We performed a GWAS for subjective response to d-amphetamine in a small sample of healthy, non-drug abusing participants who were phenotyped using a laboratory-based, double-blind, placebo-controlled, within-subjects design, using 5.4 million genotyped or imputed SNPs. Phenotypic data were summarized using SFA, which yielded ten generally interpretable factors that represented both drug-independent and drug response phenotypes. We identified a SNP in the CDH13 gene () that was associated with the degree of positive response to amphetamine. We also identified a SNP in the SRD5A1 gene () that was potentially associated with the degree of positive affect of the participants, independent of dose.
Our strongest genetic association with a drug response factor was between rs3784943 and factor F1 (). This SNP is in the 8th
intron of CDH13
, which is highly expressed in multiple brain regions 
has been implicated in drug-abuse related phenotypes by multiple studies from the same group that aggregate evidence at the gene level; specifically, methamphetamine dependence 
, alcohol dependence 
, nicotine dependence 
, successful smoking cessation 
, polysubstance dependence 
, addiction vulnerability 
, and comorbid depression and alcohol dependence 
. In addition, a meta-analysis of cigarettes smoked per day also identified multiple SNPs in CDH13
has also been implicated in attention deficit hyperactivity disorder (ADHD) at multiple SNPs 
, including rs11646411 
and rs6565113 
. A meta-analysis of several GWAS for ADHD identified rs8045006 
. Finally, a meta-analysis of linkage scans for ADHD identified a genomic region containing CDH13
. Based on these results, a candidate gene study of CDH13
and ADHD was performed that found a gene-wide significant association at one SNP (rs11150556) 
. In addition, CDH13
has also been implicated by GWAS in depression (rs10514585) 
, extraversion (rs4783307, rs8056579) 
, agreeableness (rs9940706) 
and response to antipsychotic therapy (rs17216786) 
, as well as in a meta-analysis for extraversion (rs8057458) 
. Variants in CDH13
have also been significantly associated with adiponectin levels in two GWAS 
. None of the SNPs implicated by the above studies were strongly associated with our factors, nor were any of the above-mentioned SNPs in strong LD with rs3784943. Thus, while this gene has attracted significant attention, particularly in the psychiatric genetics literature, these studies have not converged on a single genetic regulator or putative mechanism of regulation for this gene for possibly corresponding phenotypic effects. Intriguingly, it has recently been reported that CDH13
knockout mice show decreases in conditioned place preference to cocaine (J. Drgonova, SFN abstract #871.11/D64
), which is highly consistent with a difference in the subjective response to amphetamine that we observed.
The association between CDH13
and sensitivity to the subjective effects of amphetamine may provide insight into the mechanism by which an allele influences the risk for drug dependence. Drug dependence develops through many stages, including initial experimentation with drugs, continued use, dependence, withdrawal and relapse 
. Genetic variants might influence risk by impacting one or more of these stages 
. Whereas a genetic association with the diagnosis of drug dependence provides little insight into which stage is under genetic control, our intermediate phenotype approach suggests that rs3784943 affects magnitude of the initial subjective response to the drug. This would be expected to influence an early stage in the addiction process in which early experimentation with drugs progresses to more frequent drug use. Further studies are needed to assess whether this SNP might also influence the subjective response to other drugs of abuse.
Our strongest association with a baseline factor was between rs472402 and factor F2 (). This SNP is in the first intron of SRD5A1
, which is expressed in the brain 
. The protein product of this gene catalyzes the rate-limiting step in the production of the neurosteroid allopregnanolone 
, which is a GABAA
agonist. Allopregnanolone has been shown to have anxiolytic effects in animals 
and progesterone administration, perhaps because it is converted into allopregnanolone, has been shown to elicit mild sedative effects in humans 
; these are broadly similar to the phenotypes that are associated with this SNP (). Another SNP (rs248797; Table S5
) that is in strong LD with rs472402 (r2
0.73) has been identified as a cis
-eQTL for the SRD5A1
gene in human monocytes 
) and is also in moderate LD (r2
0.3–0.7) with several SNPs that have been identified as cis
-eQTLs for SRD5A1
in post-mortem samples from cerebellum and parietal cortex 
. Rs472402 is also in strong LD with rs248793 (r2
0.89), which has been associated with differences in SRD5A1 activity 
and with risk for alcohol dependence 
. Finally, SRD5A1
has also been suggested as a positional candidate gene based on a linkage study for cocaine dependence and major depressive episode 
. Taken together these results suggest that SNPs in SRD5A1
could influence subjective positive affect by modulating both expression and enzymatic activity of SRD5A1
and thereby altering allopregnanolone levels in the brain.
