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

  • Robert D Oades1Email author,

    Affiliated with

    • Jessica Lasky-Su2,

      Affiliated with

      • Hanna Christiansen1,

        Affiliated with

        • Stephen V Faraone3,

          Affiliated with

          • Edmund JS Sonuga-Barke4, 12, 16, 17,

            Affiliated with

            • Tobias Banaschewski5,

              Affiliated with

              • Wai Chen6,

                Affiliated with

                • Richard JL Anney7,

                  Affiliated with

                  • Jan K Buitelaar8,

                    Affiliated with

                    • Richard P Ebstein9,

                      Affiliated with

                      • Barbara Franke8, 10,

                        Affiliated with

                        • Michael Gill7,

                          Affiliated with

                          • Ana Miranda11,

                            Affiliated with

                            • Herbert Roeyers12,

                              Affiliated with

                              • Aribert Rothenberger13,

                                Affiliated with

                                • Joseph A Sergeant14,

                                  Affiliated with

                                  • Hans-Christoph Steinhausen15,

                                    Affiliated with

                                    • Eric A Taylor16,

                                      Affiliated with

                                      • Margaret Thompson6 and

                                        Affiliated with

                                        • Philip Asherson16

                                          Affiliated with

                                          Behavioral and Brain Functions20084:48

                                          DOI: 10.1186/1744-9081-4-48

                                          Received: 02 June 2008

                                          Accepted: 20 October 2008

                                          Published: 20 October 2008

                                          Abstract

                                          Background

                                          Low serotonergic (5-HT) activity correlates with increased impulsive-aggressive behavior, while the opposite association may apply to cognitive impulsiveness. Both types of impulsivity are associated with attention-deficit/hyperactivity disorder (ADHD), and genes of functional significance for the 5-HT system are implicated in this disorder. Here we demonstrate the separation of aggressive and cognitive components of impulsivity from symptom ratings and test their association with 5-HT and functionally related genes using a family-based association test (FBAT-PC).

                                          Methods

                                          Our sample consisted of 1180 offspring from 607 families from the International Multicenter ADHD Genetics (IMAGE) study. Impulsive symptoms were assessed using the long forms of the Conners and the Strengths and Difficulties parent and teacher questionnaires. Factor analysis showed that the symptoms aggregated into parent- and teacher-rated behavioral and cognitive impulsivity. We then selected 582 single nucleotide polymorphisms (SNPs) from 14 genes directly or indirectly related to 5-HT function. Associations between these SNPs and the behavioral/cognitive groupings of impulsive symptoms were evaluated using the FBAT-PC approach.

                                          Results

                                          In the FBAT-PC analysis for cognitive impulsivity 2 SNPs from the gene encoding phenylethanolamine N-methyltransferase (PNMT, the rate-limiting enzyme for adrenalin synthesis) attained corrected gene-wide significance. Nominal significance was shown for 12 SNPs from BDNF, DRD1, HTR1E, HTR2A, HTR3B, DAT1/SLC6A3, and TPH2 genes replicating reported associations with ADHD. For overt aggressive impulsivity nominal significance was shown for 6 SNPs from BDNF, DRD4, HTR1E, PNMT, and TPH2 genes that have also been reported to be associated with ADHD. Associations for cognitive impulsivity with a SERT/SLC6A4 variant (STin2: 12 repeats) and aggressive behavioral impulsivity with a DRD4 variant (exon 3: 3 repeats) are also described.

                                          Discussion

                                          A genetic influence on monoaminergic involvement in impulsivity shown by children with ADHD was found. There were trends for separate and overlapping influences on impulsive-aggressive behavior and cognitive impulsivity, where an association with PNMT (and arousal mechanisms affected by its activity) was more clearly involved in the latter. Serotonergic and dopaminergic mechanisms were implicated in both forms of impulsivity with a wider range of serotonergic mechanisms (each with a small effect) potentially influencing cognitive impulsivity. These preliminary results should be followed up with an examination of environmental influences and associations with performance on tests of impulsivity in the laboratory.

