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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Am J Psychiatry. 2013 Sep 1;170(9):10.1176/appi.ajp.2013.12091163. doi: 10.1176/appi.ajp.2013.12091163

DETERMINATION OF GENOTYPE COMBINATIONS THAT CAN PREDICT THE OUTCOME OF THE TREATMENT OF ALCOHOL DEPENDENCE USING THE 5-HT3 ANTAGONIST ONDANSETRON

Bankole A Johnson a, Chamindi Seneviratne a, Xin-Qun Wang b, Nassima Ait-Daoud a, Ming D Li a
PMCID: PMC3809153  NIHMSID: NIHMS487495  PMID: 23897038

Abstract

Objective

Previously, we reported that the 5′-HTTLPR-LL and rs1042173-TT (SLC6A4-LL/TT) genotypes in the serotonin transporter gene predicted a significant reduction in the severity of alcohol consumption among alcoholics receiving the 5-HT3 antagonist ondansetron. In this study, we explored additional markers of ondansetron treatment response in alcoholics by examining polymorphisms in the HTR3A and HTR3B genes, which regulate directly the function and binding of 5-HT3 receptors to ondansetron.

Method

We genotyped 1 rare and 18 common single-nucleotide polymorphisms in HTR3A and HTR3B in the same sample that we had genotyped for SLC6A4-LL/TT in the previous randomized, double-blind, 11-week clinical trial. Participants were 283 European Americans who received oral ondansetron (4 μg/kg twice daily) or placebo along with weekly cognitive behavioral therapy. Associations of individual and combined genotypes with treatment response on drinking outcomes were analyzed.

Results

Individuals carrying one or more of genotypes rs1150226-AG and rs1176713-GG in HTR3A and rs17614942-AC in HTR3B showed a significant overall mean difference between ondansetron and placebo in drinks per drinking day (−2.50; effect size (ES)=0.867), percentage of heavy drinking days (−20.58%; ES=0.780), and percentage of days abstinent (18.18%; ES=0.683). Combining these HTR3A/HTR3B and SLC6A4-LL/TT genotypes increased the target cohort from approaching 20% (identified in our previous study) to 34%.

Conclusions

We present initial evidence suggesting that a combined 5-marker genotype panel can be used to predict the outcome of treatment of alcohol dependence with ondansetron. Additional, larger pharmacogenetic studies would help to validate our results.

INTRODUCTION

Alcoholism is a heterogeneous complex disorder with multiple subtypes or endophenotypes (1). Perhaps because of this heterogeneity, the therapeutic effect sizes of the approved medicines for the treatment of alcohol dependence have been relatively small (2). A personalized approach based on a person's genetic makeup is increasingly being investigated for delivering optimum treatment to the “right” patient. Previously, in a randomized double-blind clinical trial of alcohol-dependent individuals of European descent, we showed that treatment of severe drinking with ondansetron, a specific serotonin-3 (5-HT3) antagonist, was significantly more efficacious in a subpopulation carrying the serotonin-transporter-linked polymorphic region LL (5′-HTTLPR-LL) and rs1042173-TT (SLC6A4-LL/TT) genotype combination in the serotonin transporter (5-HTT) gene, SLC6A4 (3). Briefly, in that study, we found that SLC6A4-LL/TT carriers treated with ondansetron had 2.63 fewer standard drinks per drinking day and a 16.99% higher percentage of days abstinent compared with non-carriers of the SLC6A4-LL/TT genotypes. In contrast, when subjects were not subgrouped according to their 5′-HTTLPR and rs1042173 genotypes, there was no statistically significant difference between the ondansetron and placebo groups.

Although the SLC6A4-LL/TT genotypes predicted ondansetron treatment response, ondansetron cannot bind directly to 5-HTT molecules since it is an antagonist at the 5-HT3A receptor subunit (4–7). Determination of the 5-HTT genotypes that we selected as being predictive of ondansetron treatment response in our previous study (3) was, therefore, indirect and based on previous in vitro and in vivo experimental evidence indicating that SLC6A4-LL/TT is associated with markedly reduced 5-HTT expression levels in alcohol-dependent individuals (8–11). The postsynaptic 5-HT3 receptors are formed by 5-HT3A homopentamers or 5-HT3A and 5-HT3B heteropentamer (5-HT3AB) complexes that evince even faster ion transport and conduction. The 5-HT3B subunits do not bind with serotonin or ondansetron but are important for stabilizing the receptor complex at the cell surface (12, 13). The 5-HT3 A and B subunits are encoded by the HTR3A and HTR3B genes located adjacently on chromosome 11, separated only by an intergenic region of 28 kb. Hodge and colleagues (14), using a transgenic mice model lacking the gene for the 5-HT3A subunit, showed that the reduction of alcohol consumption produced by 5-HT3 antagonism is dependent on the presence of 5-HT3A-containing receptors. Recently, a genetic association study conducted by Enoch and colleagues (15) that examined African-American male patients with a lifetime diagnosis of alcohol, cocaine, and/or heroin dependencies showed an association between these diseases and the particular genotypes of HTR3A, HTR3B, and the 5′-HTTLPR. These findings were supported by earlier work from the same group that showed an association between certain HTR3B genotypes and alcohol dependence with antisocial behaviors (16).

