Differential Dopamine Receptor D4 Allele Association with ADHD
Dependent of Proband Season of Birth
K.J. Brookes,
1*
B. Neale,
1
X. Xu,
1
A. Thapar,
2
M. Gill,
3
K. Langley,
2
Z. Hawi,
3
J. Mill,
1
E. Taylor,
1
B. Franke,
7
W. Chen,
1
R. Ebstein,
12
J. Buitelaar,
7
T. Banaschewski,
6
E. Sonuga-Barke,
10
J.
Eisenberg,
9
I. Manor,
5
A. Miranda,
8
R.D. Oades,
4
H. Roeyers,
13
A. Rothenberger,
6
J.
Sergeant,
11
H.C. Steinhausen,
14
S.V. Faraone,
15
and P. Asherson
1
2008 American Journal of Medical Genetics Part B, 147B, 94-99.
This is the reformatted manuscript submitted - prior to publication in its final form at
DOI: 10.1002/ajmg.b.30562
1 MRC Social Genetic Developmental and Psychiatry Centre, Institute of Psychiatry, London, UK
2 Department Psychological Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff,
UK
3 Department of Psychiatry, Trinity Centre for Health Sciences, St. James’s Hospital, Dublin,
Ireland
4 University Clinic for Child and Adolescent Psychiatry, Essen, Germany
5 Geha MHC, Petach-Tikva, Israel
6 Child and Adolescent Psychiatry, University of Göttingen, Göttingen, Germany
7 Department of Psychiatry, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands
8 Department of Developmental and Educational Psychology, University of Valencia, Valencia, Spain
9 ADHD Clinic, Geha Mental Health Center, Petak Tikvah, Israel
10 School of Psychology, University of Southampton, Highfield, Southampton, UK
11 Vrije Universiteit, De Boelelaan, Amsterdam, The Netherlands
12 S. Herzog Memorial Hospital, Jerusalem, Israel
13 Ghent University, Dunantlaan 2, Ghent, Belgium
14 Department of Child and Adolescent Psychiatry, University of Zurich, Zurich, Switzerland
15 Child and Adolescent Psychiatry Research, SUNY Upstate Medical University, Syracuse, New York,
USA
*Correspondence to: K.J. Brookes, MRC Social Genetic Developmental Psychiatry, Institute of
Psychiatry, De Crespigny Park, London SE5 8AF, UK. E-mail: k.brookes@iop.kcl.ac.uk
Key Words: attention deficit hyperactivity disorder (ADHD); season of birth;
Acknowledgments: We thank all the families who kindly participated in this research. Research was
funded by the MRC and Wellcome Trust in the UK, the Health Research Board and Molecular
Medicine Centre in Dublin The IMAGE project is supported by NIH grant R01MH62873 to S.V.
Faraone.
2
Abstract:
Season of birth (SOB) has been associated with attention deficit hyperactivity disorder
(ADHD) in two existing studies. One further study reported an interaction between SOB and
genotypes of the dopamine D4 receptor (DRD4) gene. It is important that these findings are
further investigated to confirm or refute the findings. In this study, we investigated the SOB
association with ADHD in four independent samples collected for molecular genetic studies
of ADHD and found a small but significant increase in summer births compared to a large
population control dataset. We also observed a significant association with the 7-repeat
allele of the DRD4 gene variable number tandem repeat polymorphism in exon three with
probands born in the winter season, with no significant differential transmission of this allele
between summer and winter seasons. Preferential transmission of the 2-repeat allele to
ADHD probands occurred in those who were born during the summer season, but did not
surpass significance for association, even though the difference in transmission between the
two seasons was nominally significant. However, following adjustment for multiple testing of
alleles none of the SOB effects remained significant. We conclude that the DRD4 7-repeat
allele is associated with ADHD but there is no association or interaction with SOB for
increased risk for ADHD. Our findings suggest that we can refute a possible effect of SOB for
ADHD.