                                          Background

                                          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 [810]. While some studies have failed to find the same relationship of low 5-HT activity with aggression in children [1113] many others do report an inverse relationship [1416] 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].

                                          Methods

                                          Participants

                                          The initial data-set contained 614 families with 776 European Caucasian cases of DSM-IV ADHD-ct and 706 siblings without the disorder. These families had been recruited as part of the International Multicenter ADHD Genetics Project (IMAGE: details in [33]). They had been included in the first stage of genotyping, which investigated 51 candidate genes for the disorder [24]. In this sample, 1180 offspring from 607 families had the necessary genotype and phenotype data to be included in the analysis. These individuals were recruited from 12 specialist clinics in 8 European countries (Belgium, Germany, Ireland, Israel, Spain, Switzerland, the Netherlands and the United Kingdom). The sample was aged 5–17 years on entry to the study (mean ages for both groups were 10.9 y: table 1). In 4 families the case did not meet the strict criteria for ADHD-ct. However, these families were included in the study because they either 1) met the criteria for inattentive or hyperactive-impulsive ADHD or 2) were just below the threshold for meeting the ADHD-ct criteria. As genetic association studies are underpowered, we felt that including these individuals in the analysis would be beneficial by keeping the sample size higher.
                                          Table 1

                                          Sample Characteristics

                                          Number of genotyped families

                                          607

                                          Total number of genotyped people

                                          2337

                                          Number of genotyped parents

                                          1157

                                          Number of genotyped offspring

                                          1180

                                          Number of offspring per family

                                           

                                          1

                                          124

                                          2

                                          402

                                          3

                                          72

                                          4

                                          9

                                          Gender of offspring:

                                           

                                             Male

                                          793 (67.2%)

                                             Female

                                          387 (32.8%)

                                          Average age of offspring (Standard Deviation)

                                          10.9 (3.0)

                                          Number of affected offspring

                                          603

                                          Origin:

                                           

                                             Northern Europe

                                          882

                                             Spain

                                          99

                                             Israel

                                          199

                                          Note: Diagnosis was restricted to children (ADHD combined type) and not systematically attempted with parents.

                                          Exclusion criteria were genetic or medical disorders associated with behavior mimicking ADHD, autism, epilepsy, IQ<70, and the absence of a medication-free period permitting evaluation in the previous 2 years. Approval for the study was obtained from ethical review boards at each clinic, which were registered with and monitored by the National Institutes of Health, and were in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).

                                          Assessments: diagnosis and impulsivity

                                          Clinical assessments were made during medication free periods or based on recall of such periods in the last 2 years. Diagnoses of ADHD-ct were based on the semi-structured Parental Account of Children's Symptoms (PACS; [34]). Interviewers received formal training in administration of the PACS. PACS includes 4 subscales, representing the different DSM-IV categories for ADHD and disruptive behaviors (hyperactivity, defiance, emotionality, and comorbid disorders). An age adjustment for symptom thresholds is built into the PACS algorithm. Detailed descriptions of the child's typical behavior in a range of specified situations defined by the context (e.g. play) or the behavior shown (e.g. crying) that had occurred in the previous week and in the previous year were rated on a 4-point scale for frequency and severity. Situational pervasiveness was determined by the different situations investigated within the interview and the presence of at least one symptom in each domain reported in the Conners teacher rating (CTRS) ADHD sub-scales. Autism spectrum disorders were screened for all children using the Social Communication Questionnaire (≥ 15) in conjunction with the pro-social scale from the Strengths and Difficulties Questionnaire (SDQ ≤ 4). Inter-rater reliability was high with product-moment correlations for pairs of interviewers ranging from 0.79 to 0.96. A mean kappa coefficient across all the sites of 0.88 (range 0.71–1.00) and an average agreement percentage of 96.6% (range 78.6–100) were obtained. Concurrent validity of the PACS diagnosis was confirmed by the biserial correlation between PACS diagnosis of ADHD-ct with the CTRS N-scale (18 DSM-items) scores at 0.68 and with Conners' Parent rating scale (CPRS) N-scale scores at 0.78 [3436].

