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Association between the 120-bp duplication of the dopamine D4 receptor gene and attention deficit hyperactivity disorder Genetic and molecular analyses.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:231 –236 (2007)
Association Between the 120-bp Duplication of the
Dopamine D4 Receptor Gene and Attention Deficit
Hyperactivity Disorder: Genetic and Molecular Analyses
Eva Kereszturi,1 Orsolya Kiraly,1 Zsolt Csapo,1 Zsanett Tarnok,2 Julia Gadoros,2
Maria Sasvari-Szekely,1 and Zsofia Nemoda1*
1
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Vadaskert Child and Adolescent Psychiatric Clinic, Budapest, Hungary
2
Abnormalities of the dopamine neurotransmission
have been hypothesized to play an important role
in the pathophysiology of attention deficit hyperactivity disorder (ADHD). Promoter variants of the
dopamine D4 receptor gene (DRD4) have attracted
particular interest due to their possible role in
regulation of gene transcription. Here we describe
the haplotype analysis of the 120 base pair duplication (120-bp dup) and three SNPs (616C/G, 615A/
G, 521C/T) in the 50 region of the DRD4 gene
among children with ADHD. We observed a trend
(x2 ¼ 14.905, df ¼ 9, P ¼ 0.093) in the four-locus haplotype distribution between ADHD probands
(N ¼ 173) and controls (N ¼ 284). The homozygote
genotype of the 1-repeat form of the 120-bp dup
(1–1) had a significantly higher frequency among
ADHD children than in controls (8.1% vs. 3.2%,
x2 ¼ 5.526, df ¼ 1, P ¼ 0.019, Odds Ratio ¼ 2.71). In
addition, a novel, 4-repeat allele was identified
among ADHD patients. This particular allele has
been cloned to the luciferase expression vector and
its transcriptional activity has been compared to
the 1- and 2-repeat allele. The number of repeats of
the 120-bp dup was found to have an effect on
transcriptional activity in both neuroblastoma
and retinoblastoma cell lines in a dose-dependent
manner (1-repeat > 2-repeat > 4-repeat). These results suggest that the 1-repeat form of the 120-bp
dup might be a risk factor of ADHD, especially in
the homozygous form and/or in the context of
certain haplotypes.
ß 2006 Wiley-Liss, Inc.
KEY WORDS:
promoter; haplotype; gene expression; tandem repeat; SNP
Please cite this article as follows: Kereszturi E, Kiraly O,
Csapo Z, Tarnok Z, Gadoros J, Sasvari-Szekely M,
Nemoda Z. 2007. Association Between the 120-bp Duplication of the Dopamine D4 Receptor Gene and Attention
Deficit Hyperactivity Disorder: Genetic and Molecular
Analyses. Am J Med Genet Part B 144B:231–236.
*Correspondence to: Zsofia Nemoda, Institute of Medical
Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, POB 260, H-1444, Hungary.
E-mail: nemzso@puskin.sote.hu
Received 31 May 2006; Accepted 23 August 2006
DOI 10.1002/ajmg.b.30444
ß 2006 Wiley-Liss, Inc.
INTRODUCTION
Attention deficit hyperactivity disorder (ADHD) is one of the
most prevalent childhood-onset psychiatric syndromes affecting 3–5% of school-age children worldwide [Swanson et al.,
1998]. It is characterized by hyperactivity, inattention,
impulsivity, and distractibility. Significant genetic component
with an estimated heritability of 0.7–0.8 has been demonstrated in the etiology of ADHD by family, twin, and adoption
studies [Faraone and Biederman, 1998]. Genetic association
analyses have been carried out to identify multiple genes of
minor effects [Comings et al., 2005]. Based on neurobiological
theories, most of the candidate genes belong to the dopamine
neurotransmitter system. In particular, the highly polymorphic dopamine D4 receptor (DRD4) gene has attracted
increasing interest. The most widely investigated polymorphism of the DRD4 gene is the 48-bp variable number of tandem
repeats (VNTR) in the third exon. A meta-analysis of casecontrol and family-based studies concluded that there is an
association between ADHD and the VNTR [Faraone et al.,
2001].
