine depen- evidence of modest long distance linkage disequili- dence only, it became apparent that the strongest brium across the two genes (Figure 1). signal derived from the alcoholics with comorbid drug dependence (Table 2). HTR3B haplotype analyses Figure 2 illustrates the results for alcohol and Six SNPs, listed from left to right in the direction of comorbid drug dependence. On either the HTR3B transcription: rs17116121, rs1176744, rs17116138, Tyr129/Tyr129 (normal activity) background or the rs2276307, rs3782025 and rs1176761 were included Ser129 carrier genotypes (enhanced activity) back- in one haplotype block (Figure 1). Within this block, ground, low 5-HTTLPR activity was associated with there were six haplotypes with X0.05 frequency that an increased liability to alcohol þ drug dependence accounted for 98% of the haplotype diversity. These compared with high 5-HTTLPR activity: OR = 2.45, haplotypes (H1–H6), together with their frequencies, 95% CI = 1.30–4.62. On either the low, medium or are shown in Figure 3a. high 5-HTTLPR activity background, the enhanced In order to increase the power of analyses with the activity HTR3B genotypes were associated with lower frequency haplotypes, we designated non- increased liability to alcohol þ drug dependence alcoholics (‘non-AD’) to include controls without compared with the normal activity genotype: addiction and non-alcoholic patients with cocaine/ OR = 1.66, 95% CI = 1.05–2.63 (Table 2). heroin addiction. The justification for this grouping When looking at the joint effects of 5-HTTLPR and was that the frequencies of the H1, H3, H4 and H5 HTR3B Tyr129Ser (Table 2; Figure 2) it can be seen haplotypes in non-AD were identical to the frequen- that, comparing the two extremes, individuals with cies in controls without addiction, and the frequen- low 5-HTTLPR activity coupled with the gain-of- cies of haplotypes H2 and H6 were very similar in function HTR3B Ser129 allele (N = 43) were signifi- both groups (Figures 3a and b). cantly more likely to have alcohol þ drug dependence There was a haplotype association with AD: global than individuals with high 5-HTTLPR activity and P-value = 0.023, 5 df, L-R w2 = 13.1. As can be seen

Molecular Psychiatry Functional serotonin gene variants and addiction M-A Enoch et al 1143 = 31). N Ser/Ser 5.95 (2.12–16.56) 4.27 (1.90–9.57) 2.81 (1.10–7.16) 6.1 (2.39–15.67) 6.57 (1.57–25.36) þ d into subsets (1) and (2). 0.0003 0.0003 0.028 0.0001 0.005 -value OR [95% CI] P low–Tyr/Ser high 5-HTTLPR activity ( 2 4.9 8.0 w þ B. 5-HTTLPR–HTR3B high–Tyr/Tyr vs 1df 12.9 13.0 15.6 <

Figure 2 Low 5-HTTLPR activity and the HTR3B -value 0.0017 0.0007 0.020 0.0005 0.0174 rs1176744 Ser129 gain-of-function allele predict alcohol P and drug dependence 5-HT3AB: ‘normal’ activity is predicted 2 A. Whole model test

w by the rs1176744 Tyr129/Tyr129 genotype, ‘enhanced’ 3df activity is predicted by the Ser129 allele. Ser/Ser genotypes. = 43); Tyr129Tyr genotype Ser/Ser þ N þ 1.66 (1.05–2.63) 15.1 1.48 (1.01–2.16) 17.1 1.73 (1.13–2.64)2.30 (1.16–4.55) 17.7 10.3 t addiction. The total group of patients are divide -value OR [95% CI] 0.0139 0.016 0.002 0.005 P Tyr/Tyr vs Tyr/Ser 2 A. Effect of HTR3B rs1176744 w Tyr/Tyr vs Tyr/Ser 1df low 5-HTTLPR activity ( HTR3B þ 2.45 (1.30–4.62) 6.0 2.49 (1.45–4.26)2.81 (1.43–5.48) 5.8 0.62.24 (1.25–4.02) 0.43 9.2 1.16 (0.68–1.86) 9.9 Figure 3 HTR3B haplotype association with alcohol rs1176744 genotypes on addiction diagnoses dependence (AD) þþP < 0.05, þ P < 0.1. (a) In the total group of alcoholics (all AD), the frequency of haplotype H5 is

low/med/high increased and H1 is decreased. (b) The signal derives from -value OR [95% CI] 0.011 0.004 0.011 0.014 P HTR3B alcoholics without drug dependence: the frequency of haplotypes H4 and H5 are increased, and H1 is decreased 2 A. Effect of 5-HTTLPR activity w