                                          For both the cases and the siblings, parents completed the long form of the revised CPRS (80 items) and teachers the long form of the revised CTRS (59 items [37]). Both also completed the SDQ (25 items). The SDQ scores are based on 5 different scales: emotional problems, behavior problems, hyperactivity/inattention, peer problems, and prosocial behavior. The scale is well validated and has good test-retest reliability (α = .85: [38, 39]). From these parent and teacher rating scales (CPRS, CTRS, SDQ) we selected symptoms with impulsive behavioral aggression and cognitive impulsiveness. The 47 items selected are listed in tables 2 and 3 where the source of each item is indicated by the code in the legend.
                                          Table 2

                                          Principle components analysis (oblique rotation) of 47 items of impulsivity: Parent and teacher ratings from Conners and SDQ questionnaires (CP, SP and CT, ST, respectively).

                                          Part 1 – The aggressive behavioral form of impulsivity (factors 1 and 2)

                                          Item

                                          Descriptor

                                          Factor 1

                                          Factor 2

                                          CP47

                                          Temper outbursts

                                          0.950

                                           

                                          CP21

                                          Loses temper

                                          0.893

                                           

                                          SP5

                                          Often has temper tantrums

                                          0.896

                                           

                                          CP31

                                          Irritable

                                          0.840

                                           

                                          CP57

                                          Touchy or easily annoyed

                                          0.859

                                           

                                          CP77

                                          Mood changes quickly

                                          0.820

                                           

                                          CP1

                                          Angry and resentful

                                          0.916

                                           

                                          CP40

                                          Actively defies or refuses to

                                          0.780

                                           

                                          CP11

                                          Argues with adults

                                          0.826

                                           

                                          CP68

                                          Demands to be met at once

                                          0.598

                                           

                                          CP28

                                          Excitable, impulsive

                                          0.356

                                           

                                          CP70

                                          Spiteful or vindictive

                                          1.000

                                           

                                          SP12

                                          Often fights with other children

                                          0.588

                                           

                                          CP66

                                          Disturbs other children

                                          0.294

                                           

                                          CP64

                                          Get upset if someone rearranges

                                          0.947

                                           

                                          SP7

                                          Generally obedient, usually ...

                                          -0.510

                                           

                                          ST5

                                          Often has temper tantrums

                                           

                                          1.000

                                          CT7

                                          Temper outbursts; explosive,

                                           

                                          0.972

                                          CT45

                                          Demands must be met at once

                                           

                                          0.898

                                          CT54

                                          Mood changes quickly, drastically

                                           

                                          0.945

                                          CT37

                                          Argues with adults

                                           

                                          0.957

                                          CT55

                                          Interrupts or intrudes on others

                                           

                                          0.655

                                          CT8

                                          Excitable, impulsive

                                           

                                          0.661

                                          CT29

                                          Has difficulty waiting in turn

                                           

                                          0.606

                                          CT46

                                          Blurts out answers to question

                                           

                                          0.674

                                          ST12

                                          Often fights with other children

                                           

                                          0.922

                                          CT47

                                          Spiteful or vindictive

                                           

                                          0.989

                                          CT20

                                          Leaves seat in classroom or

                                           

                                          0.544

                                          ST7

                                          Generally obedient, usually

                                           

                                          -0.700

                                          ST25

                                          Good attention span sees

                                           

                                          -0.958

                                          Variance Explained

                                          14.6

                                          13.2

                                          Eigen Value

                                          20.4

                                          4.42

                                          Squared Multiple Correlation

                                          0.92

                                          0.90

                                          Table 3

                                          Principle components analysis (oblique rotation) of 47 items of impulsivity: Parent and teacher ratings from Conners and SDQ questionnaires (CP, SP and CT, ST, respectively).

