The influence of serotonin- and other genes on impulsive behavioral aggression and cognitive impulsivity in children with attention-deficit/hyperactivity disorder (ADHD): Findings from a family-based association test (FBAT) analysis

Impulsive actions usually damage not only the victim but often their perpetrator. In fights, suicide or fire-setting the aggressive component is immediately apparent. But the risky and overly rapid decisions behind these actions demonstrate a cognitive component that is often revealed in gambling and seeking immediate gratification. Impulsivity is a core construct of many mental disorders [1]. In particular, it is an essential component of the diagnosis of the most prevalent form of attention-deficit/hyperactivity disorder ADHD, namely the 'combined type' (ADHD-ct: [2]).

Across a group of youngsters with ADHD-ct there are many who show abrupt bursts of impulsive aggression (related to conduct disorder), but many others who (merely) make rapid and wrong decisions in everyday life, or abhor delayed gratification to their cost [3]. Here, we consider that these two forms of impulsivity may have distinct pathophysiological features with relation to ADHD. In particular, this article focuses on serotonin activity, [4]) and heritable features of ADHD, where a 70–80% heritability has been clearly demonstrated [5].

Impulsivity has been briefly and broadly defined as "action without foresight" [6]. More generally impulsivity covers actions that appear "poorly conceived, prematurely expressed, and are unduly risky or inappropriate to the situation. They often result in undesirable consequences" [7]. Impulsivity has been linked strongly to outbreaks of aggression in adults (here considered as overt impulsive behavioral aggression) that are associated with increased 5-HT2a platelet receptor binding (Bmax and Kd), decreased 5-HT1a postmortem binding and low serotonin (5-HT) activity [8‐10]. While some studies have failed to find the same relationship of low 5-HT activity with aggression in children [11‐13] many others do report an inverse relationship [14‐16] sensitive to pharmacotherapy [17]. It may be that the phenocopy for aggression has not always fully developed at the time of study. Impulsive aggression reacts to provocation and is marked after 5-HT synthesis reduction following tryptophan depletion [18]. Indeed, Halperin's group, in showing an apparently protective effect of high 5-HT activity, recently proposed that low activity is important if not sufficient for the emergence of aggression. Consistent with the developmental expression of the trait they reported that low 5-HT responsivity in childhood predicted the development of antisocial personality disorder 9 years later [19].

More differentiated views of impulsiveness [20] suggest that, as well as aggressive and behavioral impulsiveness associated with emotional lability and motor control, there are also cognitive or attention-related elements [21]; for example, there are the cognitive expressions of impulsiveness that are associated with failures of inhibitory executive functions ("executive attention", [22]). Typically decisions are made hastily and lead to maladaptive outcomes. Such impulsivity is epitomized by errors of commission on a discrimination or Go/no-go task, and is perhaps reflected in more motivationally-based domains where response choice is driven by early reinforcement [23]. This form of cognitive impulsiveness may be regarded as a form of impaired selective attention. However, it is a result of overly rapid, inadequate information processing that leads to inappropriate action. This should be contrasted with absences of conventional attentive processes (inattentiveness) that would normally bring the searchlight, the focus of selective processing to stimulus identification. Inattention leads to the omission of a response. Thus it is important to distinguish the term inattention, as it is usually understood, from impulsive (cognitive) processing that risks non-adaptive responses. Such impulsive decisions about the moment-to-moment organization and planning of responses to stimuli are commonly made by both younger [24] and older subjects with ADHD [25].