Polymorphisms located in the 50 untranslated region of the
DRD4 gene have also been studied. A 120-bp duplication
(120-bp dup) located 1.2-kb upstream of the transcription start
site was identified by Seaman et al. [1999]. The 2-repeat allele
was twice as frequent in ADHD children compared to controls
[McCracken et al., 2000]; these results were replicated with a
larger sample size [Kustanovich et al., 2004]. However,
another group found no association between the 120-bp dup
and ADHD in a twin study [Todd et al., 2001]. There are several
single nucleotide polymorphisms (SNPs) located in the
50 regulatory region of the DRD4 gene [Mitsuyasu et al.,
1999], of which 616C/G and 521C/T were extensively
studied in association analyses of ADHD [Barr et al., 2001;
Mill et al., 2003; Lowe et al., 2004; Bellgrove et al., 2005].
Assessing the polymorphisms separately, only the 616C/G
but not the 521C/T SNP was associated with ADHD in an
Irish population [Lowe et al., 2004].
The cloning and characterization of the 50 regulatory region
of the DRD4 gene [Kamakura et al., 1997] made it possible to
test the functional effect of promoter variants. The 521T
allele reduced promoter activity by 40% relative to the C allele
in transiently transfected human retinoblastoma Y79 cells
[Okuyama et al., 1999, 2000]. The 120-bp duplicated sequence
was found to contain several transcription factor binding sites
by in silico analysis [Seaman et al., 1999], suggesting that the
duplication of this region might influence promoter activity. In
a functional study, the 1-repeat form was found to have a
higher transcriptional activity relative to the 2-repeat allele
[D’Souza et al., 2004]. However, few molecular biological
studies have been reported in the literature and the results
have not yet been confirmed in independent laboratories.
Previous studies from our group failed to detect any difference
232
Kereszturi et al.
in transcriptional activity with the 521C/T SNP [Kereszturi
et al., 2006]. In this study, we present supporting evidence
on the functional effect of the 120-bp dup. In addition, we
examined DRD4 promoter haplotypes found to be associated
with ADHD.
MATERIALS AND METHODS
retinoblastoma Y79 cells were maintained in RPMI 1640
medium (Sigma, St. Louis, MO) supplemented with 20% fetal
bovine serum and 1% Na-pyruvate. SK-N-F1 (neuroblastoma)
cells were grown in Dulbecco’s modified Eagle’s medium, High
Glucose (Gibco) supplemented with 10% fetal bovine serum
and 1% nonessential amino acids. All cell lines were grown at
378C with 5% CO2.
Subjects
Transient Transfections
In this study, 173 ADHD patients (mean age: 9.14 2.6;
87.3% male and 12.7% female) from the Vadaskert Child and
Adolescent Psychiatric Clinic were included. Diagnosis of
ADHD was based on DSM-IV criteria [American Psychiatric
Association, 1994], (combined subtype: 72%; inattentive subtype: 13%; hyperactive-impulsive subtype: 15%) and was
confirmed by the Child Behaviour Checklist [Achenbach,
1991], the Conners Rating Scale [Conners et al., 1998], and
the ADHD Rating Scale [DuPaul, 1998]. The diagnostic
procedure was conducted by two independent psychiatrists
and inter-rater reliability (kappas) reached 0.95 for all
diagnoses. Children with IQ < 80 were excluded, as well as
those who had severe medical or neurological conditions or
pervasive psychiatric disorder. IQ was estimated from the
Raven progressive matrices test [Raven, 1965]. Comorbid
conditions were assessed by a semi-structured interview, the
Hungarian child version of the Mini-International Neuropsychiatric Interview [MINI-Kid; Balazs et al., 2004]. According to
the MINI-Kid, the frequencies of comorbidities were the
following: learning disorder: 30.6%; conduct disorder: 28.9%;
anxiety disorder: 13.3%; Tourette syndrome: 12.1%. The study
was approved by the Local Ethical Committee (TUKEB). All
children and their parents provided written informed consent
for their participation. The sex-matched control group was
selected from the previously described healthy Hungarian
population [Szantai et al., 2005]. Both the clinical and the
control samples were ethnically homogenous, Caucasian and
consisted of unrelated individuals.
SK-N-F1 transfections were performed by the calcium
phosphate-DNA coprecipitation method. Y79, IMR32, and
HeLa cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The luciferase reporter constructs were
cotransfected with control pCMV-regulated b-galactosidase
vector to normalize the transcriptional activities. Luciferase
and b-galactosidase activities were detected by the Luciferase
Assay System kit (Promega) and by ONPG (O-nitrophenyl-b-Dgalactopyranoside) cleavage rate, respectively. Three parallels
were used in all transfections and all experiments were
performed in triplicate.