                                          Part 2 -The cognitive form of impulsivity (factors 3 and 4)

                                          Item

                                          Descriptor

                                          Factor 3

                                          Factor 4

                                          CP79

                                          Easily distracted by extraneous ...

                                          0.886

                                           

                                          CP37

                                          Fails to finish things started

                                          1.000

                                           

                                          SP15

                                          Easily distracted, poor concentration

                                          0.924

                                           

                                          CP12

                                          Fails to complete assignment

                                          0.938

                                           

                                          CP48

                                          Get distracted even when instructed

                                          0.827

                                           

                                          CP29

                                          Does not follow instructions through

                                          0.872

                                           

                                          CP80

                                          Blurts out answers to questions

                                          0.694

                                           

                                          CP42

                                          Has difficulty waiting in line

                                          0.601

                                           

                                          SP21

                                          Thinks things out before acting

                                          -0.818

                                           

                                          SP25

                                          Good attention span, sees ...

                                          -0.961

                                           

                                          CT26

                                          Inattentive easily distracted

                                           

                                          0.865

                                          CT52

                                          Distractibility, or attention span poor

                                           

                                          0.858

                                          ST15

                                          Easily distracted, poor concentration

                                           

                                          0.842

                                          CT58

                                          Easily distracted by extraneous

                                           

                                          0.713

                                          CT57

                                          Does not follow instructions through

                                           

                                          1.000

                                          CT17

                                          Fails to finish things started

                                           

                                          0.922

                                          ST21

                                          Thinks things out before acting

                                           

                                          -0.481

                                          ST25

                                          Good attention span sees

                                           

                                          -0.958

                                          Variance Explained

                                          16.0

                                          12.8

                                          Eigen Value

                                          2.53

                                          1.07

                                          Squared Multiple Correlation

                                          0.87

                                          0.88

                                          Item-codes from scales include C, Conners; S, Strengths and difficulties; P, Parent; T, Teacher. The combination of factors 1 and 2 relate to the behavioral aggression component, and factors 3 and 4 relate to the cognitive impulsivity component. Note: If represented as the sum, each form of impulsivity explained 28–29% of the variance. As the informants' ratings correlated highly the true contribution of the components would be approximately halved.

                                          Gene selection

                                          We selected 14 genes with 582 single nucleotide polymorphisms (SNPs) from the data available from the IMAGE study for the core analysis reported here. For the 5-HT system, markers for pre- and post-synaptic receptors (HTR1A, HTR1B, HTR1E, HTR2A, HTR3B), and synaptic availability controlled by synthesis (TPH2) and re-uptake (SERT/SLC6A4) were chosen. We reasoned that some dopaminergic (DA) biomarkers should be included in view of widespread interactions between 5-HT and DA systems in the brain (reviews in [32, 40]). These included SNPs for the receptors DRD1, DRD4 and DAT1/SLC6A3. There have been reports of associations for each of these genes with ADHD that were subsequently replicated (reviews: [4, 5, 41]). We also decided to include 4 genes for comparison that might be expected to have a less specific outcome on neural activity and behavior than those targeting neurotransmitter synthesis and receptor function (BDNF, NURR-1, FADS2, and PNMT: see discussion). For an additional analysis genotype information on repeat polymorphisms (microsatellites) was available for the DRD4 (exon 3 variable number tandem repeat [VNTR]), the DAT1/SLC6A3 (3' untranslated region VNTR), the SERT/SLC6A4 (a VNTR polymorphism in intron 2: STin2) and an in/del promoter polymorphism (5-HTTLPR), from an analysis based on 1116 ADHD families.