2df compared with controls without addiction. The numbers 1 and 2 within the blocks represent allele 1 and allele 2. For

ts. All groups were compared with controls withou each single-nucleotide polymorphism (SNP), alleles 1 and 2 are located on opposite DNA strands. The haplotypes within the HTR3B haplotype block derive from the following six SNPs (listed from left to right in the direction of transcription with allele1, allele 2 bases in parentheses): frequencies rs17116121 (A, G); rs1176744 (T, G); rs17116138 (A, G); Low Med High rs2276307 (A, G); rs3782025 (T, C); rs1176761 (A,T). As

61 0.25 0.49 0.26 2.7 0.26 1.91 (0.81–4.39) 7.8 these are haplotype analyses, the N’s are the number of 164 0.25 0.54 0.21 9.1 N 120 0.30 0.48 0.22 9.1 chromosomes. The six haplotypes, H1–H6, account for 0.98 of the haplotype diversity in the total sample. The analyses were conducted on just these six haplotypes, and therefore, the total frequencies are 1.00 for each group. ** Indicates the functional SNP rs1176744: Tyr129 (allele1), Ser129 (allele 2). drug Independent effects of 5-HTTLPR activity and þ intervals) are provided for low vs high 5-HTTLPR activity and for from Figure 3a, the most abundant haplotype H1 was dependence only results are for effect likelihood ratio (L-R) tes

(A) The whole model test(B) and the A independent model effects including of only 5-HTTLPR the following and individuals: HTR3B Ser rs1176744 129 carriers withinmore the whole model. Odds ratios (ORs) with (95% common confidence in non-AD (0.46) than in alcoholics dependence No addiction 172 0.17 0.49 0.34 Alcohol dependence only Alcohol -values in bold indicate significant effects. 2 2 All patients with addiction(1) Heroin/cocaine 345 0.27(2) All 0.51 alcoholics 0.22 10.8 225 0.25 0.53 0.22 8.5 The table shows the results from logistic regression models: P Table 2 Diagnosis 5-HTTLPR w (0.39) P = 0.044, 1 df, L-R w = 4.1. In contrast,

Molecular Psychiatry Functional serotonin gene variants and addiction M-A Enoch et al 1144 haplotype H5 was more common in alcoholics (0.15) activity had a significant effect in the group of non- compared with non-AD (0.09): P = 0.006, 1 df, L-R alcoholic patients with heroin and/or cocaine depen- w2 = 7.7; H1 vs H5: P = 0.001, 1 df, L-R w2 = 10.7. dence but not in the patients with AD only. 5-HT3 Secondary analyses showed that the signal for the receptors are rapidly potentiated by ethanol at haplotype association largely derived from the concentrations that are within the intoxicating range patients with AD only (Figure 3b). Haplotypes H4 in humans.20 Ethanol increases the rate of channel and H5 were more abundant in patients with AD only activation while decreasing the rates of deactivation compared with controls without addiction (0.17 vs and desensitization.21,22 The effects of ethanol on 0.10; 0.16 vs 0.09, respectively; H4 þ H5 vs rest: 5-HT3 receptors are similar to the effects of the 2 P = 0.003, 1 df, L-R w = 8.8). In contrast, there was HTR3B Ser129 allele on heteromeric 5-HT3AB recep- only a weak signal from the alcoholics with drug tor function.13,14 Although the effect of ethanol is dependence when compared with controls without greater in homomeric than heteromeric receptors,23,24 addiction (H5 vs rest: P = 0.086, 1 df, L-R w2 = 2.9). the potentiating effects of ethanol on the gain-of-

It can be deduced from Figure 3b that haplotypes function 5-HT3AB variant receptor may have relevance H4 and H5 together differ from haplotype H1 by only to addiction as heteromeric receptors are found in the SNP rs1176744. Our results therefore suggest that the reward circuitry.11,12 haplotype association was driven by rs176744 Furthermore, this study has shown that low Tyr129Ser. 5-HTTLPR activity and the HTR3B Ser129 allele that,