INTRODUCTION
Attention Deficit Hyperactivity Disorder
(ADHD) is one of the most prevalent and
heritable childhood behavioral disorders.
The disorder is characterized by an onset
of age inappropriate hyperactivity, impuls-
ivity and inattentiveness before the age of
7 years [American Psychiatric Association,
1994]. Familial risk is established with an
estimated sibling risk ratio (λs = risk to
siblings of ADHD probands/population
risk) for broadly defined ADHD of around
threefold to fourfold [Faraone and Doyle,
2000]. Twin studies support the view that
genetic factors are the major influence on
familial risk with heritability estimates for
ADHD symptom scores consistently
reported to be in the region of 60–90%
[Thapar et al., 1999]. In general these
studies find little evidence of shared
environmental influences on familiarity,
although the role of environment may still
be pivotal acting through mechanisms of
gene–environment interaction. Progress in
identifying some of the genes involved in
ADHD susceptibility has been relatively
fruitful over the past decade by screening
genetic variants that lie within or close to
genes that regulate neurotransmitter
systems, particularly dopamine pathways.
One of the first genetic markers
reported to be associated with ADHD was
the 7-repeat allele of a variable number
tandem repeat (VNTR) polymorphism
located within exon 3 of the Dopamine D4
Receptor gene (DRD4) [LaHoste et al.,
1996]. Subsequent studies replicated this
finding although several investigations
have also reported negative findings
[Faraone et al., 2005]. A recent meta-
analysis of available data concluded that
there was a small but significant effect of
the DRD4 polymorphism on risk for ADHD,
with a pooled odds ratio of 1.34 (95% CI
1.23–1.45, p = 2
-12
) [Li et al., 2006].
Although, genetic risk factors are
prominent in the development of ADHD,
environmental risks are also thought to be
important, acting through gene–
environmental interactions. Associated
environmental risks for ADHD include low
birth weight and maternal use of alcohol
and tobacco during pregnancy [Mick et al.,
2002a, b]. More recently specific gene–
environment interactions have been
reported between genotypes of the
3
dopamine transporter gene and maternal
use of tobacco during pregnancy on levels
of hyperactive-impulsive behavior [Kahn
et al., 2003] and maternal use of alcohol
on risk for ADHD [Brookes et al., 2006b].
Other research suggests that gene–
environment interactions may increase
the rates of antisocial behavior among
ADHD probands, rather than having a
main effect on risk for ADHD. For example
the effects of a catechol-O-methyl-
transferase (COMT) gene variant and birth
weight on the risk of early-onset antisocial
behavior in children with ADHD [Thapar et
al., 2005].
Another environmental measure that
has been investigated is the effect of
season of birth (SOB). This association is
not well established, with the two studies
reporting on this variable in relation to
ADHD giving contradictory findings. Mick
et al. [1996] concluded that winter birth
was associated with ADHD in individuals
with learning difficulties, ADHD without
psychiatric comorbidities, and ADHD with
family history of the disorder. In contrast,
an earlier study concluded that spring and
summer births increased risk for
neurodevelopment disorders, including
ADHD [Liederman and Flannery, 1994].
More recently, a report on a potential
interaction between SOB and the DRD4
exon 3 polymorphism was published
[Seeger et al., 2004]. In a sample of 64
children with comorbid hyperkinetic
disorder and conduct disorder (HD + CD)
and a matched control sample of 163
children, no main affects of the DRD4
polymorphism or SOB were observed.
However, it was found that children with
HD + CD born in the winter, had
significantly fewer 7-repeat alleles (12.5%)
compared to those born in the summer
(50%, P¼0.001, OR¼7). This suggested that
the 7-repeat allele might be a risk factor
for ADHD only for those born in the
summer months. The control population
exhibited the opposite relationship
between SOB and the 7-repeat allele, with
those born in winter having a higher allele
frequency of 7-repeat alleles (43.7%) in
comparison to those being born in the
summer (26.1%, p = 0.019, OR 2.2).