The goal of the current study is to disentangle the relationship between aggressive impulsivity of a more behavioral nature and cognitive impulsivity in ADHD by exploring the differential patterns of association of these two forms of impulsivity with genes that have significance for 5-HT function, a putative neurobiological marker of impulsivity. There is mounting evidence that polymorphisms for genes influencing several features of the 5-HT system are associated with ADHD [4], and a case has been made for a differential involvement of 5-HT activity in impulsive aggressive behavior and cognitive impulsivity [26]. For example, in one study increases of platelet 5-HT transporter affinity in 20 children with ADHD correlated with ratings of externalizing behavior, but decreases of affinity were associated with distractibility, impulsivity and the difficulty to withhold a prepotent response on the stop-task [27]. Similarly a study of adolescent prisoners described opposite correlations of 5-HT activity with crime and other measures of impulsivity [28]. On tasks assessing impulsivity (see [29]), errors of commission are typical of youngsters with ADHD who show an underactivation in the right frontal cortices [30]. An impairment is expressed in similar regions of young healthy subjects treated with a tryptophan-depleting drink [31].

We therefore explored whether there would be commonalities and differences between the two forms of impulsivity and 5-HT genes controlling the activation or suppression of activity within 5-HT systems, along with other genetic markers of neuronal activity (e.g. dopamine) which influence or are influenced by the activity in 5-HT systems [32].

After separating the 47 items from the SDQ and Conners symptom ratings into the two dimensions of aggressive and cognitive impulsivity, we used the FBAT-PC software to evaluate their possible associations with 582 SNPs distributed through 14 relevant genes. The genes were selected because their expression affects 5-HT activity and closely-related neural functions and they have previously been reported to be associated with a diagnosis of ADHD. The focus on 5-HT-related features was based on descriptions of the relevance of this transmitter to the expression of impulsivity [4, 26, 53] and for the preferential transmission of polymorphisms influencing 5-HT function in ADHD (see introduction). Gene-wide significance was achieved only for a variant of the gene encoding the enzyme for the synthesis of adrenalin (PNMT) in cognitive impulsivity. However nominal significance was shown for genes affecting a range of elements of the 5-HT and DA systems in both forms of impulsivity, and that have previously been reported to be associated with ADHD.

The absence of prominent associations for neurotransmitter markers, apart from that with adrenalin synthesis through PNMT variants, was surprising. A gene-wide significant association was found for one SNP for PNMT with cognitive impulsivity, and a second was nominally associated with behavioral aggressive impulsivity from the 4 SNPs for PNMT that were examined. In both instances the trait was over-expressed (table 4). This was partly anticipated by a similar significant finding in a candidate gene analysis for association with ADHD from the IMAGE sample [33]. PNMT is the rate-limiting enzyme in the synthesis of adrenalin that has transmitter and endocrine influences on brain function which can be stimulatory or inhibitory, respectively [54]. The association of adrenalin with CNS arousal is significant in the context of the common attribution of hypoarousal to ADHD function (review: [55]). Further, one might anticipate that arousal levels would be pertinent to both forms of impulsivity under investigation here, but especially the cognitive attention-related form that could be a precursor to the behavioral, aggression-related form. Indeed, adrenalin levels in ADHD children are usually lower than normal [56‐58] unless they show anxiety [59‐61]. Low levels are associated with restlessness and aggressive outbursts [62]. Response to treatment with methylphenidate tends to normalize levels of adrenalin [58, 63] and improve autonomic measures of low arousal (skin conductance levels: [64]). Thus there is broad support for a widespread role for impaired function of arousal and adrenalin systems in ADHD and the impulsivity that is characteristic of the disorder. From our current results, we would predict that this could be based in an altered expression or function of the PNMT gene. Considering the potential parallel involvement of 5-HT systems in impulsivity, and the demonstrated role of 5-HT in the control of autonomic function [65] it would be useful to look for tagging between variants influencing PNMT and 5-HT receptors.