Genotyping and Haplotyping
Non-invasive DNA sampling was applied as described
elsewhere [Boor et al., 2002]. Genotyping of the 616C/
G SNP was performed using methods not affected by the
615A/G SNP. The 521C/T SNP was genotyped with
methods insensitive to the 603 Tdel polymorphism. Direct
molecular haplotyping of the four promoter polymorphisms
was performed as described previously [Szantai et al., 2005].
Plasmid Constructs
The pGL3 luciferase reporter vector (Promega, Madison, WI)
was used to clone the 1,571 to 389 region of the DRD4 50 UTR
(translation start site is referred to as position þ1 according to
AC021663) using primers containing either an XhoI or an
HindIII recognition site: DR4-S1 (50 acc act cga gtg ggc tgg act
cgc cgt ttg gc 30 , corresponding to region 1,571/1,550) and
DR4-AS1 (50 aag gaa gct tcc ctc ggg cgc tca ccc tag tcc 30 , 389/
411). The templates were genomic DNA samples with 1-, 2-,
and 4-repeat alleles of the 120-bp dup. The mutagenesis in
positions 616 and 521 was generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,
CA). Constructs were verified by sequencing.
Cell Culture
The human neuroblastoma cell line IMR32 and HeLa cells
were cultured in minimal essential medium Eagle (Gibco,
Carlsbad, CA) supplemented with 10% fetal bovine serum, 1%
nonessential amino acids and 1% Na-pyruvate. The human
Statistical Analysis
SPSS 10.0 for Windows was used in the association analyses.
Statistical analysis for transcriptional data was performed
with one-way ANOVA followed by the Tukey-Kramer Multiple
Comparison Test (GraphPad InStat).
RESULTS
Genotype and Haplotype Frequencies
We first performed a single-locus analysis of four polymorphisms in the 50 regulatory region of the DRD4 gene. The
120-bp dup, 616C/G SNP, 521C/T SNP, and the newly
identified 615A/G SNP were assessed in 173 children with
ADHD. The allele and genotype frequencies were compared to
a sex-matched control population genotyped earlier [Szantai
et al., 2005]. Despite the close proximity of the investigated
polymorphisms, no linkage disequilibrium was detected
between the 120-bp dup and any of the SNPs [Szantai et al.,
2005]. No significant deviations from Hardy–Weinberg equilibrium were detected for any of the polymorphisms either in the
case or in the sex-matched control population. There was no
significant difference between the ADHD and control groups in
the allele frequency of the investigated SNPs (Table I).
However, a significant difference was detected in the allelic
distribution of the 120-bp dup (w2 ¼ 3.928, df ¼ 1, P ¼ 0.047).
The homozygote genotype of the 1-repeat form of the 120-bp
dup (1–1) was twice as frequent in the ADHD group when
compared to the pooled genotypes with at least one allele of the
2-repeat form (8.1% vs. 3.2%, w2 ¼ 5.526, df ¼ 1, P ¼ 0.019, Odds
Ratio ¼ 2.71). Interestingly, a new, 4-repeat allele of the 120-bp
dup has been detected in one ADHD patient. To confirm this
allele, the PCR amplicon of the 4-repeat allele was sequenced.
In addition, DNA samples were collected from the patient’s
parents and were genotyped for the 120-bp dup (Fig. 1).
Next, we analyzed the four-locus haplotype frequencies in
cases and controls using direct haplotyping methods (Table II).
There was a tendency for an unequal distribution (w2 ¼ 14.905,
df ¼ 9, P ¼ 0.093). An accumulation of the 1-C-A-T haplotype
(1-repeat allele of the 120-bp dup 616C 615A 521T)
was detected among ADHD children (w2 ¼ 9.326, df ¼ 1,
P ¼ 0.002, Odds Ratio ¼ 2.33). These results suggest that the
1-repeat form of the 120-bp dup might be a risk factor of ADHD,
especially in the context of certain haplotypes.
