Examining impulsivity as an endophenotype using a behavioral approach: a DRD2 TaqI A and DRD4 48-bp VNTR association study

The effort to characterize the behavioral effects of genetic polymorphisms has produced a massive web of ambiguous associations and linkages [1‐3]. One strategy to clarify the genetic bases of behavior is the endophenotype approach [2, 4, 5], which seeks to elucidate genetic associations with phenotypes of interest, typically diseases, by examining intermediary phenotypes (i.e., endophenotypes) that are more closely related to the functional influence of genetic variants. By characterizing endophenotypes, or "upstream" phenotypes that do not always result in the "downstream" disorder, progress may be made in both deconstructing the etiologies of complex psychiatric disorders and understanding the genetic and evolutionary basis for variation in non-disordered individuals [4]. In addition, endophenotypes are putatively more closely connected to genetic functionality, so larger magnitude genetic effects may be evident and thus more readily detectable in smaller samples [6, 7], cf. [8].

Impulsivity is a prototypic candidate for the endophenotype approach because it is a trait that varies considerably in the overall population [9‐12] and is associated with an array of psychiatric disorders. These include alcohol and drug dependence [13‐19], pathological gambling [20, 21], attention deficit-hyperactivity disorder (ADHD) [22, 23], borderline personality disorder [24] and antisocial personality disorder [25‐27]. Moreover, there is evidence for the heritability of impulsive behavior in both humans and non-human animals [28]. In terms of personality disorders, familial transmission of impulsive traits have been reported [24, 29]. In addition, twin studies using the Karolinska Scale of Personality (KSP), Multidimensional Personality Questionnaire (MPQ) and Barratt Impulsivity Scale, Version 11 (BIS) also found substantial heritable components to impulsivity [30‐33]. Similarly, impulsivity has also been demonstrated to be heritable in vervet monkeys as assessed by the Intruder Challenge Test [34], and in mice assessed by a delay discounting test [35]. However, impulsivity has also been found to vary with such factors as gender [36], age [37, 38], education [37, 39, 40], health [39], savings [39] and parent rearing styles [41], suggesting that other variables also have a meaningful influence. Although the relative contributions of genetic and environmental variables are unclear at this point, converging lines of evidence suggest genetic factors play an important role.

As a result, a number of studies have explored the molecular genetic basis for variation in impulsivity by examining the associations between genetic polymorphisms and measures of impulsivity. Focusing on the serotonergic system, Preuss et al. [42] reported an association between A alleles of the 5HT2A receptor – G-1438A polymorphism and increased impulsivity, but Patkar et al. [43] and Baca-Garceiro et al. [44] did not replicate that relationship. Within the dopamine system, Retz et al. [45] found an association between heterozygotes of the DRD3 single nucleotide polymorphism (SNP) and increased impulsivity, and Limosin et al. [46] found an association with the A2 alleles of the DRD2 TaqI A SNP and increased impulsivity in alcoholics, but both represent isolated reports. More broadly, in studies of the genetics of personality, impulsivity has been examined in the context of novelty seeking, a trait of which it is a cardinal feature [47]. From this perspective, a number of studies have found associations between long alleles of the DRD4 48 bp Variable Number of Tandem Repeats (VNTR) polymorphism and novelty-seeking, but many have not. One meta-analysis has found no overall association between DRD4 48 bp and novelty seeking [48], another a small effect [49] and a third review reports a positive association [50]. On balance, the current empirical literature is highly heterogeneous, in terms of the genes examined, phenotypic scales used and actual findings.

A limitation of the previous attempts to characterize genetic influences on impulsivity has been the prevailing reliance on self-report measures of impulsivity. There are a number of limitations to the self-report measures in general [51, 52] and these apply also in the case of impulsivity. For example, individuals may vary considerably in their semantic construal of impulsivity-related question content and they may also vary in their positive or negative attributions about the content of the questions, creating an implicit or explicit response bias. Moreover, there is considerable evidence that individuals' self-reports can be substantially at variance with their actual behavior [51, 52], suggesting that self-reported impulsivity may not always accurately reflect actual levels of impulsivity. This is further complicated by the fact that impulsivity is itself a multifaceted construct [28, 53, 54], including aspects of cognitive deliberation, reward valuation, behavioral inhibition and behavioral execution, among others. As such, it is unlikely that one genetic polymorphism would be pleiotropically responsible for all of these diverse facets, especially given that these different aspects are not always significantly associated with each other [e.g., [55, 56]]. Indeed, there is ongoing debate as to which represent essential features of impulsivity, and which are different constructs altogether [e.g., [28, 55, 56]].