In agreement with our expectations, analyses of the two forms of impulsivity are reflected at least nominally in similar but also in different associations with the genetic markers for monoamine activity. For the 5-HT system, association with synthesis (TPH2) and receptor-mediated mechanisms (HTR1E) may be relevant to both forms of impulsivity. Indeed the same SNP for HTR1E (rs1406946) was nominally associated with both types of impulsiveness in the dominant transmission model. It was over-expressed in the cognitive and under-expressed in the behavioral or aggressive form of impulsivity (table 4). The difference for 5-HT function is one of emphasis, with a number of markers pointing to small effects on synthesis (TPH2 3 SNPs), pre-and post-synaptic receptors (HTR1E, HTR2A, HTR3B: 5 SNPs) and reuptake (SERT: STin2) for cognitive (not behavioral) impulsivity. For cognitive impulsivity 4 SNPs for receptors (apart from HTR1E) were under-expressed in a recessive model, whereas the availability of 5-HT (e.g. TPH2 in a dominant model) was influenced by over-expression of relevant alleles. For the DA system, nominally significant markers were found for both types of impulsivity, but while the emphasis for the behavioral/aggressive form was on DRD4 (one SNP and in particular the 3-repeat allele was over-expressed), cognitive impulsivity was nominally associated with uptake (DAT1: 1 SNP) and the D1 receptor (DRD1: 2 SNPs), which were all under-expressed. Based on their anatomical distributions, this could reflect top-down mesocortical and bottom-up mesolimbic mechanisms of control, respectively [66].

The 5-HT component implies a potential influence of 5-HT activity in those children with ADHD who show aggressive behavioral and cognitive impulsivity both through its availability (TPH2-mediated synthesis and SERT-mediated uptake), and the efficacy of 5-HT action via the 5-HT2 and 5-HT1 receptor families [67]. Generalizing in this context, activation of the 5-HT1B and 5-HT2A receptors tend to be 'stimulatory', while the 5-HT1A and 5-HT2C sites are more often 'inhibitory' [68]. The 5-HT receptor mediated influence obtains especial significance with regard to the interaction of 5-HT with the DA system considering that ADHD is often considered a 'hypodopaminergic' disorder [69, 70] and the 5-HT and DA systems interact directly and widely through subcortical and cortical regions [32]. Even if the genetic effects reported here are modest, there is intriguing support for our hypothesis of opposing types of interactions of DA and 5-HT in the two forms of impulsivity. Thus, in aggressive behavioral impulsivity a DRD4 SNP was overexpressed while those for HTR1E and TPH were underexpressed. In contrast in cognitive impulsivity SNPs for DRD1 and DAT1 were underexpressed while others for TPH2 and HTR1E were overexpressed.

Corrected significant associations for the 5-HT2A gene have also been reported recently for children and adults with ADHD [71] and correlations with hyperactive/impulsive symptoms were described for a normal population [72]. However, other groups have seen no association [73, 74]. Unfortunately, little is known about the functional role of 5-HT1E sites that make up a quarter of those binding 5-HT in the forebrain in autoradiographic postmortem studies of cortical and hippocampal regions (CA3-4 >> CA1 [75]). In contrast, 5-HT3 receptors have been more widely studied, and are abundant in limbic and frontal regions [76]. In contrast to the inhibitory functions attributed to HT1 receptors, stimulation of HT3 sites enhances DA release [77]. Thus the nominal significance of the association of 4 SNPs for stimulatory receptors (HT2A and HT3B) with cognitive impulsivity is consistent with the opposing effects of 5-HT activity on the two forms of impulsiveness proposed in the introduction.

The presence of 3 SNPs for TPH2 with near gene-wide significant associations with cognitive impulsiveness along with the significant relationship for a SERT allele (as also indicated by [78]) would suggest that markers for the presynaptic availability of 5-HT merit continued attention. The importance of this is illustrated, for example, by the functional consequence of changes in SERT activity as expressed with the short allele. In this instance the binding potential of 5-HT receptors may alter, as demonstrated for the 5-HT1a binding site [79]. In turn changes at this binding site (with inhibitory function) would strongly modulate transmitter release in DA systems. Considering the well-known interactions of these two monoaminergic systems [32], the potential for tagging between genetic loci should be sought in future studies.