                                          DNA and SNP collection

                                          The DNA sample used here is a part of a larger genetic sample from the IMAGE study. Details of the DNA isolation and SNP selection can be found elsewhere [33]. Briefly, blood samples were sent to Rutgers University Cell and DNA repository, New Jersey, U.S.A., where DNA was extracted. In a limited number of cases where individuals were not able to supply a blood sample, DNA was extracted from a mouth swab sample at the SGDP laboratories in London, U.K. [42]. DNA stocks were collated in London where they were stored, organized and plated out for further analysis. Geneservice Ltd. Cambridge (UK) performed whole genome amplification on all samples with less than 100 μg stock DNA, using the REPLI-g kit (Quiagen Ltd., Crawley, UK). DNA samples were arrayed into 96-well plates at a concentration of 50 ng/μl and delivered to Illumina Inc. (San Diego, US) under dry ice. A total of 582 SNPs were genotyped throughout 14 genes, as part of a larger set of SNPs/genes described elsewhere [33]. These SNPs were selected as tag-SNPs to explain the linkage disequilibrium structure throughout the gene. Additional polymorphisms genotyped were the repeat polymorphisms in DRD4, SLC6A3/DAT1 and SLC6A4/SERT. Genotyping was by simple sequence analysis on agarose gel, as described previously [4345]. Each polymorphism was evaluated to ensure Hardy-Weinberg equilibrium, initially at α = 0.0001, then at α = 0.05. Data cleaning procedures were performed previously on the entire IMAGE set of SNP data and have been described [33].

                                          Analytical strategy: family based association test – principle components (FBAT-PC)

                                          To determine that the 47 symptoms selected for association analysis would indeed group into major factors of cognitive and aggressive behavioral impulsivity we ran a preliminary factor analysis (Promax) with an oblique rotation in the SAS statistical package. This rotation is in line with clinical experience that subjects present concurrently with symptoms from different dimensions [46]. Four factors based on an eigenvalue cutoff of = 1.0 confirmed these groupings: namely, A) impulsive aggressive behavior based on 1) parent ratings, 2) teacher ratings, and B) cognitive impulsivity rated by 3) parents, and 4) teachers (tables 2 and 3). The names of these groupings reflect the items that emphasize temper outbursts and anger (2A), and distraction (despite understanding the task) that reflects responding to inappropriate stimuli (commission rather than omission: 2B). A look at tables 2 and 3 shows that few variables loading on one factor had strong weightings on the other. Three examples for factors 1 and 2 are "excitable impulsive" (CP28), "disturbs other children" (CP66) and "generally obedient" (SP7). Of interest is the item "blurts out answers before the question is finished" that loads modestly on factor 2 (CT46) with a similar value to that on factor 3 (CP80). This item could be construed as an example of impulsive behavior overlying a cognitively impulsive decision. A previous factor analysis of ADHD symptom domains in 10–14 y-old children confirmed that behavioral impulsivity related to aggression and delinquency, while inattentive items related to hyperactivity [47].

                                          We then performed separate association analyses at each SNP for each of the two groupings while applying the FBAT-PC approach. The FBAT-PC approach generates a composite phenotype by using a weighted sum of the individual phenotypes (details in [48, 49], for applications see [50, 51]). The weights for this composite phenotype are selected to maximize the heritability at each SNP.

                                          Screening procedures were employed to limit the number of statistical tests performed using the PBAT computer program. An outline of the screening procedure can be summarized as follows: 1) the conditional mean model is used to calculate the power to detect an association using the composite phenotype; 2) SNP/genetic model combinations are selected based on the power to detect an association and then ranked; 3) for the top ten power rankings in the FBAT-PC statistics are calculated for the selected SNP/genetic model combinations and their associated phenotypes; 4) the results are adjusted for multiple comparisons using the False Discovery Rate (FDR: [52]. For these analyses, we considered additive (0), dominant (1) and recessive (2) genetic models. We required our results to have at least 20 informative families.