These limitations may be addressed by an increased emphasis on behavioral assessments of impulsivity. A number of behavioral indices of impulsivity have been developed [e.g., [57, 58]] and these measures more objectively assess narrowly defined aspects of impulsive behavior and may reduce the bias of self-report. Moreover, in some cases, animal models and cognitive neuroscience approaches have illuminated the underlying neurobiology subserving behavioral performance on such measures [59‐62], permitting more refined hypothesis testing of genetic variants that influence impulsivity. Although behavioral testing involves considerably greater experimental burden than self-report assessments, these measures may nonetheless substantially contribute to clarifying impulsivity as an endophenotype. These behavioral endophenotypes are expected to be more powerful than similar association studies which instead use broader psychological disorders as phenotypes.

The most widely studied behavioral measure of impulsivity is the delay discounting task (DDT). From a delay discounting perspective, impulsivity is defined as the relative preference for a smaller reward, sooner in time, compared to a larger reward, later in time [63]; that is, the amount a person discounts a reward based on its delay. Importantly, this measure of impulsivity has proven highly sensitive to increased impulsivity in psychiatric populations. More precipitous discounting (i.e., increased impulsivity) is associated with alcohol misuse [13, 64, 65], tobacco dependence [66, 67], opiate dependence [68, 69], stimulant dependence [70], pathological gambling [20, 55, 71], and antisocial personality disorder [27]. In addition, the DDT has been demonstrated to be stable over time [72].

Versions of the delay discounting paradigm may also be used to study impulsivity in animal models [35, 59, 60, 73]. Neurobiologically, non-human research suggests that corticostriatal-mesolimbic substrates mediate delay discounting performance [59, 61] and that dopamine is the critical neurotransmitter involved [60, 73‐75]. In addition, recent human neuroimaging findings indicate that preference for smaller immediate rewards is associated with greater mesolimbic activation, whereas preference for delayed rewards is associated with greater frontal-parietal activation [62]. Taken together, these findings suggest that impulsive decision-making from a delay-discounting perspective reflects a dynamic balance of frontal versus limbic dopaminergic activation. Importantly, there is indirect evidence that impulsivity as measured by delay discounting is heritable in humans [13] and direct evidence of its heritability in mouse strains [35].

Given the limitations to the current literature on impulsivity as an endophenotype and the potential promise of using behavioral measures, in the current study we examined impulsivity as a potential endophenotype using two dopaminergic genetic polymorphisms as candidates for observed variation in impulsivity as measured by the DDT and three traditional measures of impulsivity. These three measures include the BIS, Eysenck Impulsivity Questionnaire (EIQ), and the Sensation Seeking Scale – Form A (SSS), all of which have undergone extensive psychometric validation [11, 12, 76]. The BIS and EIQ are highly correlated and theoretically related scales, however they are associated with different neural activation profiles in a behavioral inhibition task [77], suggesting that they assess distinct facets of impulsivity. As previously mentioned, the BIS has shown a strong heritable component in a twin study [31]. Sensation seeking is a related construct to impulsivity, and has been shown to be both heritable and to potentially share genetically-mediated common biological mechanisms with impulsivity [33]. Additionally, SSS subscores are inversely related to KPS monotony avoidance, which has also been shown to be heritable [32]. In general, the empirical literature suggests performance on these measures is heritable, although this is clearly not definitive.

The two dopaminergic genetic polymorphisms we examined were the DRD2 TaqI A and DRD4 48 bp VNTR polymorphisms. Both have been associated with psychiatric disorders involving impulsivity, namely substance abuse and ADHD (for reviews, see [78, 79]). In addition, the two polymorphisms appear to functionally influence the dopamine D₂ and D₄ receptors, which are densely located in the corticostriatal-mesolimbic system [80‐87], the apparent neurobiological substrate and neurotransmitter system underlying delay discounting [59‐61, 73‐75].

The DRD2 TaqI A site is a SNP with two possible alleles, the major A2, and minor A1. The A1+ genotype (heterozygous or homozygous A1) has been most strongly associated with substance abuse, particularly alcoholism [83], albeit with some controversy. The A1+ genotype has also been related to pathological gambling, novelty seeking, and sensation seeking [88]. The DRD2 TaqI A site is 9.4 kb downstream from the coding region for the dopamine D₂ receptor gene. It is not in any known regulatory region, and although the A1 allele is associated with a decrease in dopamine D₂ binding and glucose metabolic rates in many brain regions [83, 89, 90], its mechanism for influencing DRD2 expression is unknown. The TaqI A polymorphism is also located in a nearby kinase gene, the Ankyrin Repeat and Kinase Domain Containing 1 (ANKK1) gene, where it causes a Glutamate→ Lysine substitution [91, 92]. The results of the amino acid substitution are not known, but could impact interactions of ANKK1 proteins with other proteins including the dopamine D₂ receptor [92]. No other polymorphism has been revealed in linkage disequilibrium with TaqI A that could easily account for these associations [91‐93].