Taken together, these risk association studies implicate a collective effect of 5-HT3 and 5′-HTTLPR genotypes on susceptibility for alcoholism. In the present study, we further extended our previously tested hypothesis to include functional variants in the SLC6A4, HTR3A, and HTR3B genes that would interact with each other to mediate ondansetron treatment response. This new extended hypothesis was tested in the same sample that was examined in our previously published phase ΙΙ pharmacogenetic trial with 283 alcoholics of European descent. If such predictors were found in the HTR3A and HTR3B genes, they would help identify a larger population of alcoholics who would respond to ondansetron in treating alcohol dependence.

METHOD

Participants

The study population analyzed here is identical to the sample used in our previous study that tested the genetic effects of the SLC6A4 5–-HTTLPR-LL and rs1042173-TT genotypes on ondansetron's efficacy (3). Briefly, all 283 subjects were alcohol-dependent individuals with no comorbid axis Ι diagnoses as assessed by the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (17) who scored >8 on the Alcohol Use Disorders Identification Test (18) and were enrolled in an 11-week, randomized, double-blind clinical trial in which they received either oral ondansetron (4 μg/kg twice daily) or placebo along with weekly cognitive behavioral therapy (19). Details of the inclusion and exclusion criteria for the participants have been provided previously (3).

Study Design

We performed an a priori stratification based on a subject's 5′-HTTLPR genotype, with additional genotyping of single-nucleotide polymorphism (SNP) rs1042173 in the 3′-untranslated region (3′-UTR) of the SLC6A4. Candidate genotyping for polymorphisms in HTR3A and HTR3B were performed, retrospectively, and were not used as stratification factors (see Figures 1 and 2). We assessed the effect of genotype on three different measures of alcohol consumption—drinks per drinking day (DDD; i.e., the amount of severe drinking), percentage of heavy drinking days (PHDD; i.e., the frequency of severe drinking), and percentage of days abstinent (PDA; i.e., the frequency of not drinking)—for those who received either ondansetron or placebo. Information on daily alcohol consumption was collected for the 90 days prior to enrollment and during the study period using the timeline follow-back method (20). Similar to our previous study, we employed a mixed-effects statistical model to examine genetic associations with drinking patterns throughout the 3-month treatment period, rather than at a single time point.

Figure 1.

Figure 1

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SNP selection process and statistical analyses workflow.

Footnote: SNP=single-nucleotide polymorphism; DDD=drinks per drinking day; PHDD=percentage of heavy drinking days; PDA=percentage of days abstinent.

Figure 2.

Figure 2

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CONSORT diagram of alcohol-dependent participants in the post-hoc analysis.

Footnote: 5′-HTTLPR=serotonin-transporter-linked polymorphic region; SNP=single-nucleotide polymorphism; DDD=drinks per drinking day; PHDD=percentage of heavy drinking days; PDA=percentage of days abstinent.

Genotyping

Genomic DNA was extracted from the blood of each subject at baseline with a Gentra Puregene® kit (QIAGEN Inc., Valencia, CA). For SLC6A4 polymorphisms, genotyping data for 5′-HTTLPR L/S and rs1042173 polymorphisms were obtained from our previous pharmacogenetic trial (3, 21). For HTR3A and HTR3B polymorphisms, we genotyped 10 SNPs in HTR3A and 9 SNPs in HTR3B using a commercially available TaqMan® premade genotyping assay (Applied Biosystems, Foster City, CA) on an ABI 7900 platform. Average densities of their distribution on the two genes were about 1 SNP every 2 kb in HTR3A and every 5 kb in HTR3B. Candidate SNPs were selected based on their location on the gene, having a minor allele frequency of >5%, and previously reported findings by other researchers.

Because we did not detect a significant association of ondansetron treatment response with rs25531 genotypes alone or in combination with 5′-HTTLPR genotypes in our previous study, we have not examined 5′-HTTLPR tri-allelic effects in the present study.

Genotyping procedures for ancestral informative marker SNPs to assess population stratification using a 24-ancestral marker panel have been described previously (3, 21). Biological and primer/probe sequence information for all of the above-mentioned SLC6A4, HTR3A, and HTR3B polymorphisms is shown in Supplementary Table 1.

Statistical Analysis

Departure from Hardy-Weinberg Equilibrium (HWE) was assessed using Haploview (v. 4.0) software (22). Quality of clinical data was assessed as described previously (3). Additional to the primary outcome variable, DDD, we tested two other secondary outcome measures—PHDD and PDA. One heavy drinking day is defined as a day on which the subject consumed ≥5 drinks for male or ≥4 drinks for female patients.

To study the effect of treatment and genotypes on DDD, PHDD, and PDA, we used mixed-effects linear regression models, which can accommodate missing data at random. The models included random intercept and slope (for temporal trend) and were adjusted for participants' average drinking levels prior to the study, age, gender, and proportions of genetic ancestry as covariates. Proportions of genetic ancestry were calculated using the Structure program (http://pritch.bsd.uchicago.edu/software/structure2_2.html) as described previously (3).

