Demographic and Genetic Characteristics of the Subjects
All subjects were of Caucasian origin. They were aged 18–35 years and reported 12–20 years of formal education. shows demographic characteristics including current and lifetime substance abuse of the overall sample.
Demographic characteristics for all subjects.
162 subjects completed ARCI questionnaires for all three sessions. Genotype and allele frequencies for the investigated SNPs are shown in . All SNPs were in Hardy-Weinberg-equilibrium. Allele frequency proportions did not significantly differ from those given in the Hapmap project (The International HapMap Genome Browser B36). Polymorphisms within each gene were in high intermarker linkage disequilibrium forming two haplotype blocks. Haplotype blocks and D’ values between the genetic variants are shown in . Rs1799971 and rs510769 were in high intermarker linkage disequilibrium as well as the SNPs rs1918760, rs2281617 and rs1998220 (r2<1.0). These observations were consistent with the HapMap data (The International HapMap Genome Browser B36). Analysis of ancestry informative markers (Structure 2.1.) confirmed self-reported Caucasian origin in all study participants. Caucasian origin was confirmed by score means of the analyzed 7 clusters corresponding to 7 geographic regions (Africa, Europe, Middle East, Central Asia, Far East Asia, America, Oceania). The genotype groups at locus rs2281617 did not differ in ethnicity, and because all subjects were confirmed to be Caucasian we did not repeat the ethnicity check with other polymorphisms.
Figure 1 Genomic structure of OPRM1 gene, mapped to chromosome 6, is shown to scale including 4 exons spanning 207.6 kb as well as results of linkage disequilibrium analyses: D’ values of single nucleotide polymorphisms along the OPRM1 gene, illustrating (more ...)
Correlation analyses for all demographic information with SNPs and haplotypes were performed to determine whether demographic measures should be included as covariates in statistical analyses. Genotype groups for each SNP and haplotype were similar on most demographic information. However, subjects with genotype C/C at rs660756 reported significantly higher lifetime opioid use (p<.001, χ2: 36,60, df=2) and higher lifetime sedative use (p<.001, χ2: 36,60, df=2). Participants with genotype A/G at rs1799971 reported higher marijuana consumption per month (one-way ANOVA: p<.05, F(2,152)=3.90) than people of the other genotype groups. In a separate analysis we found no relationship between lifetime opiate use or marijuana use per month and the outcome measures. Response to placebo did not differ across any of the genotype groups.
Genotype-Independent Effects of Amphetamine
The ARCI scales Euphoria, Energy and Stimulation were selected as primary outcome measures to capture the prototypic drug effects. Sedation was included as secondary outcome measure to examine whether ratings of Sedation decreased when ratings of Euphoria, Energy and Stimulation increased. Amphetamine produced the expected effects on the outcome measures with dose-dependent increases in Euphoria, Energy, Stimulation and decreases in Sedation (drug main effect from two-way ANOVA/ANCOVA, Euphoria: p<0.001, F(2, 320)=64.36, Energy: p<0.001, F(2, 320)=60.55, Stimulation: p<0.001, F(2, 322)=83.23, Sedation: p<0.001, F(2, 322)=44.94). Amphetamine also had dose-dependent effects increasing blood pressure and heart rate (drug main effect from two-way ANOVA/ANCOVA; systolic blood pressure: p<0.001, F(2, 328)=159.03, diastolic blood pressure: p<0.001, F(2, 326)=81.28, heart rate: p<0.001, F(2, 326)=40.25). For most measures the effect of capsule ingestion was significant by 60 min post-administration and peaked between 90 and 120 minutes post-administration. Males scored significantly higher on Energy than females (main effect of sex from two-way ANOVA: p<0.05, F(1,160)=5.92). Therefore, sex was used as a covariate and included as a between-subject factor in further analyses. Further demographic measures did not affect the self-reported outcome measures. Baseline scores did not differ between genotype groups.