                                          Results

                                          Sample description

                                          Table 1 summarizes the characteristics of the IMAGE sample used in this analysis. There were 607 families, each with between 1 and 4 offspring who had complete genotype and phenotype information. Two thirds of the offspring were male and 603 had a diagnosis of ADHD-ct. The predominant ethnic make-up of the sample was northern European with subsets from Spain and Israel. As family-based tests are robust to population stratification all individuals were included in the analysis. Previous analyses of these data have shown that the findings did not change substantially when the Spanish and Israeli groups were removed.

                                          FBAT-PC analysis

                                          We applied the FBAT-PC approach to the items making up 'impulsive aggression' and 'cognitive impulsivity' variables separately. For both analyses the associations were ranked by power and the first ten were selected for further consideration. No SNPs survived gene-wide corrections for an association with both forms of impulsivity.

                                          For impulsive behavioral aggression there were 6 SNPs from 5 genes that attained nominal significance (p ≤ 0.05: table 4). These genes were BDNF, DRD4, HTR1E, TPH2, and PNMT.
                                          Table 4

                                          FBAT-PC results for behavioral (factors 1 + 2) and cognitive impulsivity (factors 3 + 4)

                                          Factors 1 & 2 (behavioral impulsivity)

                                          Gene

                                          SNP

                                          Allele

                                          Frequency

                                          Model

                                          Under/Over Transmitted

                                          Inform. Families

                                          p-value

                                          Power

                                          FDR p-value

                                          Nominal significance

                                          BDNF

                                          rs7103411

                                          C

                                          0.20927

                                          2

                                          O

                                          91

                                          0.021263

                                          9

                                          0.10746

                                          BDNF

                                          rs10767664

                                          T

                                          0.20889

                                          2

                                          O

                                          87

                                          0.021492

                                          10

                                          0.10746

                                          DRD4

                                          rs3758653

                                          C

                                          0.17287

                                          2

                                          O

                                          52

                                          0.008178

                                          4

                                          0.08178

                                          HTR1E

                                          rs1406946

                                          A

                                          0.44805

                                          1

                                          U

                                          338

                                          0.031630

                                          1

                                          0.26692

                                          PNMT

                                          rs2934966

                                          T

                                          0.04997

                                          0

                                          O

                                          102

                                          0.027007

                                          2

                                          0.10349

                                          TPH2

                                          rs6582071

                                          A

                                          0.22517

                                          2

                                          U

                                          95

                                          0.010714

                                          10

                                          0.10714

                                          Factors 3 & 4 (cognitive impulsivity)