To minimize type I error, we have used two filtering steps in our study. First, we examined the drinking data for all 19 HTR3A and HTR3B SNPs (Supplementary Table 1) and restricted further analysis to only those that had a difference between the ondansetron and placebo groups of more than 1.5 DDD and a p-level <0.05 (Figure 1). The 1.5 DDD cutoff was chosen because in our primary analysis, we detected a reduction of about 1.5 standard DDD in ondansetron-treated alcoholics who possessed the 5′-HTTLPR-LL genotype, which was the randomization factor (3). Thus, the 1.5 DDD cutoff was set with the empirical goal of discovering other genetic markers that would be at least as predictive of a response to ondansetron treatment. For all 19 HTR3A and HTR3B SNPs, we tested statistical models that included 2-way interaction terms between genotypes of a given SNP. Genotype associations with drinking outcome measures for each SNP were tested using dominant (homozygous major allele genotype plus heterozygous genotype vs. homozygous minor allele genotype), recessive (homozygous minor allele genotype plus heterozygous genotype vs. homozygous major allele genotype), or heterozygous (heterozygous genotype vs. homozygous major allele genotype plus homozygous minor allele genotype) genetic models. The differences in means between “genotype × treatment” combinations for all genotypes are presented in Supplementary Table 2. Second, in addition to the above-mentioned screening process, to further minimize type I error due to multiple testing, we calculated false discovery rate (FDR) q-values (23) for all “genotype × treatment” association analyses that survived the screening process, as shown in Supplementary Table 2.

Subsequent to the screening process, the selected SNPs were subjected to a filtering process, based on their associations with the two secondary outcome measures, PHDD and PDA. Figure 1 summarizes the SNP selection process used in the study. Only the SNPs that survived filtering were analyzed for their interactive effects on the primary outcome measure—DDD—and the secondary outcome measures—PHDD and PDA. All interactions were tested at two levels: 1) the effect of treatment on DDD, PHDD, and PDA in those who possessed one, two, or all of the HTR3A and HTR3B genotypes, regardless of their previously identified 5′-HTTLPR and rs1042173 genotypes, and 2) the combined effect of the HTR3A and HTR3B genotypes together with the previously identified SLC6A4-LL/TT genotypes. The sample sizes included in the combined genotypes × treatment groups are given in Figure 2. Statistical power analyses (24) were performed as described previously (3).

Validation of detected genotype combinations using the generalized multifactor dimensionality reduction (GMDR) method

As shown above and illustrated in Figure 1, our statistical approach in identifying interactive effects of different genotype combinations was based on pooling individually significant variants and analyzing their combined effects on the drinking measures. This approach, however, could potentially fail to identify individual variants having significant epistatic effects but having no significant main genetic effects on the phenotypes of interest. To address this concern and validate our results from the primary statistical analysis, we performed an independent SNP-by-SNP interaction analysis using the GMDR method (25), one of the commonly used approaches for detecting genetic epistatic effects (26, 27). The GMDR method allowed us to test for the best interaction models consisting of 2 to 5 variants by performing an exhaustive search using all 21 variants genotyped in the present study. As the response variable for GMDR analyses, we used predicted values for the three different drinking outcome measures during the entire treatment period. These predictive values were generated by using the same mixed-effects linear regression models that we used above for detecting significant interactive effects of different genotype combinations on three outcome measures, which included random intercept and slope (for temporal trend) adjusted for age, gender, ethnicity defined by ancestral informative markers, center, and corresponding baseline drinking outcome measures.

RESULTS

Demographic Characteristics

Demographic characteristics of the sample analyzed in the current study are the same as in our previous publication (3). Briefly, the current study included all 283 participants who identified themselves as being of European descent. Within this European-American cohort, 122 Caucasian and 18 Hispanic individuals received ondansetron; 118 Caucasian and 25 Hispanic individuals received the placebo. As discussed previously (3), potential population stratification was assessed with the 24-SNP ancestral informative marker panel using the Structure program. Results from the Structure program revealed no population admixture between those who identified themselves as Hispanic and Caucasian or between ondansetron- and placebo-treated individuals. Therefore, all 283 subjects were included in the final analyses, and the individual ancestral scores were utilized as covariates in place of self-reported ancestry.

Overall genotyping failure rates for all 21 polymorphisms examined in the present study were less than 3%. For genotyping quality control, we performed HWE tests on all 21 polymorphisms in HTR3A, HTR3B, and SLC6A4, and none of the variants showed significant deviations in either the ondansetron or the placebo group (Supplementary Table 1). Thus, they were all included in the analyses detailed below. Furthermore, there were no statistically significant differences in genotype frequencies between the ondansetron and placebo groups for all genotypes included in the analyses (Supplementary Table 2).

