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ORIGINAL INVESTIGATION
The dopamine receptor D4 7-repeat allele infl uences neurocognitive
functioning, but this effect is moderated by age and ADHD status:
An exploratory study
MARIEKE E. ALTINK
1,3 , NANDA N.J. ROMMELSE
4 , DORINE I.E. SLAATS-WILLEMSE
3 ,
ALEJANDRO ARIAS V Á SQUEZ
1,2 , BARBARA FRANKE
1,2 , CATHELIJNE J.M. BUSCHGENS
1 ,
ELLEN A. FLIERS
1 , STEPHEN V. FARAONE
5 , JOSEPH A. SERGEANT
4 ,
JAAP OOSTERLAAN
4 & JAN K. BUITELAAR
1
1 Department of Psychiatry, Radboud University Nijmegen Medical Center, Donders Institute for Brain, Cognition and Behavior,
Nijmegen, The Netherlands,
2 Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The
Netherlands,
3 Karakter Child and Adolescent University Centre Nijmegen, Nijmegen, The Netherlands,
4 Department of Clinical
Neuropsychology, VU University Amsterdam, Amsterdam, The Netherlands, and
5 Departments of Psychiatry and Neuroscience &
Physiology, SUNY Upstate Medical University, NY, USA
Abstract
Objectives. Evidence suggests the involvement of the dopamine D4 receptor gene ( DRD4 ) in the pathogenesis of ADHD,
but the exact mechanism is not well understood. Earlier reports on the effects of DRD4 polymorphisms on neurocognitive
and neuroimaging measures are inconsistent. This study investigated the functional consequences of the 7-repeat allele of
DRD4 on neurocognitive endophenotypes of ADHD in the Dutch subsample of the International Multicenter ADHD
Genetics study. Methods. Participants were 350 children (5 – 11.5 years) and adolescents (11.6 – 19 years) with ADHD and
their 195 non-affected siblings. An overall measure of neuropsychological functioning was derived by principal component
analysis from fi ve neurocognitive and fi ve motor tasks. The effects of DRD4 and age were examined using Linear Mixed
Model analyses. Results. The analyses were stratifi ed for affected and non-affected participants after fi nding a signifi cant three-
way interaction between ADHD status, age and the 7-repeat allele. Apart from a main effect of age, a signifi cant interaction
effect of age and DRD4 was found in non-affected but not in affected participants, with non-affected adolescent carriers of the
7-repeat allele showing worse neuropsychological performance. In addition, carrying the 7-repeat allele of DRD4 was related
to a signifi cantly worse performance on verbal working memory in non-affected siblings, independent of age. Conclusions. These
results might indicate that the effect of the DRD4 7-repeat allele on neuropsychological functioning is dependent on age
and ADHD status.
Key words: Dopamine receptor D4 gene , attention defi cit hyperactivity disorder , neurocognitive functioning , non-affected
siblings , development
Introduction
Family, twin, adoption and molecular genetic studies
have clearly pointed out the role of genetic factors in
the etiology of attention defi cit hyperactivity disorder
(ADHD) and led to an estimated heritability around
76% (Faraone et al. 2005). Pharmacological studies
(Staller and Faraone 2007) and biochemical imaging
studies (Forssberg et al. 2006) support the involvement
of the dopamine system in ADHD. An extensively
studied candidate gene for ADHD is the dopamine
D4 receptor gene ( DRD4 ), located on chromosome
11p15.5. A specifi c variant of the DRD4 gene associ-
ated with ADHD is the functional 48 base pair (bp)
variable number of tandem repeats (VNTR) poly-
morphism in exon 3 (Li et al. 2006). The 7-repeat
allele of this VNTR, identifi ed as the risk allele for
ADHD, produces fewer or less effi cient D4 receptors
resulting in less responsiveness to dopamine stimula-
tion (Asghari et al. 1995). Although association with
Correspondence: Jan K. Buitelaar, Department of Cognitive Neuroscience (204), Donders Institute for Brain, Cognition and Behavior,
Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. Tel: ⫹ 31 24 3610750. Fax: ⫹ 31
24 3610989. E-mail: j.buitelaar@psy.umcn.nl
(Rece ived 20 October 2010 ; ac cepted 19 May 2011 )
The World Journal of Biological Psychiatry, 2011; Early Online, 1–13
ISSN 1562-2975 print/ ISSN 1814-1412 online © 2011 Informa Healt hcare
DOI : 10.3109 /156229 75.2011.595822
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2 M.E. Altink et al.
variation in design (e.g., case-control, case-only or
control-only designs), (b) the comparison of groups
with different lengths of repeats of the VNTR (7-
repeat carriers versus non-carriers, 4-repeat homozy-
gotes versus all others, or short (2 – 5 repeats) allele
carriers versus long (6 – 9 repeats) allele carriers), (c)
differences in method of sample ascertainment and
collection, and (d) small sample sizes.
An additional issue that has not been addressed is
the importance of a developmental perspective, when
studying the relationship between the DRD4 gene
and neurocognitive functioning. In both healthy and
ADHD children neurocognitive functioning improves
during development (Seidman 2006). Further, the
expression of many genes is differentially regulated
across different stages of development (Elia and
Devoto 2007), as may their infl uence on neurocogni-
tion. A well-known example is the increasing genetic
infl uence on general cognitive ability from infancy to
adolescence (Plomin et al. 1997). Similar effects
have been found for specifi c neurocognitive abilities
(Polderman et al. 2007). A recent twin study using
structural magnetic resonance imaging reported
genetic effects on cortical thickness to be most
prominent in early childhood for early developing
brain regions, such as the motor and sensory regions.