While techniques that aggregate information using dimension reduction are attractive, the results must be both interpretable and biologically meaningful. Our results here illustrate how SFA can yield more interpretable data summaries than other methods, such as PCA, facilitating the systematic aggregation of a large collection of phenotypic information without sacrificing interpretability. Regardless of the dimension reduction technique used to identify putative associations, it is important to examine the raw phenotypes to determine how a given SNP correlates with the underlying phenotypic data ( and ). In both of the potential associations presented in this study, the phenotypes that loaded onto the factor overlapped well with, but were not identical to, the phenotypes that were influenced by the identified SNPs ( and ).
This study has several strengths and limitations. One limitation is that we used a relatively small number of participants to test a large number of putative predictors (genotypes). Despite the small sample, there is credible evidence that intermediate pharmacogenomic phenotypes might be more likely to have larger effect alleles 
. Another potential limitation of this study was the use of participants that were not heavy drug abusers and thus might not have been at the highest genetic risk for developing drug abuse. Although our study was based on the idea that genetic variability in a population of healthy young adults is representative of the larger population, it is possible that our ascertainment procedures excluded relevant genetic variants. Similarly, we chose modest doses that were administered orally, whereas drug users typically ingest higher doses with faster routes of administration (intranasal, intravenous, or inhalation). Lastly, differences in drug metabolism may constitute an uncontrolled source of variability. Reducing the complex phenotype data to a small number of baseline and response factors is a strength of our study because baseline differences might otherwise confound our measurement of drug response. We chose to compute associations for factors representing both drug-independent and drug-dependent phenotypes because we believed that genetic associations with either baseline or response measurements are potentially interesting, although of course our primary motivation for the study was to study drug response.
While intermediate phenotypes have many advantages, a major limitation is that replication samples are seldom available. We were unable to identify any suitable replication dataset for the association between the SNP (rs3784943) in CDH13 and positive subjective response. Replication of this result was challenging because a suitable study would have included administration of a stimulant drug, under similar conditions and corresponding dosage, and would have had to collect the same or related measurements of subjective response. In addition, the associated SNP had a fairly low minor allele frequency and only a moderate effect on the phenotype, so a relatively large dataset would be needed to have sufficient power for replication. Alternatively, our results can be viewed as replicating prior associations with CDH13, however the lack of convergence on a single SNP or haplotype across studies is a cause for concern. Because of these limitations, the results from Cdh13 knockout mice and other biologically based studies of CDH13 function may be the best route for further investigation and replication of our results.
Replication of the association between the SNP (rs472402) in SRD5A1
and baseline positive affect appeared to be a more tractable problem because multiple GWAS datasets that include measurements of personality traits are available. Although our questionnaires were designed to detect differences in mood state, the effect persisted regardless of session or time point, which is consistent with a difference in trait independent of drug or session effects. We examined SNPs in strong LD with rs472402 in subjects from the National Institutes of Health/National Institute of Mental Health (NIH/NIMH) bipolar genetics studies with personality trait data consisting of the ZKPQ (n
1007) and TCI (n
944) subscales 
; none of our analyses provided evidence of replication. However, the differences in the phenotypes and the study design were substantial between these studies and our own; this may indicate a poor fit for replication. We also considered the baseline sessions from smaller datasets collected in our lab using similar methodology but testing different drugs, however none of these datasets showed convincing evidence for replication. Taken together these results do not support the association between rs472402 and baseline positive affect.
In conclusion we have performed the first GWAS of the subjective response to a drug of abuse and identified two interesting associations. CDH13 has previously been associated with a number of substance abuse and other psychiatric phenotypes and is also supported by data from knock-out mice. Replication in independent datasets will be important to establish the role of CDH13 in drug addiction, and to determine the extent to which initial drug responses are related to the etiology of addiction. SRD5A1 is similarly supported by prior evidence including corroborating genetic studies, gene expression and enzymatic activity data, but the lack of replication of this result is a cause for concern. We were motivated to perform this study using a relatively modest sample size because we believed that intermediate pharmacogenomic phenotypes might be influenced by alleles that contribute a larger fraction of the genetic variance. This study reflects both the utility and challenges of such phenotypes for discovery and enrichment of our understanding of complex psychiatric constructs such as drug abuse.