Association of Drinking Outcome Measures with HTR3A and HTR3B Polymorphisms at the Individual Level

The DDD estimates for all genotype groups examined in the present study are listed in Supplementary Table 2. As shown in that table, genotypes rs17614942-AC in HTR3B and rs1150226-AG, rs1176713-GG, and rs1176719-AA in HTR3A were significantly associated with DDD reductions of more than 1.5 in response to ondansetron treatment (p<0.05). Of the 4 HTR3A and HTR3B SNPs that survived the screening process, rs1176719-AA did not show a statistically significant association with PHDD (PHDD reduction=15.35%; 95% confidence interval [CI]=−37.55% to 6.86%; p=0.175); furthermore, the association of rs1176719-AA with PDA neither was statistically significant nor reached the 10% threshold (PDA improvement=3.48%; 95% CI=−2.98% to 9.94%; p=0.291). Hence, rs1176719 was excluded (Figure 1) from further analyses performed to test the interactive effects. The rs1176713 genotype GG showed only a trend with PHDD and was not significantly associated with PDA; yet, the magnitudes of PHDD and PDA (Table 1) were similar to those of rs1150226-AG and rs17614942-AC. We, therefore, included the following genotypes in the subsequent multilocus interaction analyses: SLC6A4-LL/TT, HTR3B-rs17614942-AC, and HTR3A-rs1150226-AG and -rs1176713-GG. The FDR q-values for DDD associations with the HTR3B-rs17614942-AC and HTR3A-rs1150226-AG and -rs1176713-GG genotypes ranged from 0.156 to 0.311 (Supplementary Table 2).

Table 1.

Significant Individual HTR3A and HTR3B SNP Associations with Three Drinking Measuresa

Gene SNP Genotype Population Carrying the Genotype Combination (% in Total Cohort) Outcome Measure: Estimated Mean Difference (95% CI) and p-Value
Reference Associated DDD PHDD PDA
HTR3B rs17614942 AA/CC AC O=17/135
P=19/139
(13%)		 −2.73
(−4.59 to −0.87)
0.004		 −20.45%
(−37.58% to −3.32%)
0.019		 17.95%
(0.95% to 34.95%)
0.039
HTR3A rs1150226 AA/GG AG O=20/135
P=24/139
(16%)		 −1.81
(−3.51 to −0.12)
0.036		 −20.65%
(−36.17% to −5.12%)
0.009		 19.75%
(4.30% to 35.19%)
0.012
rs1176713 AA/AG GG O=6/134
P=9/139
(5%)		 −3 87
(−6.95 to −0.78)
0.014		 −23 98%
(−51.83% to 3.87%)
0.091		 18.20%
(−9.31% to 45.70%)
0.195
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a

All comparisons are between the ondansetron and placebo groups.

SNP=single-nucleotide polymorphism; CI=confidence interval; O=ondansetron; P=placebo; DDD=drinks per drinking day; PHDD=percentage of heavy drinking days; PDA=percentage of days abstinent.

Combined Interactive Effects of Significantly Associated HTR3A, HTR3B, and Previously Reported SLC6A4-LL/TT Genotypes on Drinking

We have identified three HTR3A/HTR3B (HTR3) genotypes (i.e., HTR3B-rs176149-AC, HTR3A-rs1150226-AG, and HTR3A-rs1176713-GG) in the above single-locus analyses and the SLC6A4-LL/TT genotype combination in our previous study; all of these polymorphisms were associated with lowering alcohol consumption from heavy to non-heavy levels, reducing it by at least 2 standard drinks on a given drinking day. Yet, if these five polymorphisms are to be utilized as a marker panel for screening individual DNA samples, it is imperative to know their predictability when they are present alone or in combination with each other. To examine combined interactive effects, we adopted an analytical strategy similar to the collapsing method, which is used widely in rare-variant analyses in genetic association studies (28–32). More specifically, the collapsing method investigates for the presence or absence of several rare alleles in a region of interest and then treats them as a single common variant; similarly, in our analysis, we have investigated for the presence or absence of the HTR3B-rs176149-AC, HTR3A-rs1150226-AG, HTR3A-rs1176713-GG, and SLC6A4-LL/TT genotypes and combined them in the following two ways. First, we assessed the combined effects of HTR3 genotypes identified in the present study, regardless of the presence or absence of SLC6A4-LL/TT genotypes. Next, we assessed the combined effects of HTR3 and SLC6A4-LL/TT genotypes in both the current and previous studies.

(1) Combined effects of HTR3A and HTR3B polymorphisms on drinking outcome measures, regardless of presence or absence of SLC6A4-LL/TT genotypes

Combination-1 indicates the effects of carrying only one of the three HTR3 genotypes on ondansetron treatment response. Included subgroups are: “rs1150226-AG & no rs176149-AC or rs1176713-GG”, “rs1176713-GG & no rs1150226-AG or rs176149-AC”, and “rs176149-AC & no rs1150226-AG or rs1176713-GG”. All subgroups were present in less than 5% of the total cohort, and overall, Combination-1 represents 11% of the total study population.