respectively, increase synaptic 5-HT and 5-HT3AB HTR3A haplotype blocks receptor responsiveness to 5-HT, have significant, There was one haplotype block extending from independent effects on the risk for alcohol and drug rs897692 to rs2276302 that included eight haplotypes dependence and moreover have an additive effect on with X0.01 frequency that accounted for 0.97 of the risk. Individuals with low 5-HTTLPR activity coupled haplotype diversity. Of these, four haplotypes with the gain-of-function HTR3B Ser129 allele were X 1 occurred with 0.05 frequency: 2211 (0.41), 1122 over 2 2 times more likely to have a diagnosis of (0.26), 2212 (0.14) and 1222 (0.06). There was no alcohol and drug dependence compared with indivi- HTR3A haplotype association with alcohol or drug duals with both high 5-HTTLPR activity and the dependence. HTR3B Tyr129/Tyr129 genotype. Finally, in-depth HTR3B and HTR3A haplotype and SNP analyses HTR3B and HTR3A individual SNP analyses showed that only the HTR3B Tyr129Ser polymorph- Other than HTR3B rs1176744, none of the ism influenced alcohol and drug dependence. This SNPs showed a significant association with AD study illustrates the logic of studying genetic varia- (P = 0.11–0.99). tion that might be expected to result in additive or interactive biological effects. Comparison with earlier HTR3B haplotype analyses When we re-analyzed data from an earlier study,19 In an earlier study,19 we genotyped exactly the same we showed that in both African American and HTR3B SNPs as in our current study and reported a Finnish men, haplotype H4 was more abundant in four-SNP haplotype association with AD plus comor- individuals with AD. However, in the Finns, HTR3B bid antisocial personality disorder (ASPD) in Finnish variation was associated with AD comorbid with Caucasian men. The original four-SNP haplotype ASPD. Moreover, the earlier study showed that in the analyses did not include rs1176744. We re-did the Finns, rs3782025 and not rs1176744 was associated haplotype analyses and found that the six-SNP with AD comorbid with ASPD, nevertheless these two haplotype block (including rs1176744) that was SNPs are in strong linkage disequilibrium with each present in the African Americans was also present other. Finally, the earlier study found no HTR3B SNP in the Finns (Supplementary Figure S1). Five of the or haplotype association with AD in a community six African American haplotypes (not H5) were also sample of Plains American Indian men and women, present in the Finns but at different frequencies: H1: and we found no association with the six-SNP 0.29, H2: 0.41, H3: 0.03, H4: 0.17 and H6: 0.10. In the haplotypes (data not shown). Clearly, the association Finns, there was a haplotype association with AD of HRT3B genetic variation with AD is complex and comorbid with ASPD: global P-value = 0.0278, 4 df, may vary with ethnicity and alcoholism phenotype. L-R w2 = 10.9. Haplotype H4 was more abundant in In a study in Japanese participants, Yamada et al.25 individuals with AD þ ASPD compared with non- showed that two HTR3B Tyr129 haplotypes and alcoholics: P = 0.019, 1 df, L-R w2 = 5.5 (Supplemen- Tyr129/Tyr129 homozygotes were significantly asso- tary Figure S2). ciated with major depression in women only. There- fore, our and the Yamada et al. study together imply Discussion that the HTR3B Ser129 allele that increases the effectiveness of 5-HT transmission appears to be both Our study has shown that the predominant effect of protective against depression and also a risk factor for the HTR3B rs1176744 Ser129 allele was on risk for AD. This result might seem surprising because of the AD, and there was no effect of this allele on heroin known co-occurrence of major depression and alco- and cocaine dependence. In contrast, low 5-HTTLPR holism in epidemiological samples. The explanation

Molecular Psychiatry Functional serotonin gene variants and addiction M-A Enoch et al 1145 may lie in sex differences: the Yamada et al. study between substance dependence and other psychiatric found a significant association only in women; disorders, particularly ASPD and major depression, however, our study was conducted only in men and and the controls were free of all Axis 1 diagnoses, a population-based twin study has shown that factors it is possible that the signals for association found in underlying major depression in women and alcohol- our study are derived from hidden comorbidity. The ism in men are different.26 controls were recruited from two sources and Presynaptic 5-HT3 receptors modulate the synaptic although their mean age was appreciably lower than release of various neurotransmitters including that of patients they had largely passed through the GABA,27 whereas postsynaptic receptors are respon- peak risk age for the onset of addictive disorders. sible for the fast excitatory response to 5-HT. 5-HT3 In conclusion, our study has shown that functional receptors have a significant role in regulating the variants in the genes encoding the 5-HT3AB receptor 8 activity of VTA dopamine neurons. 5-HT3B subunits and the 5-HT transporter, respectively, had indepen- 11 can only assemble as 5-HT3AB receptors and these dent effects on risk for AD and for heroin/cocaine heteromeric receptors are expressed in several regions dependence, and furthermore had additive effects on of the human brain including the hippocampus, the the risk for alcohol þ drug dependence. It is possible amygdala and the caudate11,12,28 and have been that increased synaptic 5-HT plus increased 5-HT3 detected in the central nervous system of rodents.29 receptor responsiveness to 5-HT might result in One possible explanation for our finding is that enhanced dopamine transmission in the reward increased synaptic 5-HT coupled with increased pathway that is associated with a greater risk for