                                          Gene wide significance

                                          PNMT

                                          rs200173

                                          A

                                          0.02383

                                          0

                                          O

                                          40

                                          0.003818

                                          1

                                          0.00936

                                          PNMT

                                          rs200173

                                          A

                                          0.02383

                                          1

                                          O

                                          40

                                          0.004680

                                          4

                                          0.00936

                                          Nominal significance

                                          BDNF

                                          rs13306221

                                          A

                                          0.05762

                                          1

                                          O

                                          140

                                          0.042211

                                          6

                                          0.22758

                                          DRD1

                                          rs686

                                          G

                                          0.35975

                                          0

                                          U

                                          427

                                          0.006978

                                          6

                                          0.06978

                                          DRD1

                                          rs686

                                          G

                                          0.35975

                                          1

                                          U

                                          336

                                          0.023762

                                          8

                                          0.11881

                                          HTR1E

                                          rs1406946

                                          A

                                          0.44805

                                          1

                                          O

                                          338

                                          0.034898

                                          1

                                          0.38387

                                          HTR2A

                                          rs6561333

                                          T

                                          0.43307

                                          2

                                          U

                                          288

                                          0.012237

                                          4

                                          0.12237

                                          HTR2A

                                          rs1923886

                                          C

                                          0.41760

                                          2

                                          U

                                          278

                                          0.033097

                                          5

                                          0.16548

                                          HTR3B

                                          rs1672717

                                          C

                                          0.39106

                                          2

                                          U

                                          234

                                          0.040249

                                          4

                                          0.20125

                                          HTR3B

                                          rs3782025

                                          C

                                          0.45063

                                          2

                                          U

                                          266

                                          0.026497

                                          6

                                          0.20125

                                          SLC6A3

                                          rs13189021

                                          T

                                          0.23494

                                          2

                                          U

                                          103

                                          0.018997

                                          9

                                          0.18997

                                          TPH2

                                          rs1352250

                                          A

                                          0.43298

                                          1

                                          O

                                          359

                                          0.011413

                                          2

                                          0.06598

                                          TPH2

                                          rs10879352

                                          T

                                          0.39596

                                          1

                                          O

                                          364

                                          0.015048

                                          4

                                          0.06598

                                          TPH2

                                          rs1487275

                                          G

                                          0.28429

                                          1

                                          O

                                          356

                                          0.019794

                                          9

                                          0.06598

                                          Model: 0 = additive, 1 = dominant, 2 = recessive

                                          Under/Over Transmitted: O = over-transmitted, U = under-transmitted

                                          For cognitive impulsivity one SNP from the PNMT gene attained gene-wide statistical significance following an FDR correction for multiple testing in both additive and dominant models (p ≤ 0.05: table 4). In addition, 12 SNPs from 7 genes attained nominal significance (p ≤ 0.05) for an association with cognitive impulsivity. These genes were BDNF, DAT1/SLC6A3, DRD1, HTR1E, HTR2A, HTR3B, and TPH2.

                                          Genotype information on repeat polymorphisms (microsatellites) was available for the DRD4 (exon 3, VNTR), the DAT1/SLC6A3 (3' untranslated region VNTR) and the SERT/SLC6A4 (a VNTR in intron 2: STin2) and an in/del promoter polymorphism (5-HTTLPR) from an analysis based on 1116 ADHD families. There were nominally significant associations on an additive model for the 3-repeat DRD4 allele (p = 0.014) with impulsive aggressive behavior, and the SERT VNTR (STin2: 12 vs. 9 and 10 repeat alleles; p = 0.050) with cognitive impulsivity. Other tests for association (e.g. for the promoter region, 5-HTTLPR long and short repeats) proved not to be significant.

                                          Having identified the SNP/model combinations listed in table 4, we examined the associations with ADHD affection status. In addition to being the only gene where there was gene-wide significance, PNMT was also the only gene that was nominally associated with ADHD affection status. SNP rs200173 had associations with ADHD affection status for both the additive and dominant genetic models (unadjusted p-value = 0.0065 and 0.012 respectively) and rs2934966 had an association with the dominant model (unadjusted p-value = 0.0458).

                                          Discussion

                                          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 [5658] unless they show anxiety [5961]. 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.

                                          Limitations of the study

                                          The design of the study involved recruiting children and adolescents who showed to a greater or lesser extent the features of impulsivity we sought. Alternative approaches could contrast children with and without these features, and compare pre-, post-pubertal and young adult subjects to investigate putative developmental changes in gene expression. A comparison of groups with high comorbidity for externalizing or internalizing symptoms would be instructive both for the integrity of our concepts of impulsivity in affect and cognition, as well as for identifying the grounds for separating subgroups of ADHD with 'comorbid features'. We would also point out the severe limitations of interpreting symptoms based on lay responses to questionnaires. It is important that the preliminary work described here be extended to neuropsychological testing that would contrast not only provoked aggression with impulsive decision making, but incorporate the role of reinforcement (e.g. delayed gratification).

                                          There are also several limitations with regard to the sample and genotyping. Although this sample is reasonably sized for moderate genetic effects, the number of individuals could be larger to detect the modest genetic effects that one would expect to observe here. In addition, individuals enrolled in the IMAGE study are from several different places throughout Europe, including Spain and Israel. Although the FBAT statistic is robust to population stratification, the PBAT screening algorithm can be affected by this. Finally, current genome-wide scans are suggesting many genes that one could not have suspected of being of interest a short time ago. Therefore some candidate genes that may now seem important were not included in this analysis.