OPRM1 gene polymorphisms and ARCI scales
To reduce variability within and between subjects peak change scores within each session for the primary outcome measures Euphoria, Energy, Stimulation and the secondary outcome measure Sedation (ARCI) were used and calculated by subtracting the predrug baseline scores from the measured scores after drug ingestion. Significant associations between SNPs in OPRM1 and the peak change scores for Euphoria, Energy, Stimulation and Sedation after amphetamine ingestion are reported in . Genotype A/A of rs510769 was associated with lower Stimulation and Euphoria scores after amphetamine (two-way ANOVA/ANCOVA, Euphoria: p<0.05, F(4, 318)=2.69; Stimulation: p<0,05, F(4,318)=2.99). shows peak change scores of Euphoria and Stimulation (ARCI) between the three genotype groups of the rs510769 polymorphism. Post hoc comparisons using peak change scores of each session respectively revealed that genotype groups scored significantly different on the Stimulation scale in response to the 10 mg dose (one-way ANOVA, p=0,01, F(2, 162)=4.74). A similar trend was apparent for Euphoria (one-way ANOVA, p<0.1, F(2, 162)=2.59) after the 10 mg dose.
Results of ANOVA’s for responses to d-amphetamine for individual OPRM1 gene polymorphisms.
Figure 2 Mean±SEM peak change scores on Euphoria and Stimulation (ARCI) in the three genotype groups at rs510769 after Placebo, 10 and 20 mg of amphetamine. Amphetamine (10 mg) increased Euphoria and Stimulation more in the rs510769A/G and G/G group than (more ...)
Subjects with genotype C/C of rs2281617 felt significantly more Euphoric and Energetic after amphetamine (10 mg) compared to participants with genotypes C/T and T/T (Genotype × Drug interaction on two-way ANOVA/ANCOVA, Euphoria: p=0.01, F(2, 320)=4.73; Energy: p<0.01, F(2, 316)=4.92). shows peak change scores of Euphoria and Energy (ARCI) between the genotype groups. Post hoc analyses (one-way ANOVAs or ANCOVAs, Euphoria p<0.001, F(1, 163)=15.33; Energy p<0.001, F(1, 163)=12,88) revealed that genotype groups significantly differed in Euphoria and Energy after 10 mg amphetamine. To clarify the source of peak change scores, time courses of Euphoria, Energy, Stimulation and Sedation, are descriptively shown in as changes from baseline for the genotype groups after the 10 mg dose. Locus rs1998220 was in strong linkage disequilibrium with rs2281617 (D’=1.0) and revealed to be also significantly associated with response to amphetamine. Subjects with genotype A/A at rs1998220 had significantly higher Energy levels and felt more stimulated than subjects with alleles A/G or G/G at rs1998220 (Genotype × Drug interaction on two-way ANOVA/ ANCOVA, Energy: p<0.05, F(4, 312)=2.91; Stimulation: p<0.05, F(4, 318)=2.67), but there was no significant association between rs1998220 and Energy in Post hoc analyses (one-way ANOVA/ANCOVA).
Figure 3 Mean±SEM peak change scores on Euphoria and Energy (ARCI) in the two genotype groups at rs2281617 (C/C: N=121; C/T and T/T: N=41) after Placebo, 10 and 20 mg of amphetamine. After 10mg of amphetamine (10 mg) the rs2281617C/C group reported greater (more ...)
Figure 4 Time courses of Euphoria, Energy, Stimulation and Sedation (ARCI) after 10 mg of amphetamine administration for the rs2281617 groups. The rs2281617C/C group reported a significantly greater increase in Euphoria and Energy compared to the T/T and C/T group (more ...)
However, taking into account the number of comparisons performed, none of the results remained significant after adjustment for multiple testing.
There were no significant Genotype × Drug interactions on the outcome measures for the genotype groups at rs1799971, rs660756, rs1918760 and rs9371781. No significant associations were found between any of the seven investigated SNPs and placebo or the 20 mg amphetamine condition.
OPRM1 haplotypes and ARCI scales
Linkage disequilibrium measures (D’-values) between the seven investigated polymorphisms are shown in (Haploview software version: 4.0). Because high pairwise LD measures (D’=1.0) were found between rs1799971 and rs510769 as well as between rs1918760, rs2281617 and rs1998220, haplotype pairs from the linked SNPs were estimated for each individual. The probability of all haplotype estimates was ≥.99, except for one subject, that had an undetermined genotype at locus rs1799971. This subject was excluded from the relevant haplotype analyses. Seven reconstructed haplotypes were assessed for association with the ARCI scales by performing a two-way ANOVA/ANCOVA using peak change scores for Euphoria, Energy, Stimulation and Sedation as within subject factors. Post hoc analyses were carried out conducting one-way ANOVAs/ANCOVAs using peak change scores for each session. Results are shown in . Group peak change score means and standard error of means (SEM) for the haplotype groups after the 10 mg dose are shown in Table S1
Association of ARCI scores Euphoria, Energy, Stimulation and Sedation after amphetamine with OPRM1 gene haplotypes.
