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Association studies of 3081(AT) polymorphism of norepinephrine transporter gene with attention deficithyperactivity disorder in Korean population.

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BRIEF RESEARCH COMMUNICATION
Neuropsychiatric Genetics
Association Studies of 3081(A/T) Polymorphism of
Norepinephrine Transporter Gene With Attention
Deficit/Hyperactivity Disorder in Korean Population
Yoosook Joung,1* Chun-Hyung Kim,2 Jisook Moon,2,3 Won-Seok Jang,1 Jaewon Yang,4 Dongwon Shin,5
Soonyoung Lee,6 and Kwang-Soo Kim2
1
Department of Psychiatry, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
2
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts
3
Department of Biomedical Science, CHA University, Seoul, Korea
Department of Neuropsychiatry, Korea University College of Medicine, Guro Hospital, Seoul, Korea
4
5
Department of Psychiatry, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea
6
Department of Preventive Medicine & Public Health, Ajou University School of Medicine, Suwon, Korea
Received 4 March 2009; Accepted 19 June 2009
Recent studies showing the improvement of ADHD symptoms
obtained with the highly selective noradrenergic reuptake
inhibitor, atomoxetine, demonstrate that the noradrenergic
system plays the role of pathophysiology in this disorder. It is
revealed that the norepinephrine transporter gene (SLC6A2) is a
possible candidate gene directly related to ADHD. To determine
possible roles of the SLC6A2 as a susceptibility gene for ADHD,
we performed the genetic association study for a functional
3081(A/T) polymorphism, located in the promoter region of
SLC6A2. For the present study of association between ADHD and
the SLC6A2, 103 male patients with ADHD and 103 normal male
controls were randomly gathered. Significant differences were
found in the allele frequencies (c2 ¼ 5.60, P ¼ 0.02) and the odds
ratio for the allele T between the ADHD and normal subjects was
1.59 (95% CI: 1.08–2.34) suggesting that T allele is critical to
make the group difference. Significant group difference was also
found in AA, AT, TT genotypes (c2 ¼ 7.1, P ¼ 0.02). The odds
ratio for TT and AT genotypes was 4.57 (95% CI: 2.56–8.15) and
1.96 (95% CI: 0.96–3.78), respectively. Findings in the present
study provided further evidence of association between ADHD
and 3081(A/T) polymorphism of SLC6A2. 2009 Wiley-Liss, Inc.
Key words: attention deficit/hyperactivity disorder; association study; norepinephrine transporter gene; promoter; singlenucleotide polymorphism
Attention-deficit/hyperactivity disorder (ADHD) is a highly
heritable disorder including symptoms of inattention, hyperactivity, and impulsivity. Several lines of pharmacologic studies
have suggested that its pathophysiology is involved in the alteration
of the dopaminergic and noradrenergic pathways [Mikkelsen et al.,
1981; Zametkin et al., 1985a,b,c; Scahill et al., 2001; Hechtman,
2005; Pliszka, 2005; Wilens, 2006]. Recent studies using the highly
selective noradrenergic reuptake inhibitor, atomoxetine, demonstrate the role played by the noradrenergic system in this disorder
2009 Wiley-Liss, Inc.
How to Cite this Article:
Joung Y, Kim C-H, Moon J, Jang W-S, Yang J,
Shin D, Lee S, Kim K-S. 2010. Association
Studies of 3081(A/T) Polymorphism of
Norepinephrine Transporter Gene With
Attention Deficit/Hyperactivity Disorder in
Korean Population.
Am J Med Genet Part B 153B:691–694.
[Michelson et al., 2001, 2002, 2003; Kratochvil et al., 2002;
Thomason and Michelson, 2004]. Moreover, the latest studies
reported the association with genetic markers in SLC6A2 and
ADHD [Bobb et al., 2005; Kim et al., 2008; Xu et al., 2008]. The
reports provide the evidence that SLC6A2 is involved in the
genetic cause of ADHD. Recently, the structural–functional
relationship of SLC6A2 promoter has been characterized
[Kim et al., 1999, 2001, 2002]. The distal part at 4.0 kb to
3.1 kb contains a noradrenergic-specific enhancer and the
proximal region at 133 bp to 75 bp appears to be critical for
the noradrenergic-specific transcriptional activity of SLC6A2. We
Additional Supporting Information may be found in the online version of
this article.
All authors reported no biomedical financial interests or potential conflicts
of interest.