Discrepancies between the various
studies on SOB and ADHD mean that no
firm conclusions can be reached at this
time. We therefore set out to establish
whether in a large collaborative set of
clinical ADHD samples there was any
evidence for the association of SOB with
ADHD, and whether SOB interacts with the
DRD4 exon 3 VNTR polymorphism in the
risk for the disorder. Allowing the
confirmation or rejection of the
hypothesis that SOB may be a risk factor
for ADHD.
In the course of this research, we also
considered whether plausible biological
arguments could be made for the
association between ADHD and SOB. For
example SOB might be a proxy for risk
factors such as viral infections or amount
of daylight exposure during gestation or
birth weight [Liederman and Flannery,
1994; Mick et al., 1996]. Those born in the
winter spend most of their gestation
period in the summer months while
conversely those born in the summer have
the majority of their gestational time in
the winter months. Maternal disorders
such as seasonal affective disorder, which
might confer prenatal risk, show seasonal
variation [Chotai et al., 2003; McGrath et
al., 2005; Amons et al., 2006]. In relation
to the DRD4, the 7-repeat allele could
influence mating behavior in mammals
and the associated pattern of mating may
be part of a natural cycle with seasonal
variation observed in the general
population. The dopamine system has
been highly implicated in the development
of ADHD and it has been discussed that
this system is influenced by exogenous
factors, such as hours of sunlight, in
4
creating an endogenous daily rhythm of
dopamine receptor binding, therefore
giving credence that hours of daylight
could impact on the dopamine system
[Naber et al., 1981]. Furthermore, the
hormone melatonin is secreted from the
pineal gland, in a cyclic rhythm. This
rhythm is entrained by the length of
daylight the individual is exposed to, and
alters the timing of mammalian circadian
rhythms [Brzezinski, 1997]. Melatonin is
synthesized from serotonin by the enzyme
N-acetyl-transferase, which is entrained by
the day length cycle, and is more active
during dark periods. Therefore, melatonin
production is highest during the night and
lowest during the day [Reppert and
Weaver, 1995]. Melatonin is known to
inhibit dopamine release in numerous
brain regions including the striatum and
dopamine is thought to inhibit the
production of melatonin via the DRD4
[Zisapel and Laudon, 1983; Zisapel et al.,
1983; Zawilska and Nowak, 1994; Tosini
and Dirden, 2000; Zisapel, 2001]. Finally,
melatonin can also pass from the mother
via the placenta to the fetus, entraining
the fetus’ circadian rhythm [Goldman,
2003].
We can therefore see that is not
difficult to derive biologically plausible
explanations for the possible influence of
SOB on risk for ADHD and interaction with
components of the dopamine system.
However, on the basis of the data
presented here, we conclude that it is far
more likely that there is no effect of SOB
on risk for ADHD.
METHODS
Four independent samples were used,
collected by groups in London, Cardiff,
Dublin, and the International Multi-centre
ADHD Gene (IMAGE) project. The IMAGE
project is a multisite site with samples
collected in Belgium, England, Germany,
Holland, Ireland, Israel, Spain, and
Switzerland. Children taking part in these
studies were all of white European origin
and consisted predominantly of male
children with combined subtype ADHD
and with DNA available from both parents
(Table 1). The individual groups gathered
DRD4 exon 3 VNTR genotypes and date of
birth information separately and data was
sent for this analysis to Keeley Brookes in
London. The association findings with
DRD4 for these groups have previously
been reported [Hawi et al., 2000; Holmes
et al., 2000; Mill et al., 2001] with only the
large IMAGE sample exhibiting a trend for
excess in transmission of the 7-repeat
allele from heterozygote parents to their
affected offspring [Brookes et al., 2006a].
