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Evaluation of potential geneЦgene interactions for attention deficit hyperactivity disorder in the Han Chinese population.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:200 –206 (2007)
Evaluation of Potential Gene–Gene Interactions for
Attention Deficit Hyperactivity Disorder in
the Han Chinese Population
Qiujin Qian,1 Yufeng Wang,1* Jun Li,1 Li Yang,1 Bing Wang,1 Rulun Zhou,1
Stephen J. Glatt,2 and Stephen V. Faraone3
1
Institute of Mental Health, Peking University, China
Institute of Behavioral Genomics, Department of Psychiatry, University of California, San Diego, La Jolla, California
3
Medical Genetics Research Center and Department of Psychiatry, SUNY Upstate Medical University, Syracuse, New York
2
Several lines of evidence suggest that attentiondeficit/hyperactivity disorder (ADHD) is a polygenic disorder produced by the interaction
of several genes with minor effects. To explore
potential gene–gene interactions among candidate genes for ADHD, we studied the dopamine D2
receptor (DRD2), dopamine D4 receptor (DRD4),
dopamine transporter (DAT1), and catecholO-methyltransferase (COMT) genes in the Han
Chinese population. A sample of 340 children with
ADHD was diagnosed according to the DSM-IV
criteria. We also recruited 226 unrelated controls.
Identified polymorphisms included a 48-basepair-repeat in Exon 3 of DRD4, a 40-base-pairrepeat in the 30 untranslated region of DAT1, a
restriction-fragment-length polymorphism at
codon 158 of COMT, and a 241A > G transition
in the promoter of DRD2. Associations of polymorphisms with ADHD and its subtypes were
examined by comparing allele frequencies
between probands and controls. Binary logistic
regression analysis was used to examine the
potential gene–gene interactions. Binary logistic
regression analysis with the sample of refined
phenotypes showed that male gender and longrepeat genotypes of DRD4 and DAT1 were independent risk factors for ADHD. We found no
evidence for gene–gene interactions among
the candidate genes studied. The present study
suggests that dopamine candidate genes are
associated with increased vulnerability to
ADHD in the Han Chinese population.
ß 2006 Wiley-Liss, Inc.
KEY WORDS: attention - deficit / hyperactivity
disorder; gene; dopamine D4
receptor; dopamine transporter;
catechol-O-methyltransferase; dopamine D2 receptor;
interaction
Grant sponsor: National Natural Sciences Foundation of China;
Grant number: 30400150; Grant sponsor: Key Project for Clinical
Faculty Foundation, Ministry of Health, China; Grant number:
2004 (468); Grant sponsor: Ministry of Science and Technology,
China; Grant number: 2004BA720A20.
*Correspondence to: Yufeng Wang, Institute of Mental Health,
Peking University, Beijing 100083, People’s Republic of China.
E-mail: wangyf@bjmu.edu.cn
Received 27 January 2006; Accepted 18 July 2006
DOI 10.1002/ajmg.b.30422
ß 2006 Wiley-Liss, Inc.
Please cite this article as follows: Qian Q, Wang Y,
Li J, Yang L, Wang B, Zhou R, Glatt SJ, Faraone SV. 2007.
Evaluation of Potential Gene–Gene Interactions
for Attention Deficit Hyperactivity Disorder in the
Han Chinese Population. Am J Med Genet Part B
144B:200–206.
INTRODUCTION
Attention-deficit/hyperactivity disorder (ADHD) is a
childhood-onset, clinically heterogeneous disorder characterized by excessive motor activity, impulsiveness, and inattention. ADHD affects about 8–10% of children around the world
[Faraone et al., 2003 #15110]. In addition to the high
prevalence of the disorder, findings from family, twin, and
adoption studies have shown that ADHD is also highly
heritable [Faraone et al., 2005 #17018]. Many molecular
genetic studies have focused on dopamine genes given evidence
for dysregulated dopamine transmission in ADHD [Faraone
and Biederman, 2004 #14949]. In this report, we examine
the joint effects of two genes that have been shown through
meta-analysis to be associated with ADHD (DRD4, DAT1) and
two that have not (DRD2, COMT) [Faraone et al., 2005
#17018].