Combination-2 indicates the effects of carrying any two of the three HTR3 genotypes on ondansetron treatment response. Included subgroups are: “rs1150226-AG & rs176149-AC & no rs1176713-GG” and “rs176149-AC & rs1176713-GG & no rs1150226-AG”. All but one subject in Combination-2 belonged to the subgroup “rs1150226-AG & rs176149-AC & no rs1176713-GG”. This can be partly explainable by the low linkage disequilibrium (LD) between rs1176713 and the other two SNPs and the high LD between rs17614942 and rs1150226. (r2 values for linkage are given in the LD plot shown in Figure 3.)

Figure 3.

Figure 3

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Linkage disequilibrium analysis of all HTR3AB single-nucleotide polymorphisms (SNPs) in the total cohort and the location of significantly associated SNPs.

Footnote: Values in squares of the linkage disequilibrium plot are r2 numbers: The dotted lines point toward the location of significantly associated SNPs in reference to various splice variants of HTR3A and HTR3B primary mRNA. The numbers next to each splice variant indicate their accession numbers.

Combination-3 indicates the effects of possessing one or any combination of two or all three HTR3 genotypes. However, rs1176713-GG was found only in two of the rs17614942-AC carriers and was not detected in any rs1150226-AG carrier. This lack of individuals possessing all three HTR3 genotypes in our sample resulted in Combination-3 being more like a pooled group of Combinations-1 and -2 (23% of the total study population) rather than a new group consisting of individuals possessing 1, 2, or all 3 HTR3 genotypes. The included subgroups in Combination-3 are “rs1150226-AG & rs17614942-AC & no rs1176713-GG” (48%), “AG & no AC or rs1176713-GG” (22%), “rs1176713-GG & no rs1150226-AG or rs17614942-AC” (21%), “rs17614942-AC & no rs1150226-AG or rs1176713-GG” (6%), and “rs17614942-AC & rs1176713-GG & no rs1150226-AG” (3%). As shown in Table 2 and Figure 4A,B,C, Combination-3 was significantly associated with all three outcome measures: DDD, PHDD, and PDA. The effect sizes for DDD, PHDD, and PDA associations with ondansetron treatment outcome in Combination-3 were 0.867, 0.780, and 0.683, respectively.

Table 2.

Association of Frequent Genotype Combinations (>5%) with Primary and Secondary Outcome Measures of Ondansetron Treatment Responsea

Combination Genotype Combination Population Carrying the Genotype Combination (% in Total Cohort) Estimated Mean Difference (95% CI) and p-Value
DDD PHDD PDA
1 Possessing only one of the three identified HTR3 genotypes:
(HTR3A-rs1150226-AG alone or HTR3B-rs17614942-AC alone or HTR3A-rs1176713-GG alone)		 O=15/133
P=16/138
(11%)		 −2 35
(−4.41 to −0.29)
0.025		 −19.05%
(−37.73% to −0.36%)
0.046		 16.12%
(−2.49% to 34.74%)
(0.090)
2 Possessing a combination of any two of the three identified HTR3 genotypes:
(HTR3A-rs1150226-AG & HTR3B-rs17614942-AC or HTR3A-rs1176713-GG & HTR3B-rs17614942-AC)		 O=14/133
P=18/138
(12%)		 −2 65
(−4.65 to −0.66)
0.009		 −22.43%
(−40.77% to −4.09%)
0.017		 20.23%
(2.06% to 38.40%)
0.029
3 Possessing any one, two, or all three of the identified HTR3 genotypes:
(i.e., HTR3A-rs1150226-AG &/or HTR3A-rs1176713-GG &/or HTR3B-rs17614942-AC)		 O=29/133
P=34/138
(23%)		 −2.50
(−3.92 to −1.08)
0.0006		 −20.58%
(−33.53% to −7.62%)
0.002		 18.18%
(5.26% to 31.10%)
0.006
4 Possessing any one, two, three, or all four of the identified HTR3 and SLC6A4 genotypes:
(SLC6A4-LL/TT &/or HTR3A-rs1150226-AG &/or HTR3A-rs1176713-GG &/or HTR3B-rs17614942-AC)		 O=41/133
P=51/138
(34%)		 −1.71
(−2.88 to −0.54)
0.004		 −11.13%
(−21.94% to −0.31%)
0.044		 11.57%
(0.81% to 22.33%)
0.035
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a

All comparisons are between the ondansetron and placebo groups and are listed in descending order of magnitude of their DDD reductions; mean differences for DDD are in standard drinks; negative values indicate improvement from baseline.

CI=confidence interval; O=ondansetron; P=placebo; DDD=drinks per drinking day; PHDD=percentage of heavy drinking days; PDA=percentage of days abstinent.

Figure 4.

Figure 4

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Comparison of means for the three drinking outcome measures: drinks per drinking day (DDD) [A, D], percentage of heavy drinking days (PHDD) [B, E], and percentage of days abstinent (PDA) [C, F].