In contrast, genetic effects were most prominent
during adolescence for later developing regions, such
as the dorsolateral prefrontal and temporal areas
(Lenroot et al. 2009). This may be explained by the
involvement of new, additional genes during devel-
opment, which may infl uence ongoing brain matura-
tion. Previously we found nominally signifi cant
fi ndings of the dopamine transporter gene ( DAT1 )
on neuropsychological functioning predominantly in
adolescents in contrast to children (Rommelse et al.
2008c). With respect to the DRD4 7-repeat allele, a
longitudinal study in a community-based sample
suggested that the effects were more profound at age
11 than at age 4 – 8 (El-Faddagh et al. 2004). This is
in contrast to other longitudinal analyses that showed
that the association between the DRD4 7-repeat
allele and cortical thickness was most apparent early
in development and resolved around age 16 – 18
(Shaw et al. 2007).
The present study sought to extend previous work
by including the effect of age, which might explain
the inconsistent results of DRD4 on neurocognitive
functioning. Therefore, we examined the effect of
the DRD4 7-repeat allele on an overall measure of
neurocognitive functioning in a large sample includ-
ing both children and adolescents with combined
subtype ADHD and their non-affected siblings. The
overall-measure of neurocognitive functioning was
based on a broad range of cognitive and motor mea-
sures that met requirements for neuropsychological
ADHD is supported by a recent meta-analysis (Gizer
et al. 2009) data of individual studies on the asso-
ciation between the DRD4 7-repeat allele and ADHD
are not consistent. For example, the large family-
based association study of the International Multi-
center ADHD Genetics (IMAGE) project reported
only a trend towards association of the 7-repeat allele
( P ⬍ 0.09) and a single nucleotide polymorphism
(SNP) rs9195457 with ADHD (Brookes et al. 2006).
Other fi ndings suggest DRD4 not to be associated with
the full ADHD phenotype, but only with inattentive
(Lasky-Su et al. 2008) or hyperactive-impulsive symp-
toms (Lasky-Su et al. 2007). In addition, among
children with ADHD the presence of the 7-repeat
allele was associated with less severe instead of
more ADHD symptoms (Tahir et al. 2000). A pos-
sible explanation for the heterogeneity of fi ndings
may be that published candidate gene studies have
employed relatively small samples, which makes
interpretation of fi ndings diffi cult. Another explana-
tion is “ the presence of unmeasured sources of het-
erogeneity that modify the association between the
candidate gene and the disorder ” , such as different
genetic backgrounds of populations sampled or the
use of different assessment and diagnostic measures
(Gizer et al. 2009).
Therefore, the exact mechanism underlying the
association of the DRD4 7-repeat allele with ADHD
is not well understood. Studies on the functional
consequences of the exon 3 VNTR in DRD4 on neu-
rocognitive, neuroimaging and neurophysiological
measures show inconsistent results (see Table I). The
presence of the DRD4 7-repeat allele was associated
with defi cits on a range of these measures in some
studies (Auerbach et al. 2001; Fossella et al. 2002;
Langley et al. 2004; Kieling et al. 2006; Mill et al.
2006; Shaw et al. 2007; Froehlich et al. 2007;
Herrmann et al. 2007; Boonstra et al. 2008; Loo et al.
2008) whereas others found no relationship (Langley
et al. 2004; Szobot et al. 2005; Barkley et al. 2006;
Genro et al. 2006; Demiralp et al. 2007; Herrmann
et al. 2007; Sonuga-Barke et al. 2008; Monuteaux
et al. 2008). In contrast to expectations, carriers of
at least one 7-repeat allele performed even better than
non-carriers on a verbal working memory task
(Boonstra et al. 2008), inhibitory control (Kramer et
al. 2009) and IQ test (Gornick et al. 2007). In a
case – control design, ADHD 7-repeat non-carriers
rather than ADHD 7-repeat carriers performed
worse on attention and inhibition tests and measures
of speed and variability (Swanson et al. 2000). Fur-
ther, in a longitudinal study the DRD4
7-repeat allele
was associated with a normalization of the right pari-
etal cortex thickness, a higher IQ, and with a better
clinical outcome among ADHD patients (Shaw et al.
2007). These inconsistent results may be due to (a)
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D R D 4 a n d n e u r o c o g n i t i v e f u n c t i o n i n g i n A D H D 3
Table I. Overview of studies reporting on the 48-bp VNTR of the DRD4 in exon 3 and functional (neurocognitive, neuroimaging and neurophysiological) measures.
Authors N Age (years) Functional Measure (s) Test Results
Neurocognitive measures
Swanson et al. 2000 96 ADHD
48 controls
M ⫽ 11.7
M ⫽ 11.2
EF, orienting, alerting,
Shifting, maintenance of
attention
7R ⫹ vs. 7R – 7R – more cognitive problems (more slowly and
variable), 7R ⫹ not different from controls.
Fossella et al. 2002 200 normals Adults Attention 4R ⫹ vs. 4R – 4R absent (2/2 and 7/7) have lower (effi cient) executive
attention scores.
Manor et al. 2002 180 ADHD M ⫽ 10.68 Impulsivity, response
variability
Long (6-8R) vs.
short (2-5 R)
Short allele is related to poorer performance on
impulsivity and response variability. Dose-response
effect with number of repeats, signifi cant difference
2 vs. 7 repeat.
Langley et al. 2004 133 ADHD M ⫽ 9.2 Sustained attention, impulse
control
7R ⫹ vs. 7R – 7R ⫹ greater impulsiveness, faster and less
Accurate control, inhibition
Bellgrove et al. 2005 54 ADHD
39 controls
M ⫽ 12.3
M ⫽ 11.3
Sustained attention, response
variability
7R ⫹ vs. 7R – 7R – more errors and greater variability.
7R ⫹ did not differ from controls.
Barkley et al. 2006 122 ADHD followed
longitudinally
67 controls followed
longitudinally
12-20 → 19-25
12-20 → 19-25
Attention, EF (vigilance,
problem solving,
impulsiveness)
7R ⫹ vs. 7R – No differences on any measures.