Dopamine D4 Receptor Gene (DRD4)
DRD4 maps to chromosome 11p15.5 [Gelernter et al., 1992
#3440] and encodes one of the five known dopamine receptors
[Civelli, 1991 #10855]. Mice lacking DRD4 have been found to
be supersensitive to ethanol, cocaine, and methamphetamine.
In these mice, synthesis and clearance of dopamine were
elevated in the dorsal striatum [Rubinstein et al., 1997
#10859].
Most molecular genetic studies of DRD4 and ADHD have
focused on a variable-number tandem-repeat (VNTR) polymorphism consisting of a 48-base-pair (bp) repeat unit coding
for an amino-acid sequence located in the third cytoplasmic
loop of the receptor [Van Tol, 1992 #5826]. The most prevalent
of these alleles contain either four, seven, or two repeats, with
global mean allele frequencies of 64.3, 20.6, and 8.2%,
respectively [Chang et al., 1996 #6315]. The 4- and 7-repeat
alleles demonstrate considerable variability in prevalence
across populations, ranging from 0.16 to 0.96 and 0.01 to 0.78
in frequency, respectively. Researchers have focused particular attention on the 7-repeat allele, given the low frequency of
this allele and the similarly low prevalence of ADHD in Asian
populations where this allele is quite rare [Leung et al., 1996
4928]. The product of the 7-repeat allele mediates a blunted
response to dopamine compared with other forms of the
receptor [Asghari et al., 1995 #6899].
Evaluation of Potential Gene – Gene Interactions
A considerable number of studies, including both casecontrol and family-based association studies, have examined
the association between the 7-repeat allele of this DRD4
polymorphism and ADHD, and a meta-analysis of this
relationship concluded that, as a group, existent studies
showed a small but statistically significant association
[Faraone et al., 2001 #10013].
Dopamine Transporter Gene (DAT1)
Medications that inhibit the dopamine transporter, including methylphenidate, pemoline, and amphetamine, increase
synaptic dopamine levels and alleviate the symptoms of
ADHD. This treatment approach arose from research showing
that the dopamine transporter knock-out mouse displayed
behavioral traits highly reminiscent of ADHD characteristics
observed in humans [Giros et al., 1996 #5629].
DAT1 (or SLC6A3) is localized to chromosome 5p15.3, and a
40 bp VNTR in the 30 untranslated region of the gene has
variants with a range of 3–11 copies [Vandenbergh et al., 1992
#10874]. Many association studies of DAT1 and ADHD have
focused on this marker, with particular focus on the 10-repeat
putative high-risk allele. A meta-analysis found a nearly
significant association between this DAT1 polymorphism and
ADHD (P ¼ 0.05), with an estimated odds ratio of 1.63 [Maher
et al., 2002 #10910].
Catechol-O-Methyltransferase (COMT)
COMT has been of recent interest in ADHD because it is
involved in the metabolism of neurotransmitters such as
dopamine, epinephrine, and norepinephrine, all of which are
believed to be related to the etiology of ADHD. COMT
localizes to chromosome 22q11.2 [Grossman et al., 1992
#8755], and contains a polymorphism in Exon 4 wherein a G/
A transition at codon 158 results in a valine to methionine
substitution [Lotta et al., 1995 #8763]. Given the role of
COMT in catecholaminergic transmission and the hypothesized role of catecholaminergic dysfunction in ADHD [Faraone
and Biederman, 1998 #6960], it is reasonable to hypothesize
that the activity of COMT may contribute to the etiology of
ADHD.