Footnote: Graphs in the first row are for ondansetron- or placebo-treated subjects carrying any one or a combination of the HTR3A-rs1150226-AG, HTR3A-rs1176713-GG, and/or HTR3B-rs17614942-AC genotypes (genotype Combination-3, represented by the purple bars) who showed the best treatment response and for those not carrying the above-mentioned genotypes (“All Others”, represented by the green bars). Graphs in the second row are for ondansetron- or placebo-treated subjects carrying any one or a combination of the SLC6A4-LL/TT, HTR3A-rs1150226-AG, HTR3A-rs1176713-GG, and/or HTR3B-rs17614942-AC genotypes (genotype Combination-4, represented by the red bars) and for those not possessing any of the above-mentioned genotypes (“All Others”, represented by the yellow bars).

(2) Combined effects of HTR3A, HTR3B, and previously reported SLC6A4-LL/TT on drinking outcome measures

Our previously reported 5-HTT gene SLC6A4-LL/TT genotype combination had a 2.08 (95% CI=−3.75 to −0.41; p=0.015) DDD reduction between SLC6A4-LL/TT carriers who received ondansetron and placebo regardless of their genotypes at the 3 HTR3 SNPs; there were no statistically significant differences for PHDD or PDA between SLC6A4-LL/TT carriers who received ondansetron and placebo. However, within the group of subjects who received ondansetron, SLC6A4-LL/TT carriers, compared with non-carriers, were significantly more likely to have more abstinent days (12.68%; 95% CI=0.48 to 24.89; p=0.042). Next, to study the combined effects of the HTR3 and SLC6A4-LL/TT genotypes on the three outcome variables, we pooled all subjects carrying the above-mentioned Combination-3 and/or SLC6A4-LL/TT genotypes (Combination-4) and compared them with the non-carriers of Combination-4.

Combination-4 indicates the effects of possessing the following genotype subgroups: “LL/TT & no HTR3 genotypes” (32%), “any two HTR3 genotypes & no LL/TT” (26%), “any one HTR3 genotype & no LL/TT” (25%), “LL/TT combined with two HTR3 genotypes” (8.5%), and “LL/TT combined with only one HTR3 genotype” (8.5%). As shown in Table 2, the DDD reductions in Combination-4 were close to two standard drinks in ondansetron-treated individuals, compared with the placebo group (p=0.004) (Figure 4D). In addition, carriers of Combination-4 who were treated with ondansetron also had significantly improved PHDD (Figure 4E) and PDA (Figure 4F). The effect sizes for DDD, PHDD, and PDA associations with ondansetron treatment outcome in Combination-4 carriers were 0.593, 0.416, and 0.428, respectively.

Notably, in all of the above combinations, the ondansetron group not carrying the targeted genotype group (i.e., groups designated as “All Others” in Figure 4), compared with their placebo counterparts, had a trend toward worse outcomes on our three measures of alcohol consumption (i.e., DDD, PHDD, and PDA).

The Combination-4 group included four infrequent (<5% of the total study population) subgroups carrying SLC6A4-LL/TT and one or two of the HTR3 genotypes. Associations of these infrequent genotype groups with the primary outcome measure, DDD, are given in Supplementary Table 3. As seen in Supplementary Table 3, DDD reductions in these groups of individuals with markers for both HTR3 and SLC6A4 were around 4 standard drinks, which was greater than the DDD reductions detected for Combinations 1–4 in Table 2. The greatest reduction in DDD was seen in the combination of SLC6A4-LL/TT and rs1176713-GG (Supplementary Table 3; Combination-1).

Independent Determination of SNP-by-SNP Interactive Effects Using the GMDR Method

To validate the significant interactive effects of various genotype combinations detected by the above-mentioned statistical approach, we conducted an independent SNP-by-SNP interaction analysis on all SNPs of the three genes using the GMDR method. As shown in Table 3, the detected significant 5-variant genotype combination for the three outcome measures, determined by the primary statistical approach above, appeared also to be significant based on the parameters of test accuracy, cross-validation consistency, and permutated p-value used commonly in the evaluation of GMDR results (25, 33, 34). This provides an independent determination of the best genotype combinations that can be used to predict treatment outcomes for the three alcohol dependence measures.

Table 3.

SNP-by-SNP Interaction Models Detected to Be Significant Using the GMDR Method, with the Closest Resemblance to Combinations 1–4 from the Primary Analysisa

Outcome Measure Interaction Model Test Accuracy Cross-Validation Consistency Permutated p-Valueb
DDD HTR3A: rs1176713–SLC6A4: 5′-HTTLPR 0.674 10/10 0.0001
HTR3A: rs1176713–SLC6A4: 5′-HTTLPR, rs1042173 0.595 10/10 0.039
HTR3A: rs1150226,rs1176713–HTR3B:rs17614942–SLC6A4: 5′-HTTLPR, 0.483
rs1042173 10/10 0.576
PHDD HTR3A: rs1176713–SLC6A4: 5′-HTTLPR 0.612 7/10 0.003
HTR3A: rs1176713–SLC6A4: 5′-HTTLPR, rs1042173 0.594 10/10 0.031
HTR3A: rs1150226,rs1176713–HTR3B:rs17614942–SLC6A4: 5′-HTTLPR, 0.565
rs1042173 10/10 0.094
PDA HTR3A: rs1176713–SLC6A4: 5′-HTTLPR 0.593 8/10 0.033
HTR3A: rs1176713–SLC6A4: 5′-HTTLPR, rs1042173 0.628 10/10 0.003
HTR3A: rs1150226-rs1176713–HTR3B:rs17614942–SLC6A4: 5′-HTTLPR, 0.598 10/10
rs1042173 0.029
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a

SNP=single-nucleotide polymorphism; GMDR=generalized multifactor dimensionality reduction; DDD=drinks per drinking day; PHDD=percentage of heavy drinking days; PDA=percentage of days abstinent.

b

p-Values were generated with 106 permutations.