Froehlich et al. 2007 174 normals M ⫽ 5.5 EF measures (spatial working
memory, rule learning and
reversal, spatial span and
planning)
7R ⫹ vs. 7R – 7R ⫹ impaired spatial working memory.
Boonstra et al. 2008 45 ADHD M ⫽ 39.1 EF (fl uency, planning, working,
memory, set shifting,
inhibition) and non-EF
7R ⫹ vs. 7R – 7R ⫹ performed better on verbal short term memory.
7R – better performance on visuo-constructive ability
and set shifting.
Loo et al. 2008 540 ADHD affected
sibling pairs
M ⫽ 12.3,
M ⫽ 9.7
EF measures (inhibition, working
memory, interference control,
set-shifting) & intelligence
7R ⫹ vs. 7R-
240 bp allele vs.
120 bp allele
7R ⫹ lower intelligence functioning, impaired speeded
naming, interference control and working memory.
Homozygous 240-bp allele: slower reaction time.
Kr ä mer et al. 2009 656 students (EEG
paradigm N ⫽ 20)
M ⫽ 21.7 Inhibitory control & EEG
paradigm
7R ⫹ vs. 4R ⫹
(EEG paradigm
7/7R vs. 4/4R)
7R ⫹ more accurate in the Go/Nogo-task than
4R ⫹ (marginal signifi cance). No genetic effects on
ERPs.
Non-executive measure: IQ
Genro et al. 2006 242 normals
100 normals
220 normals
Children
Children
Adults
IQ 7R ⫹ vs. 7R – No differences in IQ.
Sonuga-Barke et al. 702 ADHD
694 Unaffected siblings
5-17 IQ 7R ⫹ vs. 7R – In both groups, carriers versus non-carriers did not
differ in IQ.
Mill et al. 2006 171 ADHD (sample 1)
1758 controls (sample 1)
49 ADHD (sample 2)
45 controls (sample 2)
5
5
7, 9, 11, 13
7, 9, 11, 13
IQ (combined score of 4
assessments in sample 2)
7R ⫹ vs. 7R – In both independent samples, 7R ⫹
lower IQ, but only in ADHD subjects and
not in controls.
(Continued)
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4 M.E. Altink et al.
Table I. (Continued).
Authors N Age (years) Functional Measure(s) Test Results
Neuroimaging measures
Durston et al. 2005 26 ADHD
26 non-affected siblings
20 controls
Children
Children
Children
MRI volume analyses of
prefrontal gray matter and
caudate nucleus
4/4 versus
others
4/4 less volume for prefrontal gray matter volume, most
signifi cantly in the unaffected siblings.
Shaw et al. 2007 105 ADHD
67 ADHD at follow-up
103 controls
47 controls at follow-up
M ⫽ 10.1
M ⫽ 16
M ⫽ 10.2
M ⫽ 14.5
MRI, IQ 7R ⫹ vs. 7R – 7R ⫹ thinner right orbitofrontal and posterior parieto-
occipital cortex. Possession of 7-repeat affects cortical
thickness similarly in ADHD and controls. ADHD
7R ⫹ had better clinical outcome and normalization
of right parietal cortex.
Gornick et al. 2007 166 ADHD
282 controls
M ⫽ 9.0
M ⫽ 15.9
IQ, inhibition, MRI scan 7R ⫹ vs. 7R – Cases with 7R ⫹ higher scores on 3 WISC subscales.
Monuteaux et al. 2008 24 ADHD
19 ADHD & BPD
20 controls
M ⫽ 37.8
M ⫽ 33.6
M ⫽ 36
Brain regions (superior frontal,
middle frontal, anterior
cingulate, and cerebellum
cortices) by MRI
7R ⫹ vs. 7R – Only within ADHD group: 7R ⫹ reduction volume in
the superior frontal cortex and cerebellum cortex.
Neurophysiological measures
Szobot et al. 2005 34 ADHD M ⫽ 11.6 Vigilance using SPECT 7R ⫹ vs. 7R – No differences in regional cerebral blood fl ow during
the task.
Muller et al. 2006 87 normals M ⫽ 22.1 EBR refl ecting fl exibility 4/4 vs. 4/7 No main effect of 7R carriership.
Herrmann et al. 2007 40 normals M ⫽ 22.6 Working memory, fNIRS (brain
activation)
7R ⫹ vs. 7R – No differences for working memory. 7R ⫹ higher/
ineffective brain activity in dorsolateral PFC.
Demiralp et al. 2007 50 normals M ⫽ 21.5 Auditory target detection 7R ⫹ vs. 7R – No differences on 2 of 2 variables. Main effect of
evoked and induced gamma response activity in
subjects with the 7-repeat.
ADHD, attention-defi cit/hyperactivity disorder; R ⫹ , 7-repeat carrier; R – , 7-repeat non-carrier; IQ, Intelligence Quotient; EF, Executive Functioning; EBR, Eye Blink Rate; (f)MRI, (functional)
magnetic resonance imaging; (f)NIRS, near-infrared spectroscopy; EEG, ElectroEncephaloGram; ERP, event related potential; SPECT, single photon emission computed tomography; PFC,
prefrontal cortex; BPD, bipolar disorder.