Haplotype AA from rs1799971 and rs510769 was found to be significantly associated with levels of Euphoria and Stimulation (two-way ANOVA, Euphoria: p<0.05, F(4, 310)=3.34; Stimulation: p=0.01, F(4, 310)=3.38) and haplotype AG from rs1799971 and rs510769 was associated with Stimulation scores (two-way ANOVA, p<0.05, F(4, 310)=2.90). Both of these findings were confined to the 10 mg dose condition (one-way ANOVA, p<0.05, F(2, 157)=5.36). Subjects with 0 copies of haplotype AG scored lower on the associated ARCI scale than subjects with one or two haplotype copies. Participants with two copies of haplotype AA felt less euphoric and stimulated after 10mg of amphetamine than subjects with one or zero haplotype copies (one-way ANOVA, Euphoria: p<.0.05, F(2, 157)=3.26; Stimulation: p=0.01, F(2, 157)=4.76).
Haplotype ATA rs1918760, rs2281617 and rs1998220 was significantly associated with Euphoria and Energy (two-way ANOVA/ANCOVA, Euphoria: p<0.01, F(4, 312)=3.47; Energy: p<0.05, F(4 312), =3.18) after the 10 mg dose (one-way ANOVA/ANCOVA, Euphoria: p<0.001, F(2, 158)=8.17; Energy: p=0.001, F(2, 155)=7.01). People having zero copies of haplotype ATA from rs1918760, rs2281617 and rs1998220 felt significantly more energetic and euphoric after 10 mg amphetamine than participants with one or two copies. Along the same lines the haplotype GCG was significantly associated with increased Energy (two-way ANCOVA, p<0.05, F(4, 306)=2.78) after 10 mg amphetamine; however this effect was not significant in any of the post-hoc analyses.
There were few associations between the haplotypes and baseline scores. Haplotype ATA groups from rs1918760, rs2281617 and rs1998220 differed at baseline on the Euphoria and Stimulation scales (two-way ANOVA, Euphoria: p<0.05, F(4, 312)=2.69; Stimulation: p<0.01, F(4, 312)=3.77). Subjects with two copies of haplotype ATA scored higher on Euphoria and Stimulation at baseline at the 10 mg session, which was significant in post hoc analyses for Euphoria only (one-way ANOVA, p: p<0.05, F(2, 158)=3.37). Haplotype GCG groups from rs1918760, rs2281617 and rs1998220 differed at baseline on the Sedation scale (two-way ANOVAs, p<0.05, F(4, 312)=2.85). Subjects with 2 copies of haplotype GCG appeared to score higher on Sedation at baseline of the 10 and 20 mg amphetamine session. However, these differences were not significant in post-hoc analyses.
OPRM1 gene polymorphisms, haplotypes and physiological measures
Association analyses were performed between peak change scores of heart rate, systolic and diastolic blood pressure at the different doses and the investigated SNPs and haplotypes (two-way ANOVA/ANCOVA). Because subjects with a higher BMI had significantly higher diastolic blood pressure BMI was included as a covariate in analyses involving this measure.
The SNP rs2281617 and one haplotype were associated with some of the physiological measures (Table S2
). Specifically, genotype C/C of rs2281617 was associated with higher diastolic blood pressure after 10 mg amphetamine (two-way ANCOVA, p<0.05, F(2, 310)=3.84, post hoc analysis: one-way ANCOVA, 10 mg: p<0.05, F(1, 159)=5.76). Increases in diastolic blood pressure after 10 mg amphetamine were also significantly associated with haplotype ATA from rs1918760, rs2281617 and rs1998220 (two-way ANCOVA, p<0.05, F(4, 298)=3.07 post hoc analysis: one-way ANCOVA, 10mg: p<0.05, F(2, 153)=3.45) and haplotype GCG (two-way ANCOVA, p=0.05, F(4, 296)=3.27, post hoc analysis: one-way ANCOVA, 10 mg, p<0.05, F(2, 153)=4.58). There was a significant Drug×Genotype interaction between haplotype AA from rs1799971 and rs510769 and diastolic blood pressure (two-way ANOVA, p<0.05, F(2, 296)=2.89), but post hoc analyses revealed that haplotype groups significantly differed after placebo only (one-way ANOVA, p F(2, 155)=3.79). Other polymorphisms and haplotypes did not differ at baseline, after placebo, 10 mg or 20 mg dose for any of the investigated physiological measures.