The association analyses were also carried out with
estimated haplotypes using the EH program elaborated by
Ott [2003]. The estimated haplotype frequencies of the control
DRD4 Promoter Polymorphisms and ADHD
233
TABLE I. Genotype Frequencies of the DRD4 Promoter Polymorphisms in ADHD Patients and Controls
Polymorphic sites
616 C/G SNP
120-bp dup
615 A/G SNP
521 C/T SNP
Genotype
1–1
1–2
1–4
2–2
CC
CG
GG
AA
AG
GG
CC
CT
TT
Control (N ¼ 284)
Frequency (%)
ADHD (N ¼ 173)
Frequency (%)
9
3.2
14
8.1
73
25.7
45
26.0
0
0.0
1
0.6
202
71.1
113
65.3
81
28.5
43
24.9
131
46.1
86
49.7
72
25.4
44
25.4
218
76.8
121
69.9
62
21.8
49
28.3
4
1.4
3
1.7
55
19.4
27
15.6
139
48.9
92
53.2
90
31.7
54
31.2
1–1, 1–2, 1–4, and 2–2 refer to the genotypes; 1–1: homozygous for the 1-repeat allele, 1–2: heterozygous for the 1 and 2 repeat alleles, 1–4: heterozygous for
the 1 and 4 repeat alleles, 2–2: homozygous for the 2-repeat allele of the 120-bp dup. The P-values for the allele distribution (df ¼ 1) were the following: 120-bp
dup: 0.0475, 616C/G: 0.583, 615A/G: 0.127, 521C/T: 0.627.
and the ADHD samples did not differ significantly from the
original frequencies detected with direct haplotype methods
(w2 ¼ 9.45, df ¼ 9, P ¼ 0.40 for the control group; w2 ¼ 4.91,
df ¼ 10, P ¼ 0.90 for the ADHD group). On the other hand,
comparison of the estimated haplotype frequencies resulted in
a significant difference of haplotype distribution between the
case and control groups (w2 ¼ 19.55, df ¼ 9, P ¼ 0.021), while the
data originating from the direct haplotype method showed only
a trend (P ¼ 0.093). This observation supports the importance
of direct haplotyping methods.
Functional Analysis of Promoter
Polymorphisms and Haplotypes
We also examined whether these promoter haplotypes are
functionally different at the molecular level. To this end, we
characterized the previously studied 120-bp dup and 521C/T
SNP in two neuronal cell lines: IMR32 neuroblastoma and Y79
retinoblastoma. Since the 616C and 615A alleles are more
frequent, reporter constructs contained haplotypes of the 120bp dup and 521C/T with a background of 616C and 615A
(Fig. 2A). Transient transfection experiments were carried out
using the luciferase reporter assay with the pGL3-Basic vector
serving as a negative control. Figure 2B shows the relative
transcriptional activity normalized to the activity of the
promoter-less vector. In this experimental setup, there was
no significant difference in the transcriptional activities of the
four promoter haplotypes in either cell lines tested, although
there was a trend for haplotypes with the 2-repeat allele of the
120-bp dup to have a lower activity (1-C-A-C > 2-C-A-C).
To further investigate the effect of the 120-bp dup on gene
expression, we constructed reporter plasmids containing
different numbers of repeats with a background of 616C,
615A, and 521C. Since we detected a 4-repeat allele in our
ADHD sample, we also constructed reporter plasmids harboring four repeats of the 120-bp sequence. The DRD4 regulatory
region containing the single copy of the 120-bp sequence
revealed a significantly higher transcriptional activity in Y79
(P < 0.01), SK-N-F1 (P < 0.001), and HeLa (P < 0.05) cell lines
compared with that having 2-repeat allele (Fig. 3). Additionally, the 2-repeat allele showed significantly higher transcriptional activity than the 4-repeat form in SK-N-F1 and HeLa
cells (P < 0.001, Fig. 3). All cell lines have been found to express
DRD4 mRNA endogenously from previous studies [Kereszturi
et al., 2006]. These results show that the 120-bp sequence
represses gene expression and the increasing copy number
results in a further decrease in transcriptional activity
(1-repeat > 2-repeat > 4-repeat).