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D R D 4 a n d n e u r o c o g n i t i v e f u n c t i o n i n g i n A D H D 5
all raw PACS data to yield diagnosis based on oper-
ational 18 DSM-IV criteria for ADHD (Brookes
et al. 2006). These were combined with items that
were scored 2 (pretty much true) or 3 (very much
true) in the teacher-rated Conners ADHD subscales
(L, M, and N) to generate the total number of hyper-
active-impulsive and inattentive symptoms of the
DSM-IV symptom list. Situational pervasiveness
was defi ned as at least one symptom occurring in
two or more different situations, as indicated by the
parents in the PACS interview, as well as the pres-
ence of at least one symptom scoring 2 or 3 from
the ADHD subscales (L, M, and N), as indicated by
the teachers on the Conners questionnaire. For pur-
poses of analysis here, siblings were regarded as non-
affected if they obtained scores in the nonclinical
range on both the parent and teacher questionnaires
(Conners N scale: T score ⬍ 63, SDQ: ⬍ 90th per-
centile) and had no formal or suspicion of ADHD
diagnosis. No PACS interview was administered
concerning non-affected siblings. The “ proband ”
and “ affected sibling ” both have a formal clinical
ADHD diagnosis, passed screening procedures and
fulfi lled the PACS-ADHD diagnosis (probands had
the combined type).
This study reports on a Dutch part of the larger
IMAGE sample of which neuropsychological data
also was obtained. A total of 238 families partici-
pated. This resulted in the participation of 238
ADHD affected probands and 112 affected siblings
(64 with combined subtype, 28 with inattentive sub-
type and 20 with hyperactive-impulsive subtype) and
195 non-affected siblings. Two groups were formed:
one group of affected participants ( N ⫽ 350, mean
age ⫽ 12.0, %boys ⫽ 75.7) and one group of non-
affected participants ( N ⫽ 195, mean age ⫽ 11.5,
%boys ⫽ 45.6).
Neuropsychological measures
To be selected as a cognitive endophenotype, mea-
sures should have satisfi ed several criteria outlined
in the literature, such as being associated with
ADHD, showing familiality, and being observable in
non-affected fi rst-degree relatives of an affected
individual (Gottesman and Gould 2003). Variables
of 10 neuropsychological tasks (fi ve cognitive and
fi ve motor tasks) satisfi ed these criteria and were
selected (Rommelse et al. 2008a). For a description
of these tasks, see Table II. The cognitive tests were:
Time Test (time reproduction; Barkley 1998), Visuo-
Spatial Sequencing (visuo-spatial working memory),
Digit Span (verbal working memory; Wechsler 2002),
Stop Task (motor inhibition of an ongoing response;
Logan 1994) and Shifting Attentional Set (motor
inhibition and cognitive fl exibility). The fi ve motor
endophenotypes of ADHD in earlier reports
(Rommelse et al. 2008a). We tested the hypotheses
that the effect of the DRD4 7-repeat allele on
neurocognitive functioning would differ between
children and adolescents, and between ADHD
affected and non-affected participants, since -accord-
ing to earlier studies by our group (Altink et al. 2008;
Boonstra et al. 2008) the impact of the DRD4
7-repeat allele might differ depending on affected
status.
Materials and methods
Participants
Families with at least one child with the combined
subtype of ADHD (proband) and at least one addi-
tional sibling (regardless of possible ADHD status)
were recruited from clinics in order to participate in
the International Multi-Centre ADHD Genetics
(IMAGE) project. This is an international collabora-
tive study in eight European countries (Belgium,
Germany, Ireland, Israel, Spain, Switzerland, The
Netherlands and the United Kingdom) that aims to
identify genes that increase the risk for ADHD using
linkage and association strategies (Brookes et al.
2006). Ethical approval was obtained from National
Institutes of Health recognized local ethical review
boards, and all families gave written informed con-
sent prior to participation. All participants were aged
5 – 19 and of European Caucasian descent. Exclusion
criteria included IQ ⬍ 70, presence of autism, epi-
lepsy, brain disorders and any genetic or medical
disorder associated with externalizing behaviours
that might mimic ADHD.
Details of the screening procedures and measures
for phenotyping are described elsewhere (Brookes
et al. 2006). In short, both the children already clin-
ically diagnosed with ADHD and their siblings were
similarly screened with the following rating scales:
parent and teacher Conners long version rating
scales (Conners 2003) and the Strength and Diffi -
culties Questionnaire (SDQ; Goodman 2001). Only
clinical cases with an average T score of the DSM-IV
total symptom score (N-scale) ⱖ 63 on the Conners
scales and scores ⬎ 90th percentile on the SDQ-hy-
peractivity scale were recruited into IMAGE. Subse-
quently, all children in the family with clinical scores
on any of these questionnaires, the clinical diagnosis
(of the probands and affected siblings) was verifi ed
with the Parental Account of Childhood Symptoms
(PACS; Taylor et al. 1986). PACS is a semi-struc-
tured, standardized, investigator-based, parent-in-
formed interview developed as an instrument to
provide an objective measure of children’s behaviour.
A standardized algorithm for PACS was applied to
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6 M.E. Altink et al.
Table II. Description of the neuropsychological tasks.
Task Aim of measurement Performance measures
Cognitive tasks
Stop task
a Motor inhibition of an ongoing response Stop signal reaction time (SSRT)
Shifting attentional set
b Motor inhibition and cognitive fl exibility Percentage of errors across blocks
Time test
c Time reproduction Accuracy in seconds (absolute discrepancy)
Visuo-spatial Sequencing
b Visuo-spatial working memory Total number of correct targets in the
correct order
Digit span
d Verbal working memory Maximum span backwards
Motor tasks
Pursuit
b Motor control under continuous adaptation Precision (mean distance cursor and target
in mm)
Tracking
b Motor control without continuous
adaptation required
Precision (mean distance to midline in mm)
Tapping
b Self-generated motor output Variability (SD in ms) in tapping rate
Baseline speed
b Motor output as response to an external
cue
Variability (SD in ms) in reaction times
Motor timing
e Timing of motor output Variability (SD in ms) in reaction times
SD, standard deviation; mm, millimeter; ms, milliseconds.
a Logan 1994.
b Subtests of the Amsterdam Neuropsychological Tasks (ANT; De Sonneville 1999).
c Barkley 1998.
d Subtest WISC-III (Wechsler 2002).
e Van Meel et al. 2005.
tasks were: Tapping (self-generated motor output),
Pursuit (motor control under continuous adapta-
tion), Tracking (motor control without continuous
adaptation required), Baseline Speed (motor output
as response to an external cue; De Sonneville 1999)
and Motor Timing (timing of motor output; van
Meel et al. 2005). To normalize the dependent vari-
ables, a Van der Waerden transformation was applied
(Statistical Package for the Social Sciences [SPSS]
version 16). A principal component analysis was per-
formed on the 10 task measures, to reduce error
variance and to create a stronger familial neuropsy-
chological composite of the individual task measures.