Clinical procedures for making research
diagnoses of ADHD across the different
studies used comparable approaches since
probands were all ascertained from
specialist ADHD clinics and research
interviews were the main source of data
capture. DSM-IV operational criteria were
applied in each case however no direct
comparisons were made to check
reliability of diagnosis between the
different sites and slightly different
protocols applied. Detailed descriptions of
the sample ascertainment and assessment
procedures can be found in the original
articles for the DRD4 VNTR [Hawi et al.,
2000; Holmes et al., 2000; Mill et al., 2001;
Brookes et al., 2006a] and the measures
used in each study are listed in Table 1.
In this study, we investigated each
sample separately before combining the
data into a single set of 1,110 ADHD-
parent trios. Each cohort was stratified
into two subsets dependent on the date of
birth of the proband. Following the
seasonal definitions used by Seeger et al.
[2004] those born between the 22nd
March and 22nd September were
classified as the summer season group,
whereas those born between the 23
rd
September and the 21
st
March were
classified as the winter season group. Each
seasonal subset was analyzed using the
Table 1:
Description of the 4 independently collected ADHD family data sets utilized in this analysis
Sample N Trios % Males Age Range
(Mean/SD)
Clinical
Procedure
Diagnosis Comorbidity
(ODD and
CD) %
London
137 90% 5 – 15 (10.4/2.3) Conners, CAPA,
Hypescheme
DSM IV 53.3%
Cardiff
128 92% 6 – 12 (9.3/1.8)
CAPA DSM IV 88%
Dublin
174 85% 4 – 14 (11.7/3.9) Conners, CBCL,
ACTeRS
DSM IV 80%
IMAGE
671 89% 5 – 15 (11.2/2.7) Conners, SDQ,
PACS
DSM IV 78.7%
Transmission Disequilibrium Test (TDT)
implemented in the UNPHASED program
[Dudbridge, 2003; http://portal.litbio.org/
Registered/Menu/]. Allele-specific tests of
association were calculated from the
number of transmissions and non-
transmission of the 2-, 4-, and 7-repeat
alleles from heterozygote parents to their
affected offspring for the two seasonal
groups.
Significant differences between the
seasonal groups were tested using the Chi-
square test on the number of transmitted
(T) and un-transmitted (NT) transmissions
for each allele. Since there are several
alleles that could show transmission ratio
differences between the two seasons, we
adjusted for the number of tests by
permuting the data to derive an empirical
distribution of p-values in the following
way. The SOB group status for each family
was permuted a total 10,000 times and
the T/NT ratio and significance re-
calculated for each allele. We then took
the most significant Chi-square value from
the analysis of the various alleles, to
derive the empirical distribution of
maximum Chi-square values. This enabled
us to determine how frequently the most
significant Chi-square values occur by
chance in our sample.
For the combined dataset, 95%
confidence intervals were derived using
the t-test statistic (T-statistic [Mitchell et
al., 2003]). The T-statistic represents the
TDT information in terms of the
proportion of transmitted alleles to the
total number of transmissions from
heterozygote parents. Under the null
hypothesis of no association the
proportion of the transmitted alleles to
the total number of transmissions is
expected to be 0.5. The general formula
for confidence intervals is then applied:
z*√((T(1-T)/(M1+M2)).
RESULTS
Season of Birth Effect
UK Census data over the last decade
(www.statistics.gov.uk/statbase/Product.a
sp?vlnk=5768) of 6,919,604 live births
during the period 1994–2004 suggests
that there is no bias in the SOB for babies
born in the UK. Close to 50% of live births
occurs in the summer months (March to
August = 50.72%) and in the winter
months (September to February =
49.28%). Since the ADHD samples are
predominantly male (>95%), we further
investigated whether males were
predominantly born in either the summer
months or the winter months. The UK
census data showed no difference in the
proportions of male birth between
seasons with near 50% of males being
born in the summer and winter seasons.
The IMAGE sample consists of a
combination of ADHD probands