*Correspondence to:
Dr. Yoosook Joung, M.D., Ph.D., Department of Psychiatry, Samsung
Medical Center, Sungkyunkwan University School of Medicine, #50 IlwonDong, Kangnam-Gu, Seoul, Korea. E-mail: yschoung@skku.edu
Published online 14 August 2009 in Wiley InterScience
(www.interscience.wiley.com)
DOI 10.1002/ajmg.b.31012
691
692
hypothesized that a polymorphism in SLC6A2 promoter region
might be involved in the genetic susceptibility to the development
of ADHD. We performed an association study to contrast allele
and genotype frequencies for the 3081(A/T) polymorphism
(rs28386840) in ADHD cases and normal controls. The frequency
of the 3081(T) allele in ADHD patients was significantly higher
than in controls [Kim et al., 2006]. Given that the sample size was
relatively small, more extensive investigation would be warranted
to test an association between ADHD and 3081(A/T) polymorphism. The objective of this study was to examine an association between ADHD and a novel polymorphism located in the
promoter region of SLC6A2 [Kim et al., 2006] in Asian ethnic group
including lager sample size. The association of SLC6A2 allelic
variants in ADHD diagnostic subtypes was also examined to
investigate whether the evidences of the association between the
allelic variants of the SLC6A2 and ADHD subtype, clinical severity,
or dimensional measures might be found.
A total of 103 unrelated males with ADHD and 103 normal male
controls were included in this study, all of whom were Korean. They
were selected from among the patients treated at the Samsung
Medical Center ADHD Clinic between March 2004 and
February 2007. The patients were children and adolescents, with
range from 6 to 14 years, who met the DSM-IV-TR [American
Psychiatric Association, 2000] criteria for ADHD, were determined
by clinical assessment and semi-structured interview of Korean
version of K-SADS-PL [Kaufman et al., 1997; Kim et al., 2004].
ADHD diagnostic subtypes were determined according to the
DSM-IV diagnostic classification. The symptom severity and the
symptom dimension were evaluated using the ADHD Rating
Scale-IV—Parent version-scored scale (ADHDRS-IV-Parent)
[DuPaul et al., 1998; Kim et al., 2003]. The patients were required
to have at least borderline intelligence (IQ > 70) as assessed by the
full KEDI-WISC-III. The exclusion criteria included any serious
medical illnesses, psychosis, bipolar disorder, a history of a seizure
disorder, learning disorder, and autism spectrum disorder including Asperger’s disorder. The study protocol was approved by the
institutional review board of Samsung Medical Center, and appropriate written informed consent was obtained from the parents of
all subjects (ADHD patients and normal controls). The normal
controls were volunteers randomly recruited from a middle school
in Seoul. All of the control subjects were screened for apparent
medical illness, ADHD, learning disorder, or mental retardation
by means of a teacher’s and parent’s informal report, the ADHDRSIV-teacher scale (teacher’s rating score <18), and a school record.
None of the control had any history of medical or psychiatric illness.
The analysis of genomic DNA samples was carried out under
the conditions used in our previous study [Kim et al., 2006]. Chisquare analysis with a two-tailed P-value was used to compare the
allele and genotype frequencies between the patients and controls.
Fisher’s exact test with the permutation method was used for
multiple testings to compare the frequencies of the three types of
genotypes. Allele frequencies were calculated and analyzed using the
SPSS statistical package version 17.0 (Cornell University mainframe computer). The tests for Hardy–Weinberg equilibrium
(HWE) using R genetic package are conducted in patient and
control groups separately. The genotype and allele odds ratios were
estimated by logistic regression using additive model. The genotype
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
TABLE I. Demographic and Clinical Characteristics of Subjects
With ADHD
Subjects with Normal control
Variable
ADHD (n ¼ 103)
(n ¼ 103)
Age in year, mean (SD)
9.7 (2.5)
13.6 (0.9)
Intelligence
Total IQ
99.2 (15.2)
Verbal IQ
99.7 (15.1)
Performance IQ
98.4 (15.0)
ADHDRS-P, mean (SD)
Total score
28.7 (10.3)
6.2 (7.4)
Inattentive score
16.3 (5.8)
Hyperactive/impulsive score 12.3 (5.8)
Subtype, n (%)
103 (100)
Combined
81 (78.6)
Inattentive
16 (15.5)
NOS
6 (5.8)
ADHD, attention-deficit/hyperactivity disorder; ADHDRS-P, attention-deficit/hyperactivity disorder rating scale-IV—parent version-scored scale; NOS, not otherwise specified.
distribution and allele frequencies in the three subgroups of ADHD
were analyzed by using chi-square test. The ANOVA was used to
compare the symptom severity among the subjects with three
genotypes.
The demographic characteristics of the ADHD patients are listed
in Table I. Testing for deviation from HWE was performed using a
Fisher’s exact test (R Genetics Package) in the full sample (c2 ¼ 0.59,
P ¼ 0.57), as well as separately in cases (c2 ¼ 0.35, P ¼ 0.71) and
controls (c2 ¼ 0.001, P ¼ 1.000) indicating that there is no possibility of a deletion polymorphism or a segmental duplication
caused by a mutant PCR primer site or due to a tendency to
miscall heterozygotes as homozygotes. We first evaluated
allele frequency between groups using a Fisher’s exact test.