Although fi ve of the tasks were theoretically judged
as belonging to the cognitive domain and the other
fi ve as belonging to the motor domain, all 10 task
measures were found to load on one major compo-
nent, explaining 46.5% of the variance in the task
measures (see Table III), which is in concordance
with an earlier report (Rommelse et al. 2008a).
DNA collection
Directly after collection, blood samples were sent to
Rutgers University Cell and DNA Repository, NJ,
USA, where DNA was extracted from part of the
blood or from immortalized cell lines. DNA stocks
were collated at the Social, Genetic and Develop-
mental Psychiatry Center (SGDP) laboratories in
London where they were stored, organized and
plated out for further analysis. Geneservice Ltd.,
Table III. Component matrix with factor loadings (principal
component analysis).
Neurocognitive variables Factor 1
Time test 0.734
Visuo-spatial sequencing 0.789
Digit span 0.651
Stop task 0.652
Shifting attentional set 0.650
Tapping 0.543
Pursuit 0.792
Tracking 0.591
Motor timing 0.742
Baseline speed 0.634
Cambridge (UK) performed whole genome amplifi -
cation on all samples with ⬍ 100 μ g stock DNA, using
the REPLI-g kit (Quiagen Ltd, Crawley, UK).
Statistical analyses
Hardy – Weinberg equilibrium (HWE) proportions
were estimated from parental DRD4 genotype infor-
mation using the Markov – Chain Monte-Carlo
approximation of the exact test implemented in the
GENEPOP package V 3.3. (Raymond and Rousset
1995). Genotyping errors leading to Mendelian incon-
sistencies were detected using PedCheck (O’Connell
and Weeks 1998) and inconsistencies that could not
be resolved were excluded (nine participants). The
DRD4 48 bp VNTR is a multiallelic polymorphism,
for which we identifi ed seven different alleles in our
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D R D 4 a n d n e u r o c o g n i t i v e f u n c t i o n i n g i n A D H D 7
sample. The most common genotype was the one
with two copies of the 4-repeat (39.7%), followed by
the 4/7 genotype (23.7%), 2/4 genotype (12.5%), 3/4
genotype (7%), 7/7 genotype (3.1%) and 2/2 geno-
type (2.2%). Other genotypes were even less fre-
quent. In order to maximize the power of our
analysis, we recoded the genotypes according to
absence or presence of the 7-repeat allele into carri-
ers (one or two 7-repeat alleles) and non-carriers
(zero 7-repeat alleles) of the allele. No genotypic
information was available for 47 participants, due to
refusal of blood extraction.
The effects of DRD4 and age on neuropsycho-
logical functioning were examined using linear mixed
model (LMM) analyses, which accounted for within
family correlation. Age was recoded into two cate-
gories using median age as a threshold, “ children ”
(age ⱕ 11.5 years) and “ adolescents ” (age ⬎ 11.5
years). The reason for the median as a cut-off score
is to ensure that we the age groups are similar in size.
Another reason is that at age 11.5 years children go
to high school. In high school more independence of
the child is asked for (such as homework, planning)
which may refl ect a different developmental phase.
The LMM included the fi xed factors 7-repeat allele
(present versus absent), and age (children versus ado-
lescents) and the interaction between both factors.
The overall neurocognitive composite score was used
as dependent measure. The model was adjusted for
IQ and gender, given the difference in gender ratio in
the affected and non-affected children.
After an omnibus test, analyses were separately
conducted for affected and non-affected participants,
given our hypotheses, and because previous studies
suggest that genetic effects may be different in
these groups (Altink et al. 2008). Correction for
multiple comparisons according to the false discov-
ery rate (FDR) controlling procedure was applied to
the analyses with a P value setting of 0.05.
Results
No deviations from HWE were detected for the DRD4
7-repeat allele. More than one-third of the total sam-
ple ( N ⫽ 168; 34.4%) was carrier of the 7-repeat
allele (ADHD children: 31.4%; non-affected: 39.7%).
The 7-repeat carriers and non-carriers did not sig-
nifi cantly differ in age, gender or IQ ( F (1,498) ⫽ 2.16,
χ ² (1,489 ⫽ 0.01, F (1,498) ⫽ 1.02, all P ⬎ 0.05). The
association of ADHD diagnosis with the 7-repeat allele
was not signifi cant ( χ ² (1,489) ⫽ 3.36, P ⫽ 0.067), as
previously also found in the IMAGE sample (Brookes
et al. 2006). Inspired by fi ndings in a larger IMAGE
sample (Lasky-Su et al. 2007; Altink et al. 2008) we
also examined the association between the 7-repeat
allele and ADHD symptoms , measured as the averaged
teacher and parent rating on the Conners scales.
ADHD affected participants carrying the 7-repeat
allele showed signifi cantly lower scores on the hyper-
activity scale compared to ADHD participants with-
out the 7-repeat allele (60.8 vs. 63.9; F (1,487) ⫽ 7.54,
P ⬍ 0.05). No signifi cant differences were detected
for inattention and the total ADHD scale
( F (1,487) ⫽ 3.32, F (1,488) ⫽ 4.93; P ⬎ 0.05).
