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Association of the DAT1 polymorphism with attention deficit hyperactivity disorder (ADHD) A family-based approach.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:309 –311 (2006)
Association of the DAT1 Polymorphism
With Attention Deficit Hyperactivity
Disorder (ADHD): A Family-Based Approach
Myung-Ho Lim,1* Hyun Woo Kim,1 Ki-Chung Paik,1 Soo Churl Cho,2 Do Young Yoon,3 and Heon-Jeong Lee4
1
Department of Psychiatry, College of Medicine, Dankook University, Cheonan, Korea
Department of Child and Adolescent Psychiatry, Seoul National University Hospital, Seoul, Korea
3
Cell Biology Laboratory, Korean Research Institute of Bioscience and Biotechnology, Daejeon, Korea
4
Department of Psychiatry, Korea University College of Medicine, Seoul, Korea
2
The dopamine transporter gene (DAT1) is
of particular interest in the genetic study of
attention deficit hyperactivity disorder (ADHD),
because psychostimulants interact directly with
the dopamine transporter protein. Association
between ADHD and the 10-repeat allele of a 40 base
pair (bp) variable number of tandem repeats
(VNTR) polymorphism of DAT1 was first reported in 1995 [Cook et al. (1995); Am J Hum Genet
56:993–998]. Subsequently, several investigators
have also confirmed this association, although
others reported conflicting results. We analyzed
the DAT1 polymorphism in a sample of 33 Korean
probands with a Diagnostic and Statistical
Manual of Mental Disorders version IV (DSM-IV)
diagnosis of ADHD and found evidence of
increased transmission of the 10-repeat allele
using transmission disequilibrium test (TDT)
(P ¼ 0.001; OR ¼ 7.88, CI ¼ 2.20–28.29). These data
support the role of DAT1 in ADHD susceptibility
among Asian populations.
ß 2006 Wiley-Liss, Inc.
KEY WORDS:
association;
attention
deficit
hyperactivity
disorder(ADHD);
DAT1;
dopamine transporter
gene; genetics; polymorphism;
transmission disequilibrium test
INTRODUCTION
Attention deficit hyperactivity disorder (ADHD) is a common
disorder that has about 3–7% prevalence rate among prepubertal elementary school children. Recent family study
shows that ADHD has an 80–90% hereditability [Eaves et al.,
1997]. Accordingly molecular genetic studies are being actively
carried out. Most of the studies are being done on the enzyme or
receptor related genes in the dopaminergic neurotransmission
pathways. Recently, genetic studies of ADHD have focused on
dopaminergic neurotransmission related genes. Symptoms of
*Correspondence to: Myung-Ho Lim, M.D., Ph.D., Department
of Psychiatry, College of Medicine, Dankook University, Cheonan,
Chungnam Province, Korea. E-mail: paperose@dku.edu
Received 16 November 2004; Accepted 21 April 2005
DOI 10.1002/ajmg.b.30282
ß 2006 Wiley-Liss, Inc.
ADHD show a 70% improvement upon treatment with the
psychostimulants such as methylphenidate [Elia et al., 1999].
These medications act on the dopamine transporter gene
(DAT1), which is believed to prolong dopamine activity within
the synapses.
In a knockout-gene study in mice, Giros et al. [1996] reported
that mice that were homozygous for deactivation of the DAT1
allele were 5–6 times more active and had dopamine remaining active in the synaptic cleft 100 times longer than
heterozygous and wild-type mice.
Vandenbergh et al. [1992] reported a 40 base pair (bp)
variable number of tandem repeats (VNTR) polymorphism in
the 30 -untranslated region (UTR) of the DAT1 gene located at
5q15.3. Association of DAT1 VNTR polymorphism and ADHD
has been reported in several studies [Cook et al., 1995; Gill
et al., 1997; Waldman et al., 1998; Daly et al., 1999; Barr et al.,
2001; Chen et al., 2003], they indicated the allele with 10 copies
of the 40-bp sequence (10-repeat allele) as the risk allele for
ADHD. However, this association was not replicated in some
studies [Palmer et al., 1999; Holmes et al., 2000; Curran et al.,
2001; Roman et al., 2001; Qian et al., 2004].