DISCUSSION
In the present study, we performed an association analysis of
the DRD4 promoter polymorphisms and ADHD. In addition to
the 521C/T SNP, the 616C/G SNP and the 120-bp dup, the
newly described 615A/G SNP [Ronai et al., 2004a] was also
studied in a Hungarian sample. Using a case-control approach,
no association was found between ADHD and any of the SNPs
mentioned above. We did observe, however, an overrepresentation of the 1-repeat form of the 120-bp dup in the clinical
TABLE II. Haplotype Frequencies of the DRD4 Promoter
Polymorphisms in ADHD Patients and Controls
Control
Haplotype
1-C-A-C
1-C-A-T
1-G-A-C
1-G-A-T
1-G-G-C
1-G-G-T
2-C-A-C
2-C-A-T
2-G-A-C
2-G-A-T
2-G-G-C
2-G-G-T
4-G-A-C
Fig. 1. Genotyping for the 4-repeat allele of the 120-bp dup in an ADHD
family. The gel-insert shows the newly identified 1–4 genotype of an ADHD
patient (first lane). The 4-repeat allele was transmitted from his father (2–
4 genotype, second lane), while the 1-repeat allele came from his mother
(1–2 genotype, third lane). Please note that the PCR product of the longer
allele is faint because of the preferential amplification of the shorter allele.
ADHD
2N ¼ 568
Frequency
(%)
2N ¼ 346
Frequency
(%)
22
23
17
24
3
3
86
162
76
88
46
18
0
3.9
4.0
3.0
4.2
0.5
0.5
15.1
28.5
13.4
15.5
8.1
3.2
0.0
14
31
11
8
5
5
40
87
42
57
33
12
1
4.0
9.0
3.2
2.3
1.4
1.4
11.6
25.1
12.1
16.5
9.5
3.5
0.3
The number of chromosomes (2N) is 568 and 346 for the control and the
ADHD sample, respectively. The relative chromosomal localization of the
alleles could be identified unambiguously by using direct haplotyping
methods [Szantai et al., 2005]. The alleles in the haplotypes (in cis phase, i.e.,
on the same chromosome) are presented in the following order: 120-bp dup,
616C/G, 615A/G, and 521C/T.
234
Kereszturi et al.
Fig. 2. Functional analysis of the DRD4 promoter haplotypes. A: Four haplotypes of the 120-bp dup and the 521C/T SNP were constructed in pGL3
luciferase reporter plasmid containing the human DRD4 gene promoter region (1,571 to 389) with the 616C and 615A background. The promoter
polymorphisms are indicated in the following order: 120-bp dup (1- or 2-repeat), 616C/G, 615A/G, and 521C/T SNPs. Transient transfection was carried
out in IMR32 and Y79 cells. B: Luciferase activity was normalized to the b-galactosidase activity. Data are presented as fold increments over the pGL3-Basic
activity and shown as mean SD. Results of a representative experiment are shown as measured in triplicates. Similar data were obtained from three
independent transfection experiments.
group (Table I). One weakness of the present study is that
population admixture was not controlled for (since the
studied populations were ethnically homogenous, of Caucasian
origin).
Although an association between the promoter SNPs and
ADHD was reported recently [Lowe et al., 2004], several
negative findings have also been published [Barr et al., 2001;
Mill et al., 2003]. Likewise, data presented in the literature
regarding the 120-bp dup have been controversial. A few
studies indicated the 2-repeat form as a risk factor for ADHD
[McCracken et al., 2000; Kustanovich et al., 2004], while others
did not [Todd et al., 2001; Barr et al., 2001; Mill et al., 2003;
Brookes et al., 2005]. Our results suggest that the 1-repeat
form of the 120-bp dup might be the risk allele in ADHD. This
assumption is supported by an association between the 1repeat allele and novelty seeking, a personality trait sharing
common characteristics with ADHD [Rogers et al., 2004].
Contradictory findings in the literature necessitate a
broader approach in the analyses of these polymorphisms.
Studies of separate polymorphisms are being replaced by
haplotype analyses, given that the effect of each polymorphism
is small and can be modulated by other variants [Ebstein,
2006]. Among association studies of ADHD, many used familybased approach to investigate DRD4 promoter haplotypes.
Barr et al. [2001] found no association between promoter
haplotypes and ADHD using Transmission Disequilibrium
Test. However, after including the 48-bp exon III VNTR in the
haplotype, one of the combinations (2-repeat of the 120-bp dup,
616C, 521T, 7-repeat allele of the 48-bp VNTR) was found to
be preferentially transmitted. Mill et al. [2003] reported a weak
but significant association with the haplotype of the 2-repeat
allele of the 120-bp dup, 616C and 521C. Lowe et al. [2004]
studied variations of the 120-bp dup and 616 C/G SNP only,
and observed preferential transmission of the 2-C haplotype.