Effects of DRD4 7-repeat allele and age on
neuropsychological functioning
The omnibus analysis of the overall neuropsycho-
logical component in the whole sample showed
main effects of age ( F (1,402.61) ⫽ 294.22, P ⬍ 0.001)
and ADHD status ( F (1,365.21) ⫽ 7.62, P ⫽ 0.006).
Older children performed better than younger chil-
dren, and children with ADHD performed worse than
their non-affected siblings. These variables also sig-
nifi cantly interacted ( F (1,396.50) ⫽ 8.31, P ⫽ 0.004).
The 7-repeat allele had no signifi cant main effect
( F (1,327.98) ⫽ 0.286, P ⫽ 0.593). The allele did sig-
nifi cantly interact with age ( F (1,402.98) ⫽ 4.78,
P ⫽ 0.029), but not with ADHD ( F (1,388.22) ⫽ 0.20,
P ⫽ 0.655). Importantly, we found a signifi cant
three-way interaction between age, ADHD and the
7-repeat allele ( F (2,401.35) ⫽ 4.62, P ⫽ 0.032).
These results showed that the effects of the 7-repeat
allele and age were different in ADHD affected chil-
dren and their non-affected siblings. This gave us
enough evidence to further perform stratifi ed analy-
ses in affected and non-affected participants.
As expected, a main effect of age was found in the
separate analysis for both ADHD affected and non-
affected participants, with adolescents performing
better than children on the overall neuropsycholo-
gical component. There was no main effect of the
DRD4 7-repeat allele on the overall neuropsycho-
logical component in the separate analysis for either
affected or non-affected participants. Further, the
7-repeat allele was found to interact signifi cantly
with age in the non-affected (
F (1,140.00) ⫽ 5.95,
P ⫽ 0.016) but not in the affected participants. To
further localize this large modifying effect of age, we
stratifi ed the sample in four groups: affected and
non-affected children (age ⱕ 11.5 years; N ⫽ 125,
N ⫽ 77 respectively), and affected and non-affected
adolescents (age ⬎ 11.5 years; N ⫽ 137, N ⫽ 63 respec-
tively). Post-hoc analyses showed that non-affected
adolescents carrying the 7-repeat allele performed
signifi cantly worse on overall neuropsychological
functioning than non-affected adolescents without
the risk allele ( P ⫽ 0.008; Figure 1), whereas this
effect was not apparent in affected adolescents or
children (affected or non-affected).
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8 M.E. Altink et al.
Figure 1. Z scores and standard error of the mean of the effect of
the DRD4 7-repeat allele on the overall neurocognitive component
stratifi ed according to ADHD status and age. A higher score
refl ects a worse performance. Non-affected adolescents carrying
the 7-repeat allele performed signifi cantly worse on the overall
neurocognitive component compared to non-affected adolescent
non-carriers ( P ⫽ 0.008).
To further identify the single neurocognitive mea-
sures contributing to the interaction effect of DRD4
with age, the LMM analyses were repeated for all
10 neurocognitive measures separately. Again, age
showed signifi cant effects on all neurocognitive mea-
sures in affected and non-affected participants. In
addition, the 7-repeat allele was associated with a
signifi cantly worse performance on Digit Span in the
non-affected siblings ( F (1,157.16) ⫽ 7.24, P ⫽ 0.008,
N ⫽ 172), independent of age. The signifi cant inter-
action between 7-repeat allele carriership and age in
the non-affected group was found on four tasks,
namely Visuo-Spatial sequencing ( F (1,172.00) ⫽ 6.19,
Figure 2. For explanation, see Figure 1. Non-affected adolescents
carrying the 7-repeat allele performed signifi cantly worse on
Visuo-Spatial Sequencing compared to non-affected adolescent
noncarriers ( P ⫽ 0.019). An opposite effect was found in
ADHD affected children, with a signifi cantly better performance
for 7-repeat allele carriers compared to the non-carriers
( P ⫽ 0.041).
Figure 3. For explanation, see Figure 1. Non-affected adolescents
carrying the 7-repeat allele performed signifi cantly worse on
Digit Span compared to non-affected adolescent non-carriers
( P ⬍ 0.001).
Figure 4. For explanation, see Figure 1. Non-affected adolescents
carrying the 7-repeat allele performed signifi cantly worse on
Shifting Attentional Set compared to non-affected adolescent
non-carriers ( P ⫽ 0.023). An opposite effect was found in
ADHD affected children, with a signifi cantly better performance
for 7-repeat allele carriers compared to the non-carriers
( P ⫽ 0.034).
P ⫽ 0.014), Digit Span ( F (1,169.89) ⫽ 6.04, P ⫽
0.015), Stop Task ( F (1,172.00) ⫽ 6.38, P ⫽ 0.012)
and Shifting Attentional Set ( F (1,146.47) ⫽ 4.30,
P ⫽ 0.040; nominally signifi cant). The latter result
did not survive multiple testing. Within the ADHD
affected group no signifi cant main or interaction
effects of 7-repeat allele carriership were found.
Post-hoc analyses showed that non-affected
adolescents carrying the 7-repeat allele performed
signifi cantly worse than those without the risk allele
on Visuo-Spatial Sequencing ( P ⫽ 0.019, N ⫽ 78;
Figure 2), Digit Span ( P ⬍ 0.001, N ⫽ 78; Figure
3) and Shifting Attentional Set ( P ⫽ 0.023, N ⫽ 78;
Figure 4). In contrast, affected participants carrying
the 7-repeat allele appeared to perform better than
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D R D 4 a n d n e u r o c o g n i t i v e f u n c t i o n i n g i n A D H D 9
of the brain during development (Polderman et al.