As reported previously, except for fatigue (p=0.019), no other adverse events (including QT-interval prolongations) occurred more frequently in the ondansetron-treated group than in the placebo group (3); nor did they occur more frequently among carriers of the above-mentioned genotype combinations than among non-carriers (data not shown). There were no life-threatening events, and the five most commonly reported adverse events were (ondansetron vs. placebo): insomnia (20.5% vs. 22.3%), headache (20.9% vs. 19.4%), appetite disturbance (18.0% vs. 20.1%), fatigue (18.0% vs. 11.7%), and diarrhea (13.1% vs. 15.2%).

DISCUSSION

We identified three genotypes in the HTR3A and HTR3B (collectively designated as HTR3) genes that were significantly associated with efficacy of ondansetron treatment for alcohol dependence in individuals with European ancestry. We showed that possession of at least one of the HTR3A-rs1150226-AG, HTR3A-rs1176713-GG, and/or HTR3B-rs17614942-AC genotypes, along with the previously identified SLC6A4-LL/TT genotypes (i.e., Combination-4), was predictive of ondansetron treatment response; approximately 34% of the cohort carried these genotypes.

A unique strength of our findings was that the 5-genotype Combination-4 (Table 2) was associated not only with a reduction in the amount of severe drinking (DDD) but also with two other measures of treatment response—the frequency of heavy drinking and of abstinence (PHDD and PDA, respectively). Additional to these widely used outcome measures, we also performed an exploratory analysis for the genotype associations with an extreme dichotomous responder vs. non-responder endpoint efficacy variable, percentage of subjects with no heavy drinking days (PSNHDD), which may be used by regulatory agencies as an important outcome measure for larger phase III trials (35). When only 1 heavy drinking day during the final 4 weeks was allowed, we detected at least a 2-fold increase in PSNHDD in the ondansetron-treated population carrying one or more of the Combination-4 genotypes, compared with the placebo group or the ondansetron-treated non-carriers. Notably, however, carriers of Combination-3 that constituted a subset of Combination-4 (approximately 25% of the cohort) had more than a 5-fold increase in PSNHDD when the ondansetron and placebo groups were compared, and they might define a group of “super-responders”.

Furthermore, the greatest DDD reductions were seen in small subpopulations possessing both SLC6A4-LL/TT genotypes and any one or more of the HTR3 genotypes. This finding supports our hypothesis that the combined effects of genetic variants for the presynaptic 5-HTT and postsynaptic 5-HT3 would predict a greater response than genetic variations within each individual gene, as they represent the state of both synaptic availability of serotonin and its postsynaptic receptor levels. Another factor supporting this hypothesis is the greater effect of SLC6A4 + HTR3 than the combined effect of the HTR3A and HTR3B genotypes. However, results for the combined effect of SLC6A4 and HTR3 should be interpreted cautiously as they were limited by the small sample size within each placebo and ondansetron “treatment × genotype” group (Supplementary Table 3), thereby reducing the statistical power to draw definitive conclusions. Nevertheless, these initial statistical trends that generally support a molecular basis for the use of ondansetron as a treatment for a particular cohort of alcohol-dependent individuals warrant further corroboration with studies of larger sample size. The second-best response for DDD was seen in the group that pooled individuals carrying any one or a combination of the three HTR3 genotypes, regardless of LL/TT carrier status (Combination-3 in Table 2). From a biological perspective, this is quite explainable as 5-HT3AB receptors are the primary target of ondansetron, which in fact led us to select HTR3A and HTR3B as candidate genes for the present study. Even though ondansetron's therapeutic effect on the selected SLC6A4 genotypes might appear to be smaller than that on the target HTR3 genotypes, presumably because the biological effect of the 5-HTT on ondansetron pharmacodynamics is an indirect one, their interaction is most intriguing in showing a more robust response across different outcome measures of alcohol consumption.

Notably, the significant associations that we detected with SNPs rs1150226 and rs17614942 were through their heterozygous genotypes; i.e., the heterozygotes showed significantly improved response to ondansetron compared with both homozygous genotype carriers. Although it is argued that such a comparison utilizing the heterozygous genetic model may not reflect underlying biological mechanism(s), a dominant heterozygous effect over both homozygotes, which also is referred to as molecular heterosis, is an increasingly recognized phenomenon in many human gene-disease association studies including addictions (36–40). Another possible explanation for detecting heterozygous associations could be the small sample size in the homozygous minor allele genotype groups, rather than the effects of molecular heterosis.