We could not replicate any of these findings; our case-control
analysis showed an association between the 1-C-A-T haplotype
and ADHD. It has to be noted that all previous haplotype
studies used either statistics software or derived genotypes
from family data to construct haplotypes. Statistical programs
can only give probabilities for individual haplotypes. From
family genotype data, not all haplotype combinations can be
constructed, resulting in a loss of data if both parents are
heterozygous for multiple polymorphisms. In this study, allele
combinations were determined by molecular haplotyping
methods previously developed in our laboratory [Ronai et al.,
2004a; Szantai et al., 2005].
The haplotype variations in the DRD4 promoter may
influence transcriptional activity, which in turn may result
in different receptor densities. Thus, promoter polymorphisms
that affect transcriptional activity can contribute to the
molecular background of neuropsychiatric disorder. Characterization studies of the 50 regulatory region of the DRD4 gene
were performed with transiently transfected SK-N-F1 and
IMR32 (neuroblastoma) and Y79 (retinoblastoma) cell lines.
These studies revealed the presence of a putative negative
modulatory region [Kamakura et al., 1997; Kereszturi et al.,
2006], but the exact position of this region is still unclear
[Kereszturi et al., 2006]. Functional studies of the DRD4
promoter polymorphisms previously associated with psychiatric disorders have also been performed. Okuyama et al. [1999,
2000] found that the 521T allele had a 40% lower transcriptional activity compared to the C allele. Our group could not
replicate these results as we found that promoter activities of
the 521 alleles were essentially identical in three neuronal
cell lines [Kereszturi et al., 2006]. The 120-bp dup contains
consensus sequences of several transcription factor binding
sites such as MEP-1, CEB/P, and Sp1 [Seaman et al., 1999].
The 2-repeat allele showed enhanced binding capacity for Sp1
in a mobility shift assay [Ronai et al., 2004b]. A study
addressing the functional effect of the 120-bp dup found that
the 1-repeat allele had a higher promoter activity [D’Souza
et al., 2004]. Thus, several lines of evidence suggest that the
120-bp dup has a role in transcriptional regulation of the DRD4
gene.
In the present study, a functional analysis of the DRD4
promoter haplotypes associated with ADHD was carried out.
Transcriptional activity of the haplotypes was essentially
identical in neuroblastoma as well as in retinoblastoma cell
lines. However, there was a trend for combinations containing
the 2-repeat allele of the 120-bp dup to have reduced promoter
activity (Fig. 2). It has to be noted here that several other
polymorphisms have been identified in the 50 regulatory region
of the DRD4 gene [Mitsuyasu et al., 1999], some of which have
not yet been included in either association or functional
studies. Thus, it cannot be ruled out that further haplotype
variations could eventually show functional differences. To
test the effect of the 120-bp dup, we used reporter plasmids
containing different numbers of these repeats. Since we
DRD4 Promoter Polymorphisms and ADHD
235
ities in all three cell lines tested (Fig. 3). These results support
the findings of D’Souza et al. [2004], supplemented with the
study of the 4-repeat variant. It should be noted that the study
of D’Souza et al. [2004] used a short DRD4 promoter fragment
containing only the 120-bp dup to detect functional differences
between alleles. In our experiments, we used reporter
constructs comprising the major part of the 50 regulatory
region which previously demonstrated promoter activity
[Kereszturi et al., 2006].
In summary, we did not find an association between ADHD
and three SNPs (616C/G, 615A/G, and 521C/T) in the
promoter region of the DRD4 gene with a case-control approach.
However, the 1-repeat form of the 120-bp dup was significantly
higher in the ADHD group compared to controls. We also found
that the 1-C-A-T haplotype was overrepresented in the cases.
The 120-bp dup had a negative effect which was also dosedependent: increasing copy number of the duplication resulted in
decreasing transcriptional efficiency. Regarding the haplotypes,
the transcriptional activities of the tested allele combinations
were essentially identical. These results add further support to
the role of the 120-bp dup in DRD4 gene expression.
ACKNOWLEDGMENTS
This work was supported by Hungarian funds OTKA
T048576, D048430, and F46788. The luminometer used for
the luciferase assay was provided by OTKA M045341. The
authors thank Gabriella Kolmann for her valuable technical
assistance.
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a representative experiment are shown as measured in triplicates. Similar
data were obtained from three independent transfection experiments.
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