2007). Genetic effects on later developing regions,
such as the dorsolateral prefrontal and temporal
areas, are most prominent during adolescence. These
are the regions that express the highest levels of
DRD4 (Meador-Woodruff et al. 1996). Differences
in the weight of genetic infl uences over age on neuro-
cognitive performance might therefore explain why
DRD4 effects are restricted to adolescents in our
study. Another related and partly overlapping pos-
sibility are age-related changes of the dopamine sys-
tem (Spear 2000). Whereas the density of dopamine
receptors appears to decline over late childhood and
adolescence, at the same time the dopamine activity
in the prefrontal cortex tends to increase while
improving neurocognitive performance, such as
working memory (Thompson et al. 2004). Carrying
the DRD4 7 repeat allele may interfere with a sub-
optimal functioning of the dopamine system in the
prefrontal cortex. Thus normally developing adoles-
cents may be much more sensitive than children
to this effect of carrying the DRD4 7-repeat allele.
The differential effects of the gene in the adolescents
and children could explain why earlier studies
have reported inconsistent results with regard to
the association between DRD4 and neurocognitive
functioning.
The effect of DRD4 was not comparable in affected
participants and non-affected siblings, with signifi -
cant effects only found in the non-affected siblings.
If anything, the direction of effects was reversed in
the affected individuals, i.e. those with the 7-repeat
allele had less hyperactive-impulsive symptoms and
better neurocognitive functioning. This remains a
puzzle in need of solving. A possible explanation why
DRD4 effects were seen in the non-affected siblings
only may be given by the fact that ADHD is a
multifactorial disorder. Multiple genes and environ-
mental risk factors, each of small effect, are thought
to underlie these complex disorders (Crosbie et al.
2008). When these multiple factors together reach a
certain threshold, pathology occurs, in our case
ADHD. Therefore, in the affected individuals, most
of whom were affected with the most severe, com-
bined subtype of ADHD, the dopamine neurotrans-
mission might be rather impaired. Hence, the effects
of a single gene on neurocognitive functioning
can be distinguished less easily. In the non-affected
siblings, less genetic load and/or gene – gene and
gene – environmental interactions might be present,
and single gene effects might therefore be easier to
observe. Anyhow, the developmentally expected
greater sensitivity to suboptimal functioning of
the dopaminergic system as discussed above is obvi-
ously absent in adolescents with ADHD. This would
lead to the conclusion that DRD4 may serve as a
those without the 7-repeat allele on Shifting Atten-
tional Set ( P ⫽ 0.034, N ⫽ 166; Figure 4) and
Visuo-Spatial Sequencing ( P ⫽ 0.041, N ⫽ 166;
Figure 2), but these effects were not signifi cant upon
correction for multiple testing. For raw means, stan-
dard deviations of each task see Table IV.
To summarize, the results show a clear effect of
age and ADHD: older children and children without
an ADHD diagnosis perform better on the overall
neurocognitive construct. The effect of DRD4 is not
straightforward and appears to be dependent on age
and ADHD diagnosis. In other words, the effect of
DRD4 differs for younger children compared to
older children, and children with the disorder versus
without. The signifi cant effects are most pronounced
in the non-affected adolescents; carrying the 7-repeat
allele results in a worse performance on the overall
neurocognitive construct, Visuo-Spatial Sequencing
task, Digit Span and Shifting attentional Set. Indepen-
dent of age the results show an effect of DRD4 on Digit
Span, but only in the non-affected participants.
Discussion
Studies on the functional consequences of DRD4 on
neurocognitive, neuroimaging and neurophysiologi-
cal measures show inconsistent results. Apart from
variation in sample size, ascertainment and study
design, the heterogeneity of fi ndings might be
explained by ignoring a development perspective.
Therefore this study tested the hypothesis that the
effect of the DRD4 7-repeat allele on neurocognitive
functioning would differ between children and ado-
lescents, and also between ADHD affected and non-
affected participants, since the impact of the DRD4
7-repeat allele might differ depending on affected
status within families. Apart from a main effect of
age on neurocognitive functioning, the effect of the
DRD4 on neurocognitive functioning indeed dif-
fered between children and adolescents. Among ado-
lescents, carriers of the 7-repeat allele performed
worse than non-carriers, but this was not observed
in children. This effect was not due to a greater vari-
ability of neurocognitive performance in children.
Possible explanations for the main effect of age
remain speculative but should be sought in brain
maturation processes taking place during adolescence.
Decrease of grey matter density and increase of white
matter density occurs globally in the brain during
adolescence, with specifi c increase in the frontal, pari-
etal and occipital lobes (Krain and Castellanos 2006).
This is refl ected by improvements in neurocognitive
functioning, as also observed in our study.
As mentioned in the introduction, the heritability
of cortical thickness changes across different regions
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10 M.E. Altink et al.
Table IV. Non-transformed multiple testing means, standard deviations and number of each task and the overall neuropsychological
component for the affected, non-affected children stratifi ed according to age and 7-repeat carriership.