Lastly, we studied SNPs representing 3 other genes (BDNF, FADS2, NURR1/NR4A2) on the grounds that their activity could influence neurotransmission including that based on 5-HT, and that some associations with ADHD have been reported [33]. Brain derived neurotrophic factor (BDNF) facilitates the development of the release mechanisms for several neurotransmitters. It is known to have age-dependent effects on 5-HT transporter function [80]. In BDNF-knockout mice this led to reduced 5-HT metabolism [81] and aggressive impulsive behavior [82]. A nominal association with both forms of impulsiveness emerged from the present analysis, that may reflect a small effect on the development of monoamine systems in general for cases of ADHD that particularly reflect an etiology of delayed maturation [83]. However, there is not much support for an association of BDNF markers with the diagnosis of ADHD [84]. NURR1/NR4A2 is an orphan nuclear receptor and transcription factor crucial to the genetic control of development of DA neurons [85, 86]. The expression of NURR1/NR4A2 is essential for the induction of several markers of DA activity (e.g. tyrosine hydroxylase). However, there was no evidence here for the altered expression or function of NURR1/NR4A2. Finally, fatty-acid desaturase gene-2 (FADS2) is involved in regulating membrane synthesis and function and at least one polymorphism has been closely associated with ADHD [87]. The ratio of omega-3 to omega-6 unsaturated fatty acids in neural membranes certainly influences their fluidity and the ability of neurons to conduct efficiently: this is a problem for ADHD with documented white matter anomalies [88] and potentially for the function of oligodendrocytes responsible for membrane synthesis [89]. But we found no evidence that the FADS2 markers investigated were associated with the types of impulsivity studied here.

ADHD is a highly heterogeneous disorder: it has been argued that there are potentially multiple variants of the condition with different etiological bases, pathophysiological signatures and neuropsychological and behavioral markers [90]. Consequently, it has been suggested that one of the key goals for ADHD research should be to parse that heterogeneity, to create more neurobiologically homogeneous sub-groupings so as to provide more refined diagnostic criteria and more specific treatment targets [91]. One manifestation of such a strategy is the search for endophenotypes, for pathophysiologic markers that mediate the pathways from genetic and environmental risk to disorder [92]. From this perspective the current differentiation between two types of impulsiveness on the basis of their associated genes and their behavioral characteristics both highlight these constructs as putative endophenotypes for ADHD and encourage further research to explore the possibility that two 'subtypes' of ADHD might exist marked by different types of impulsiveness. The one has a strong basis in motivation and emotion with expression dependent on a degree of motor disinhibition (aggressive-behavioral impulsivity), and the other reflects poor executive control of endogenous attention-related processes that can underlie risky decision-making in several functional domains (cognitive impulsivity).

In particular, this study has taken an exploratory look at whether some SNPs from genes related to 5-HT function are associated with symptom ratings that reflect aggressive behavioral and cognitive impulsivity. Clearly different results could emerge from the study of other genes/SNPs as they become available. These SNPs cover genes for only four of the 22 types 5-HT receptor identified. There are indications that other receptors could be involved (e.g. 5-HT1D, 5-HT4, 5-HT6, 5-HT7: [71, 93]. Complementary to the present study, one using cognitive impulsivity measures from neuropsychological tests (e.g. continuous performance and flanker tasks) would be necessary before one could draw firm conclusions about the influence of heritable features of the 5-HT system on cognitive decision-making processes.

Nonetheless, based on ratings of impulsive symptoms, in the first instance this study strongly implicates genetic influences in ADHD on the synthesis of adrenalin and by implication on the degree of arousal influencing the organization and expression of behavior. In the second instance we report a genetic influence on the expression of SERT, along with potential, small effects of TPH2, 5-HT1E, 5-HT2A, DAT1, DA D1 and D4 receptors prominently expressed in limbic and association cortices and implicated in the initiation of impulsive processes and their expression by young people with ADHD. These features of 3 monoamine systems and two types of impulsivity may be regarded as 'intermediate phenotypes' for the psychopathology of combined-type ADHD. The usefulness of the FBAT-PC approach in the context of dissecting impulsivity in ADHD has been demonstrated, and warrants further, closer study using measures of impulsivity defined by neuropsychological task performance.