5-HT3AB receptor responsiveness to 5-HT might result addiction. The results of our study may have in enhanced dopamine transmission in the reward pharmacogenetic implications for therapeutic 5-HT3 pathway that is associated with a greater risk for antagonists such as ondansetron. addiction. Moreover, 5-HT is a less potent activator of heteromeric receptors than homomeric receptors.11,30 Conflict of interest Therefore, genetic variants that increase the responsiveness of heteromeric receptors in the reward The authors declare no conflict of interest. pathway may make an impact on addiction vulnerability. Acknowledgments Studies in humans have shown that the 5-HT3 receptor antagonist ondansetron decreases alcohol This research was supported by the Intramural cue-induced activation of the ventral striatum31 and Research Program of the National Institute on Alcohol influences drinking behavior.32–34 The results of our Abuse and Alcoholism, NIH and in part by grant RO1 study may have pharmacogenetic implications for DA 10336-02 to AR from the National Institute ondansetron. It is conceivable that alcoholics with of Drug Abuse, NIH. and without the gain-of-function heteromeric receptor may respond differently to this therapeutic agent. References The rs1176744 Ser129 variant is common in all ethnic groups. The frequencies in non-addicted 1 Pezawas L, Meyer-Lindenberg A, Drabant EM, Verchinski BA, controls in the current and earlier study19 are: African Munoz KE, Kolachana BS et al. 5-HTTLPR polymorphism impacts Americans: 0.38, Plains American Indians: 0.38, human cingulate-amygdala interactions: a genetic susceptibility mechanism for depression. Nat Neurosci 2005; 8: 828–834. Finnish Caucasians: 0.29. Curiously, the more abun- 2 Feinn R, Nellissery M, Kranzler HR. Meta-analysis of the dant Tyr129 allele is only found in humans, ranging association of a functional serotonin transporter promoter in frequency in HapMap populations from 0.57 in polymorphism with alcohol dependence. Am J Med Genet B Africans to 0.83 in Chinese. From Figure 3, it can be Neuropsychiatr Genet 2005; 133B: 79–84. seen that the Ser129 allele is found on four different 3 Gerra G, Garofano L, Santoro G, Bosari S, Pellegrini C, Zaimovic A et al. Association between low-activity serotonin transporter haplotype backgrounds (H3–H6), whereas the Tyr129 genotype and heroin dependence: behavioral and personality allele is found on two almost identical haplotypes correlates. Am J Med Genet B Neuropsychiatr Genet 2004; 126B: (H1 and H2), suggesting that the Tyr129 allele is a 37–42. relatively recent development. The fact that the 4 Mannelli P, Patkar AA, Murray HW, Certa K, Peindl K, Mattila-Evenden M et al. Polymorphism in the serotonin trans- ancestral Ser129 allele is at a lower frequency porter gene and response to treatment in African American suggests that selective pressure unique to humans cocaine and alcohol-abusing individuals. Addict Biol 2005; 10: maintains the Tyr129 allele in all ethnic groups. 261–268.

5-HT3AB receptors are abundant in the gut and are 5 Patkar AA, Berrettini WH, Hoehe M, Hill KP, Sterling RC, implicated in the neurocircuitry associated with Gottheil E et al. Serotonin transporter (5-HTT) gene polymorph- 12 isms and susceptibility to cocaine dependence among African- emesis. It is conceivable that the Tyr129 allele has American individuals. Addict Biol 2001; 6: 337–345. a selective advantage in this context, perhaps by 6 Saiz PA, Garcia-Portilla MP, Florez G, Arango C, Corcoran P, inducing protective vomiting in response to toxic Morales B et al. Differential role of serotonergic polymorphisms in substances.35 alcohol and heroin dependence. Prog Neuropsychopharmacol Biol Psychiatry 2009; 33: 695–700. There are some limitations to this study. Data on 7 Cravchik A, Goldman D. Neurochemical individuality: genetic other Axis 1 diagnoses were not available for the diversity among human dopamine and serotonin receptors and patients. As it is known that there is high comorbidity transporters. Arch Gen Psychiatry 2000; 57: 1105–1114.

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Molecular Psychiatry