7-repeat non carrier 7-repeat carrier
⬍ 11.5 ⬎ 11.5 ⬍ 11.5 ⬎ 11.5
MSD N MSD N MSD N MSD N
ADHD participants
Main component ( Z score) 0.67 0.74 82 ⫺0.48 0.74 99 0.66 0.81 43 ⫺0.45 0.72 38
Time reproduction (s) 3.56 1.96 92 2.32 2.16 120 3.42 1.82 51 2.28 1.73 46
Visual spatial sequencing ( N ) 80.99 13.05 92 94.20 8.26 120 82.80 12.80 51 92.07 9.84 46
Digit span backwards ( N ) 3.41 0.87 92 4.33 1.21 120 3.41 0.92 51 4.28 1.15 46
Stop task (SSRT) 328.56 77.80 82 255.42 62.27 105 351.73 105.65 43 259.69 54.53 40
Shifting attentional set (%) 16.78 9.99 92 10.26 8.26 120 13.86 8.80 51 9.71 8.03 46
Tapping (ms) 78.18 34.39 92 56.01 19.73 120 74.11 26.42 51 58.11 27.79 46
Pursuit (mm) 7.84 5.34 92 4.24 1.36 120 8.16 5.41 51 4.25 1.61 46
Tracking (mm) 3.58 2.40 92 2.39 1.73 120 3.45 3.13 51 2.14 0.94 46
Time estimation (ms) 541.78 428.00 92 304.19 216.86 120 612.67 518.18 51 329.46 193.22 46
Baseline speed (ms) 177.57 107.35 92 123.81 100.81 120 188.25 119.08 51 101.56 79.02 46
Unaffected siblings
Main component ( Z score) 0.74 0.93 43 ⫺1.16 0.58 40 0.49 0.77 34 ⫺0.80 0.68 23
Time reproduction (s) 3.33 2.13 54 1.52 0.62 50 2.86 1.33 40 1.78 1.33 28
Visual spatial sequencing ( N ) 80.19 17.02 54 98.74 5.28 50 86.55 11.04 40 96.12 5.69 28
Digit span backwards ( N ) 3.28 1.04 54 4.90 0.93 50 3.23 0.77 40 4.11 0.88 28
Stop task (SSRT) 348.14 116.50 43 223.31 35.52 45 313.73 102.90 34 241.53 41.41 27
Shifting attentional set (%) 16.99 10.03 54 6.62 5.08 50 14.82 10.43 40 8.78 7.06 28
Tapping (ms) 90.51 63.36 54 48.93 16.91 50 86.36 88.12 40 49.62 22.24 28
Pursuit (mm) 8.26 4.13 54 3.69 1.10 50 7.18 4.38 40 3.83 1.19 28
Tracking (mm) 618.14 563.12 54 1.64 1.53 50 2.99 1.58 40 1.85 1.11 28
Time estimation (ms) 3.27 2.26 54 258.10 304.28 50 593.24 522.51 40 231.65 73.76 28
Baseline speed (ms) 183.50 114.45 54 99.09 81.83 50 184.50 130.60 40 124.30 103.24 28
M, mean; SD, standard deviation; s, second; N , number; SSRT, stop signal reaction time; ms, millisecond; mm, millimeter.
modifying gene, acting against a background of
other genetic and environmental etiological factors,
rather than as a gene merely increasing the risk for
ADHD.
Post-hoc analyses suggested that the effect of
DRD4 on overall neurocognitive functioning in
non-affected adolescents was related to effects on
neurocognitive rather than to motor functioning
tests. For four of fi ve neurocognitive tasks the same
effect of DRD4 was observed that non-affected ado-
lescents carrying the 7-repeat allele performed worse
than non-affected adolescents not carrying the allele.
This interpretation would be in keeping with evi-
dence showing that DRD4 is mainly expressed in
the frontal lobe which subserves neurocognitive
functions, and not in subcortical structures like the
striatum which are involved in motor functions
(Durston et al. 2005). However, other explanations
for this fi nding cannot be ruled out, such as measure-
ment error or neurocognitive load differing between
the single tasks.
This is the fi rst study to report a main effect of the
DRD4 7-repeat allele on verbal working memory in
children and adolescents. Carrying the 7-repeat was
associated with a worse performance of verbal work-
ing memory in non-affected siblings. Working memory
is one of the core defi cits of ADHD (Klingberg et al.
2005), and genetic factors were found to explain
36% of the variance of working memory perfor-
mance according to a recent twin study (Kuntsi
et al. 2006). Importantly, in one of our previous
studies non-affected siblings displayed verbal work-
ing memory defi cits, whereas they did not manifest
clinical ADHD symptoms (Rommelse et al. 2008b).
Other evidence also suggests that DRD4 plays a role
in working memory in healthy ADHD non-affected
individuals. Working memory is regulated by the pre-
frontal cortex and DRD4 is highly expressed in this
brain region (Ariano et al. 1997). Indeed, the exon
3 VNTR polymorphism of DRD4 was associated
with the volume of the prefrontal cortex (Durston
et al. 2005). Dopamine plays a crucial role in work-
ing memory performance (Diamond 2007). Several
animal studies have also indicated the importance of
the D4 receptor in working memory using specifi c
agonists and antagonists for the receptor (Arnsten
et al. 2000; Zhang et al. 2004; Browman et al. 2005).
Working memory is often regarded as a foundation
for other executive functions (Klingberg et al. 2005).
That we did not observe main effects of the 7-repeat
allele for the other cognitive tasks associated with the
prefrontal cortex (e.g., inhibition, cognitive fl exibil-
ity, visuo-spatial working memory) may most likely
be due to these other cognitive processes being less
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D R D 4 a n d n e u r o c o g n i t i v e f u n c t i o n i n g i n A D H D 11
using LMM analyses. Another limitation is the
cross-sectional design of our study and the lack of
longitudinal measures of neurocognitive function-
ing. Other potential limitations are the assessment
of only one polymorphism at the DRD4 gene and
the use of a statistically derived global measure of
neurocognitive functioning.
In conclusion, our results indicate that the effect
of the DRD4 7-repeat allele on neurocognitive func-
tioning may be dependent on age and ADHD status
and may relate to neurocognitive rather than to
motor functioning. Increased activity of the dop-
amine system in adolescence may make adolescents
more vulnerable to carrying the DRD4 7-repeat
allele which interferes with optimal functioning of
dopamine transmission in the prefrontal cortex. This
study thus may give new insights into how a devel-
opmental perspective is inextricably bound up with
genetics. As such, our study may be viewed as explor-
atory, with the results presented to be considered a
valuable dataset for replication.
Acknowledgements
The authors thank all of the parents, teachers, and
children who participated. We thank Keeley Brookes
and Xiaohui Xu for genotyping. This study was
partly funded by a grant assigned to Stephen Fara-
one by the National Institute of Mental Health (NIH
grant # R01 MH62873-01A1).
Statement of interest
None to declare.
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