The DRD4 48-bp VNTR polymorphism is in exon 3 of the gene coding for the dopamine D₄ receptor. The VNTR polymorphism varies between 2 and 11 repeats of a similar 48 bp coding region sequence, with a trimodal distribution of 2, 4 and 7 repeat alleles (2R, 4R and 7R) in most, but not all, populations [94]. Although the functional significance of the DRD4 VNTR polymorphism has not been definitively characterized, long alleles (typically 7R as opposed to 4R) have been generally found to be functionally less reactive in in-vitro expression experiments [95‐99], with some heterogeneity [100‐104]. Additionally, in-vivo pharmacological treatments are also generally consistent with 7R alleles resulting in less responsive D₄ receptors than 4R alleles [105‐109].

We predicted that possession of at least one A1 allele for the DRD2 TaqI A and at least one long allele (7-repeats or longer) of the DRD4 VNTR genotype would be associated with greater impulsivity. Moreover, we predicted that the delay discounting task would be more sensitive than the self-report measures, as reflected in larger magnitude effects. However, we did not predict one polymorphism to be more likely to exhibit significant associations relative to the other. Finally, based on previous findings reporting interactions between D₂ and D₄ receptor genes [e.g., [110]], we also examined both potential interactive effects (i.e., quantitatively disproportionate effects based on a combination of polymorphisms of both genes) and potential additive effects (i.e., linearly increasing effects based on a combination of polymorphisms of both genes).

The premises of this study were that the ambiguity in understanding genetic contributions to impulsivity may be due to limitations of self-report measures and that behavioral tasks measuring discrete facets of impulsivity may address this issue. This hypothesis was broadly supported by the results. Performance on the DDT revealed a significant main effect of DRD2 TaqI A status, such that A1+ individuals exhibited greater impulsivity, and a significant DRD2 TaqI A by DRD4 48 bp VNTR interaction, such that possession of at least one A1+ allele and at least one L+ allele was associated with a pronounced increase in impulsivity. These differences in delay discounting were quite dramatic; for example, A1+/L+ subjects valued $100 less than half as much as all the other allelic combinations at a 5-year delay (Figure 2). Importantly, these relationships did not appear to be a function of population stratification or gender.

Beyond delay discounting, it was notable that there were no significant genetic associations evident for any of the survey-based self-report measures, with the exception of a trend toward greater sensation seeking reflecting the same DRD2 TaqI A by DRD4 VNTR interaction. This could be because the survey-based scales generated scores that were based on the individual's subjective perception of a variety of distal behavioral tendencies and the relationships were muddied by self-report biases. Alternatively, it is possible that the facets of impulsivity assessed by the survey-based measures are simply not relevant endophenotypes of the genes examined in this study. Consistent with evidence of impulsivity as a multifaceted construct [12, 53], mixed correlations were evident across the various measures (Table 5), revealing moderate associations among the self-report measures, but generally negligible associations between the DDT and the other indices. Previous studies have reported both significant and nonsignificant correlations between delay discounting and self-report measures of impulsivity [65, 68, 125‐127]. The overlap between these different facets and methods of assessing impulsivity appears to be variable by sample.

There are a number of aspects of these findings that warrant further discussion, particularly the delay discounting findings. The DRD2 TaqI A main effect was consistent with our hypotheses, but we also predicted a main effect for DRD4 VNTR genotype, which was not evident, and an interaction between the two was examined based on its plausibility, but was not predicted a priori. In terms of the DRD2 TaqI A main effect, subsequent analyses revealed that the most meaningful role of A1+/A1- status was actually in combination with DRD4 VNTR genotype in an interaction effect on performance. However, in understanding this effect, an important distinction must be made between additive and interactive genetic effects. In this case, an additive effect would refer to possession of each allele of interest being associated with greater impulsivity, and possession of alleles at both loci being associated with a linearly greater level of impulsivity. A clear example of an additive genetic effect is exemplified in two recent studies [128, 129], in which polymorphisms of the DRD2 TaqI A and SLC6A3 genes were examined in reference to stress- and cue-elicited craving in African-American smokers. Both polymorphisms of interest were associated with greater craving, and exhibited an additive effect such that possession of neither polymorphism was associated with the least amount of craving, possession of either was associated with greater craving, and possession of both was associated with the greatest amount of craving.

In contrast, an interactive effect in this case would refer to the case in which possession of the two alleles would be associated with quantitatively disproportionate level of impulsivity, which was the case in the current study. Individuals who were A1+/L+ exhibited disproportionately more precipitous delay discounting than all other groups, whereas all other allelic combinations exhibited highly similar levels of discounting (Figure 2). An additive effect, which would have been evident if both polymorphisms of interest exhibited main effects and a linear increase in performance based on the possession of both candidate alleles, was not evident.