In Korea, ADHD is a common disorder with a mortality rate
of about 5%, recently there has been a few research on its
imaging study and genetic study. [Cho, 1999].
Recently, using [123I]IPT SPECT, Cheon et al. [2004]
reported that ADHD in seven children with 10/10 genotype
had a significantly higher DAT density in basal ganglia than
four children without 10/10 genotype. [Cheon et al., 2004].
By using extended transmission disequilibrium test (TDT),
we studied 33 Korean trio families to test the allelic association
between ADHD and DAT1 VNTR polymorphism.
METHODS
Subjects
The subjects were recruited in the ADHD outpatient clinic at
the Child and Adolescent Psychiatric Division of the Psychiatric Department of Dankook University Hospital, Cheonan,
Korea, between March and December 2001. We diagnosed
ADHD by the Diagnostic and Statistical Manual of Mental
Disorders version IV (DSM-IV) [American Psychiatric
Association, 1994]. All subjects were Korean. Subjects were
excluded if there was any evidence of conduct disorder, mood
disorder, anxiety disorder, Tourette disorder, pervasive development disorder, mental retardation (IQ < 70), and neurological conditions including epilepsy. All the interviews were done
by a well-trained child psychiatrist (M.H. Lim). The ADHD
patients were administrated the attention deficit diagnosis
system (ADS) [Shin et al., 2000], a Korean version of modified
310
Lim et al.
computerized test of variables of attention (TOVA) [Greenberg
and Waldman, 1993] for the accurate diagnosis. The Korean
version of the child behavior checklist (CBCL) [Oh and Lee,
1990; Achenbach, 1991] and the Korean version of Conners
rating scales [Conners and Barkley, 1985; Oh et al., 1997] were
used in the present study to obtain parental reports of child
behavior. Subjects were included from our sample if the
subjects performed over two standard deviation of norm in
the tests of ADS. Subjects with high-anxiety scores (Spielberger trait/state anxiety scale score >47/49) in Korean version of
Spielberger trait/state anxiety scale for children [Spielberger,
1973; Cho and Choi, 1989] were also excluded.
Finally, 33 families consisting of father, mother, and affected
child (trios) were included in our study. The parents of subjects
provided written informed consent. The study was approved by
the Institutional Review Board of the Dankook University
Hospital.
Genotypings
Standard high salt extraction methods were used to isolate
DNA from whole blood lymphocytes. The primer sequence used
to amplify the 40-bp sequence of the VNTR polymorphic loci was
as follows [Cook et al., 1995; Holmes et al., 2000]: PCR
amplification of the DAT1 40-bp VNTR in the 30 -UTR was
carried out using the following pair of primers, upstream:
50 -TGT GGT GTA GGG AAC GGC CTG AG-30 and downstream:
50 -CTT CCT GGA GGT CAC GGC TCA AGG-30 . Thirty-five
cycles were conducted consisting of denaturation at 948C for
60 sec, annealing at 608C for 60 sec, and extension at 728C for
60 sec. An initial denaturing step at 958C for 10 min and a last
extension step at 728C for 9 min were also added. The reactions
were 50 ml that consisted of 0.1 mg of genomic DNA, 20 pmol of
each primer, 10 mM dNTPs, 2.5 U Taq polymerase, and 10 Taq
buffer, and distilled water. Ten-microliters of PCR products
were run on 1.5% agarose gel. A 50-bp DNA ladder was used
to identify the various repeat alleles by size: 7-repeat (360 bp),
9-repeat (440 bp), 10-repeat (480 bp), and 11-repeat (520 bp).
Statistical Analyses
Allele and genotype frequencies were compared using the
Chi-square test. For the family-based analyses, we used the
TDT [Sham and Curtis, 1995] instead of haplotype-based
haplotype relative risk (HHRR) method to have any potential
population stratification [Terwilliger and Ott, 1992].
RESULTS
Of the total 33 cases, 24 (72.7%) were boys and 9 (27.3%)
girls. Their ages ranged from 7 to 13 years with a mean age of
9.3 2.4 (SD) years. CBCL mean score of ADHD patients was
68.4 9.3 (SD), while Connors scores was 47.8 18.2 (SD),
Spielberger trait/state anxiety scale scores was 31.8 8.19/
33.79 7.45.