An additional caveat of our work is that the preclinical studies on the functionality of rs1150226 and rs1176713 of HTR3A and rs17614942 of HTR3B have not been reported in the literature, and, therefore, the exact molecular mechanism for their interactive effects remains to be determined. However, given their location within the genes, it is possible that all three of these polymorphisms may alter mRNA expression levels. The rs1150226 is located in the promoter region of HTR3A within 1 kb of putative binding sites for several transcription factors that have been shown to be modulated by ethanol (41, 42). Furthermore, genetic association analyses have shown an association of rs1150226 with alcoholism comorbid with antisocial personality disorder (16), altered temporal lobe activity (43), and clozapine treatment outcome for schizophrenia (44). Both rs1176713, which is a synonymous SNP located on exon 8 (Leu459Leu) of HTR3A, and rs17614942, located in intron 8 of HTR3B, could alter expression levels of functional 5-HT3AB subunits through altered splicing of primary mRNA to form nonfunctional secondary mRNA. Interestingly, rs17614942 is located in different regions in different mRNA isoforms (Figure 3). In primary mRNA isoforms AM293589 and NM.006028.3 (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?c=geneid&org=9606&l=9177), the two longer HTR3B mRNA transcripts that are found to be present in the brain, rs17614942 is located in intron 8. In rodents, intron 8 consists of a splice acceptor site that results in the long isoform (45); even though this site is not known to be present in humans, it underlines the importance of the intron 8 region in the formation of various mRNA splice variants. In the shorter primary mRNA isoform BF942819 (http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/av.cgi?c=geneid&org=9606&l=9177), which has not yet been reported to be present in the brain, rs17614942 is located in the 3′-UTR, which also can lead to functional 5-HT3B subunit expression level changes through different molecular mechanisms such as altered mRNA polyadenylation, miRNA binding, and decay. In vivo experiments would be required to characterize accurately the functional effects of the variants in Combination-4, both individually and collectively. We, therefore, propose our theorization as a framework upon which to launch further molecular investigations (3).

Another caveat of our findings is the relatively small sample size to perform statistical comparisons for all different genotype combinations constituted by the 5 variants included in Combination-4. It is, therefore, not possible to dissect fully the relative contribution of each individual genotype with treatment response. However, such an analysis that tested for all 16 different combinations of the 5 variants was well beyond the scope of this phase II clinical trial and would require a multicenter clinical trial that enrolled several thousand treatment-seeking, alcohol-dependent individuals.

Another consideration was that we used a descriptive filtering process to control for type I error, by minimizing the number of inferential contrasts, to understand the relationship between ondansetron treatment response and genotype. Thus, an additional replication study is needed in a much larger sample to validate our results. Furthermore, as the frequency of the identified alleles may vary in different ethnic populations, additional studies are needed to test the applicability of the current findings in other ethnic populations. Nevertheless, our findings were bolstered by the fact that the Combination-4 variants also were included in the detected significant genetic epistatic models by GMDR independent epistatic analyses, which were performed to validate the findings from the primary analysis.

Interestingly, a previous association study conducted by Enoch and colleagues (15) had suggested that rs1176744 polymorphisms in HTR3B and SLC6A4 (LALA) were associated with the development of alcohol dependence and, speculatively, ondansetron treatment response. Confirming these findings, an independent association analysis conducted by our team (46) also showed a highly significant association between alcohol dependence and a genotype combination comprising HTR3A-rs10160548, HTR3B-rs1176744 and -rs3782025, SLC6A4-rs1042173, and 5′-HTTLPR polymorphisms.

In conclusion, we have shown that polymorphisms in the SLC6A4, HTR3A, and HTR3B genes are predictors of reduced drinking in response to ondansetron. Based on these findings, we hypothesize that the Combination-4 genotypes can be utilized for screening individuals who would respond to ondansetron. Furthermore, the effect sizes of Combination-4 across different measures of alcohol consumption appear to be medium to high. Clinical trials randomized based on Combination-4 genotypes are thus needed to test the replicability of our findings.

Supplementary Material

01

Acknowledgments

The authors thank the National Institute on Alcohol Abuse and Alcoholism for its generous support through grants 5 R01 AA010522-17 and 1 R01 AA021163-01 to B. A. Johnson and grant 5 R01 AA019720-02 to N. Ait-Daoud; the National Institute on Drug Abuse for its support of M. D. Li through grant 5 R01 DA012844-12, and Robert H. Cormier, Jr., BA, for his assistance with manuscript preparation.

Footnotes

Disclosures: B. A. Johnson served as a consultant to Johnson & Johnson (Ortho-McNeil Janssen Scientific Affairs, LLC) 5 years ago, Transcept Pharmaceuticals, Inc. 4 years ago, Eli Lilly and Company 3 years ago, and Organon 3 years ago; he currently consults for D&A Pharma, ADial Pharmaceuticals, LLC (with which he also serves as Chairman), and Psychological Education Publishing Company (PEPCo), LLC. M. D. Li has served as a consultant and board member of ADial Pharmaceuticals, LLC. The other authors report no financial relationships with commercial interests.

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