With regard to the mechanisms of underlying the interaction between the two loci, any discussion must be speculative given that the two polymorphisms under consideration have not been definitively characterized. Acknowledging this ambiguity, the observed associations may be understood in the context of the relative neuroanatomical localization of the D₂ and D₄ receptors. Dopamine D₂ receptors seem to play a more prominent role in the striatum than D₄ receptors, whereas D₄ receptors seem to be more influential in the prefrontal cortex and cortex than D₂ receptors [80, 84‐87, 130‐134], however: [135, 136]. As observed with functional magnetic resonance imaging (fMRI), choices for immediate smaller rewards is associated with greater peak activation in the ventral striatum and regions of the medial prefrontal cortex and less relative activation in regions of the lateral prefrontal and parietal cortex [62]. In contrast, for choices for delayed rewards, the relative activations are reversed, with generally greater lateral prefrontal and parietal activation and decreased activation of the ventral striatum and medial prefrontal cortex [62]. Other studies suggest more generally that behavioral inhibition, a fundamental part of impulsivity is modulated via dopamine systems in the striatum and prefrontal cortex [28]. Together, this suggests that the D₂ polymorphism may be modifying more primitive limbic neural systems involved in reward salience while the D₄ polymorphism may be modifying higher-level systems involved in abstract thinking, deliberation and behavioral inhibition. Thus, particularly steep delay discounting (increased impulsivity) in those with both long DRD4 and A1 DRD2 alleles may reflect the concurrent variation in two key points in the reward decision-making loops spanning the corticostriatal-mesolimbic axis. Specifically, the pattern of findings suggest that for those individuals who have a greater sensitivity to reward based on possession of the DRD2 A1 allele, decreased frontal-cortical inhibition resulting from possession of a DRD4 VNTR long allele results in substantially greater discounting of delayed rewards.

Of interest, however, the contrapositive relationship by genotype was not indicated. Considered together, the presence of a DRD2 TaqI A main effect, the absence of a DRD4 VNTR main effect, and a significant interaction suggests that the influence of A1+-mediated functional variation in striatal D₂ receptors has primacy over the influence of L+-mediated cortical D₄ receptor variation in modulating inter-temporal choice. That is, for individuals with a mesolimbic dopamine system that is predisposed to favor dopaiminergic rewards (A1+), the presence of an allele that may be associated with reduced frontal cortical inhibitory control (L+) may result in disproportionately greater discounting of delayed rewards, but not the other way around. In cognitive terms, the results suggest that the effects of inhibitory considerations of the future are superimposed on the limbic signals of the incentive salience of immediate rewards. However, we reiterate that given that the functional roles of both genetic polymorphisms are far from fully understood, this explanation must be speculative at this point. Future studies using neuroimaging techniques may directly address this interpretation.

Assuming these findings are genuine and can be replicated, they have potential implications for the ambiguity in association studies of genetic variables in psychiatric disorders [1‐3]. To date, single locus association studies have typically reported mixed findings of significant associations and failures to replicate. The current data, and other studies revealing interactions between unlinked loci [e.g., [110, 137, 138]], suggest that single gene association studies may overlook the fact that important facets of a disorder may depend not only on independent effects of polymorphism, but also on interactions between multiple genes within a given system.

There are a number of qualifications of these findings worth noting. Although they provide preliminary support for the notion that behavioral aspects of impulsivity may be more amenable to investigations of genetic influences, to our knowledge they represent the first report of specific genetic influences on delay discounting. As such, they must be replicated to affirm their empirical validity and to more conclusively affirm the potential of a task-based approach to generate more reliable findings than self-report measures. In addition, it should be noted that these findings most clearly apply to the European ancestry subsample, which both represented the largest proportion of subjects and was sufficiently large to affirm the findings independent of other ethnic subsamples. As such, although we found no evidence of population stratification, studies of delay discounting as a potential endophenotype in larger samples of non-European descendants will be important to address the generalizability of these findings.

In summary, the current study sought to examine the influences of two genetic polymorphisms on impulsivity using a behavioral task and traditional self-report measures. The findings indicated both independent and interactive genetic effects on the behavioral task, suggesting that genetically-based functional differences in the corticostriatal-mesolimbic dopamine system are responsible for variation in impulsivity from a delay discounting perspective. Rather than being a function of a single locus, these data suggest that the most prominent effect was as result of an interaction between polymorphisms of the DRD2 TaqI A and DRD4 VNTR genes. Although these findings must be replicated, they may contribute to understanding the genetic basis of impulsivity and provide support for the notion of using behavioral tasks in doing so.