Allele frequencies in the ADHD sample were 1.5% 360-bp
alleles (7 repeats), 1.5% 440-bp alleles (9 repeats), 1.5% 520-bp
alleles (11 repeats), and 95.5% 480-bp alleles (10 repeats).
Allele frequencies of the parents’ sample were 6.1% 360-bp
alleles, 6.1% 440-bp alleles, 3.8% 520-bp alleles, and 84.1% 480bp alleles. The transmission of DAT1 VNTR 10-repeat allele in
33 ADHD trios is presented in Table I. The HHRR result
suggested that the 10-repeat allele was preferentially transmitted to ADHD children. (Fisher exact test, P ¼ 0.001;
OR ¼ 7.88, CI ¼ 2.20–28.29).
DISCUSSION
The allele frequency of the DAT1 10-repeat allele are 95.5%
in our ADHD sample and 84.1% in their parents, and this
TABLE I. Transmission Tests for Individual Alleles of DAT1
VNTR Polymorphism Transmission in 33 ADHD Triads Using the
ETDT Program
T
NT
X2
P value
360
440
480
520
1
7
4.50
0.036
1
7
4.50
0.036
18
3
10.71
0.0011
1
4
1.80
NS
T, Transmitted Number; NT, Nontransmitted Number.
result is quite different from the frequency around 70% in the
Caucasian population [Doucette-Stamm et al., 1995]. However, ours is similar to those observed in Asian populations
[Chen et al., 2003; Qian et al., 2004].
Genetic variation of DAT1 is of particular interest in the
study of ADHD, since stimulant drugs for ADHD inhibit
directly dopamine transporter function. Association between
ADHD and the 10-repeat allele of a 40-bp VNTR polymorphism that lies within the 30 -UTR of DAT1 was first reported in
1995 [Cook et al., 1995], To date, in Caucasian population, at
least five studies supported the positive association between
DAT1 10-repeat allele and ADHD [Gill et al., 1997; Waldman
et al., 1998; Daly et al., 1999; Barr et al., 2001; Curran et al.,
2001]. However, at least five did not support an association
[Swanson et al., 1998; Palmer et al., 1999; Holmes et al., 2000;
Curran et al., 2001; Roman et al., 2001]. In Asian population,
Chen et al. [2003] have analyzed the same association in
a sample of 110 Taiwanese probands with ADHD and found
evidence of increased transmission of the 10-repeat allele.
However, Qian et al. [2004] did not replicate prior findings of
an association between the DAT1 10-repeat allele and ADHD.
Instead, they found that long repeat alleles (11–12 repeats)
were associated with the disorder. However, our data did
not show this finding. Therefore, our result confirmed
the previous reports of the association between the DAT1
10-repeat allele and ADHD.
Here we wish to acknowledge some study limitations. First,
although we excluded comorbid major psychiatric illness, it is
difficult to convince that controlling of comorbidity was
complete. Second, we could not consider the ADHD subtype,
gender difference, and stimulant treatment response.
Recently, Kirley et al. [2003] reported an association between
the DAT1 10-repeat VNTR and favorable response to methylphenidate in ADHD children. Considering those variables
might give more informative data. Third, we could not consider
association of ADHD gene using haplotypes other than single
VNTR. Barr et al. [2001] analyzed using a TDT on the dopamine transporter gene whose haplotype is made of the three
parts VNTR and exon 9, intron. The results showed that in
ADHD patients, a haplotype made of a specific allele existing in
the 10 VNTR and exon 9, intron 9 was highly transmitted.
Similar to our study, it is believed that only following the VNTR
in studies of ADHD will have varying results depending on the
haplotype of the control group. However, larger sample size is
necessary for controlling the confounding factors. Lastly, the
relatively small sample size limits the generalizability of our
findings. Our study showed a high-odds ratio and wide
confidence interval compared to previous studies [Daly et al.,
1999; Holmes et al., 2000]. This is most likely due to the small
effect size as a result of the small number of samples.
Taking these problems into consideration, further study
with a larger sample size whilst controlling for subtype, gender
difference, and drug response is needed. It is also necessary to
evaluate the possible involvement of as-yet-uncovered gene(s)
that influence ADHD other than DAT1, and also the possibility
of gene–gene and gene–environment interactions.
Association Study of DAT1 Polymorphism With ADHD
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