                                          Conclusion

                                          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.

                                          Abbreviations

                                          ADHD-ct: 

                                          Attention-deficit/hyperactivity disorder-combined type

                                          BDNF: 

                                          brain derived neurotrophic factor

                                          CP/TRS: 

                                          Conners parent/teacher rating scales

                                          DA: 

                                          dopamine (receptors DRD1, DRD4)

                                          DAT1 (SLC6A3): 

                                          dopamine transporter

                                          FADS2: 

                                          Fatty-acid desaturase gene-2

                                          FBAT-PC: 

                                          family-based association test with principle components

                                          IMAGE: 

                                          International Multicenter ADHD Genetics study

                                          NURR1/NR4A2: 

                                          orphan nuclear receptor/transcription factor

                                          PACS: 

                                          Parental account of children's symptoms

                                          PNMT: 

                                          phenylethanolamine-N-methyl transferase

                                          SDQ: 

                                          Strengths and difficulties questionnaire

                                          5-HT: 

                                          Serotonin (receptors 5-HT1a, b, 5-HT2a, c, 5-HT3b)

                                          SERT: 

                                          serotonin transporter

                                          SNP: 

                                          single nucleotide polymorphism

                                          STin2: 

                                          Serotonin transporter [VNTR] in intron 2

                                          TPH: 

                                          tryptophan hydroxylase

                                          VNTR: 

                                          variable number tandem repeat.

                                          Declarations

                                          Acknowledgements

                                          The IMAGE project is a Multisite, international effort supported by National Institutes of Health (NIH) Grant R01MH62873 to Stephen V. Faraone. Additional sources of financial support for data collection reported in this paper included the Medical Research Council (London; Kuntsi); Economic and Social Research Council, Southampton Primary Care Trust, HOPE and the University of Southampton (Southampton; Sounga-Barke and Thompson); Catherijne Foundation and the Radboud University Medical Centre (Nijmegen; Buitelaar). Chief Investigators (CIs) at each site were Marieke Altink, Wai Chen, Hanna Christiansen, Rafaela Marco, Ueli C. Mueller, Fernando Mulas, Aisling Mulligan, Nanda Rommelse, Lamprini Psychogiou and Henrik Uebel. There are no further financial disclosures to report. We sincerely thank all the families who kindly participated in this research.

                                          Authors’ Affiliations

                                          (1)
                                          Clinic for Child and Adolescent Psychiatry and Psychotherapy, University of Duisburg-Essen
                                          (2)
                                          Channing Laboratory, Brigham and Women's Hospital
                                          (3)
                                          Departments of Psychiatry, Neuroscience and Physiology, SUNY Upstate Medical University
                                          (4)
                                          Developmental Brain-Behavior Unit, School of Psychology, University of Southampton
                                          (5)
                                          Department of Child and Adolescent Psychiatry and Psychotherapy, Central Institute of Mental Health
                                          (6)
                                          Developmental Brain-Behavior Unit, School of Psychology, University of Southampton
                                          (7)
                                          Department of Psychiatry, Trinity Centre for Health Sciences, St. James's Hospital
                                          (8)
                                          Department of Psychiatry, Radboud University Nijmegen Medical Center
                                          (9)
                                          S. Herzog Memorial Hospital
                                          (10)
                                          Department of Human Genetics, Radboud University Nijmegen Medical Center
                                          (11)
                                          Department of Developmental & Educational Psychology, University of Valencia
                                          (12)
                                          Department of Experimental Clinical and Health Psychology, Ghent University
                                          (13)
                                          Department of Child and Adolescent Psychiatry, University of Göttingen
                                          (14)
                                          Vrije Universiteit
                                          (15)
                                          Department of Child & Adolescent Psychiatry, University of Zurich
                                          (16)
                                          MRC Social Genetic Developmental & Psychiatry Centre, Institute of Psychiatry
                                          (17)
                                          Child Study Center, New York University

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