Significant differences were found in the allele frequencies
(c2 ¼ 5.60, P ¼ 0.02). Cochran–Armitage test was performed to
apply more conservative/robust analysis and not to rely on a HWE
assumption. The findings confirm the results from Fisher’s exact
test (c2 ¼ 5.60, P ¼ 0.02). The recurrence of the 3,081 T allele is
higher in ADHD patients compared to controls (Table II). To
further investigate that T allele is critical to make group difference,
we performed another analysis using logistic regression model for
the linear trend contrasting the number of T alleles. It is revealed
that the frequency of T alleles in ADHD patients is significantly
higher than that from control group [c2 ¼ 5.60, P ¼ 0.02, log odds
ratio ¼ 1.59, 95% confidence interval (CI) ¼ 1.08–2.34] (Table II).
Differences in the genotypes of ADHD cases and controls were
further examined. Significant differences were found in the AA, AT,
and TT genotypes (c2 ¼ 7.1, P ¼ 0.02). Similar to the allele-wise
results, the TT genotypes were more presented in ADHD cases
whereas the AT genotypes were slightly overrepresented in
ADHD cases compared to control cases (Table II). Logistic analyses
with T-dominant model confirmed the results that TT genotypes
were overrepresented in ADHD [c2 ¼ 26.41, P < 0.0001, log odds
ratio ¼ 4.57, 95% CI ¼ 2.56–8.15] (Table II). We also examined the
significance by using permutation tests given in our sample size.
JOUNG ET AL.
693
TABLE II. Distribution of Alleles and Genotype Frequencies of SLC6A2 Polymorphism in ADHD and Normal Control and Odds Ratio by
Logistic Regression Model for Genotype and Allele Frequency
Genotype distribution,* n (%)
Group
Control (n ¼ 103)
ADHD (n ¼ 103)
OR (95% CI)
AA
32 (31.1)
18 (17.5)
1.0
AT
55 (53.4)
59 (57.3)
1.96 (0.96–3.78)
TT
16 (15.5)
26 (25.2)
4.57 (2.56–8.15)
Allele frequency,* n (%)
A
119 (57.8)
95 (46.1)
1.0
T
87 (42.2)
111 (53.9)
1.59 (1.08–2.34)
SLC6A2, norepinephrine transporter gene; CI, confidence interval; OR, odds ratio; ADHD, attention-deficit/hyperactivity disorder.aStatistically significant (P < 0.05) by using chi-square test.
The permutation-based significance levels were comparable with
standard P-values further confirming our conclusions regarding
the associations between ADHD and the 3,081(A/T) SNP. From
the total ADHD subjects of 103, 81 children were a combined type.
Sixteen children were an inattentive type. Six children were a not
otherwise specified (NOS) type (Table I). There was no significant
association between genotypes and allele variants and subtypes of
ADHD. Among ADHD subgroups with the three genotypes (AA,
AT, and TT), there were no significant differences of total,
hyperactive–impulsive, and inattentive symptom severity scores.
In this study, we examined the novel polymorphism, which was
recently discovered in the promoter region of SLC6A2 [Kim et al.,
2006]. Significant differences in the allele and genotype frequencies
of this variant were found between the patients and controls. In
particular, the odds ratio for ADHD with homozygous TT was
significant. Our data provided further evidence for the existence
of an association between ADHD and the A/T variants in the
50 untranslated region.
Polymorphisms in the promoter region of the SLC6A2 may be
functional or nonfunctional. It is important to note that the A/T
polymorphism is a functional one. We found that the 3081T
alleles significantly decreases promoter function compared with the
A allele [Kim et al., 2006]. The presence of a functional polymorphism in the promoter region means that a genetic variation may
cause the alteration of protein functions or the level of gene
expression. Down-regulated promoter function of SLC6A2 results
in decreased transcriptional activities, which may give rise to
reduced level of norepinephrine transporter. The study that found
a significantly reduced level of norepinephrine transporter in
the locus coeruleus of major depressive subjects compared with
age-matched normal control subjects has suggested that the transcriptional activities of the SLC6A2 may play an important role in the
development of psychiatric illnesses [Klimek et al., 1997], though
the findings have not made mention of ADHD.
Because ADHD affects multiple domains and heterogenetic
populations, it is very important for the sample group to have
highly homogenous characteristics in an association study. All of
the subjects were of Korean descent, which is generally assumed to
constitute a very homogenous ethnic group.
Although the current findings represent a significant step forward
in the quest to identify the specific genetic influences on ADHD,
there are limitations to this study. There was a significant difference
in the mean ages of the ADHD and normal control groups.
Nevertheless, the research findings reported the continuity of
diagnosis or impairment of ADHD between childhood and
adolescence impaired by their symptoms till adolescence
[Gittelman et al., 1985; Lambert et al., 1987; Barkely, 1996; Biederman et al., 1998]. These data suggest that adolescents with ADHD
from childhood may not miss the diagnosis of ADHD when they
evaluate first in later stage. Therefore, the probability of such a
problem that normal control group may include ADHD patients
arising is very low. To our knowledge, as the first report of
association between promoter region of SLC6A2 and ADHD in
Asian population with homogenous ethnic group, the current study
provides further insight into the role of SLC6A2 in ADHD.
ACKNOWLEDGMENTS
The authors are thankful to the patients with ADHD who participated in the study.
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