Eisenberg et al. [1999 #10867] found an association between
the Val/Met polymorphism of COMT and ADHD where the
high-activity Val allele was preferentially transmitted to
ADHD probands, especially those with the hyperactiveimpulsive type of ADHD. In contrast, within our sample of
202 ADHD trios, family-based association testing suggested
that the low-activity Met allele was preferentially transmitted
to ADHD boys, especially among those with the inattentive
subtype [Qian et al., 2003 #10539]. However, several other
studies found no association between COMT and ADHD [Barr
et al., 1999 #10868; Hawi et al., 2000 #8878; Tahir et al., 2000
#7230].
Dopamine D2 Receptor (DRD2)
The dopaminergic system, particularly the dopamine D2
receptor, has been implicated in the mediation of reward.
Impaired D2 receptor function is believed to place an
individual at high risk for engaging in multiple impulsive,
addictive, and compulsive behaviors, such as severe alcoholism, cocaine, heroin, and nicotine use, pathological gambling,
ADHD, Tourette’s Syndrome, and antisocial behavior. Indeed,
meta-analysis has confirmed an association of DRD2 with
impulsive, addicitive, and compulsive behaviors [Blum et al.,
1995, #4628].
DRD2 has been localized to chromosome 11q23. Previous
research suggested that the DRD2 A1 allele might be a risk
201
allele for ADHD. For example, Comings et al. [1996 #4695]
reported an association between the A1 allele of DRD2 and
ADHD as a comorbid behavior with Tourette’s syndrome, while
Rowe et al. [1999 #10215] found that higher counts of ADHD
symptoms were associated with decreasing frequencies of the
A1 allele.
These prior genetic studies of ADHD suggest it is a complex
genetic disorder caused by the cumulative effects of several
genes along with environmental risk factors. This view of
ADHD suggests it would be worthwhile to explore potential
gene–gene interactions among candidate genes. To address
this issue, we tested for the association of ADHD with
polymorphisms in DRD4, DAT1, COMT, and the promoter
region of DRD2 in the Han Chinese population.
METHODS
Subjects
Subjects with ADHD were recruited from the child psychiatric clinics at Peking University Institute of Mental Health
between September 1999 and August 2001. Families, who
provided consent to participate in the study, underwent a
comprehensive assessment process. To be included in
the study, children had to meet the following three criteria:
(1) have a diagnosis of ADHD classified according to the
Diagnostic and Statistical Manual of Mental Disorders, Fourth
Edition (DSM-IV) [American Psychiatric Association, 1994
#7949]; (2) have a full scale IQ above 70 (mean ¼ 100.6,
standard deviation (SD) ¼ 13.6) according to the Wechsler
Intelligence Scale for Chinese Children (standardized by Gong
Yaoxian); and (3) originate from the Han population. Individuals were excluded for any evidence of major neurological
conditions or a primary diagnosis of schizophrenia, affective
disorder, pervasive developmental disorder, or epilepsy. The
sample consisted of 340 Chinese children between 6 and
17 years old (mean ¼ 10.4, SD ¼ 2.6), with boys accounting for
86.8% of the sample. A large percentage of children with ADHD
(71.2%) also met diagnostic criteria for other disorders: 33.5%
had comorbid oppositional defiant disorder (ODD) and/or
conduct disorder (CD), 15.6% had an emotional disorder (such
as specific phobia, separation anxiety disorder, social phobia,
obsessive-compulsive disorder, and generalized anxiety disorder), 14.4% had a tic disorder, 6.8% had a depressive
disorder, 3.5% had a bipolar bisorder, and 39.1% had a learning
disability (LD). We also recruited a control group (n ¼ 226),
which was drawn from the Han population, and was comprised
of healthy blood donors and individuals from our institute. The
study was approved by the Ethics Committee of the Health
Science Center, Peking University.
Diagnoses
Consensus diagnoses were made according to the American
Clinical Diagnostic Interviewing Scales (CDIS) [Barkley, 1998
#6574], a structured interview derived from the DSM-IV. The
CDIS assesses some behavioral and emotional disorders
present during childhood, such as ADHD, ODD, CD, emotional
disorders, affective disorders, tics, and LD. The CDIS was
translated into Chinese by one of our native-speaking group
members. This rating scale was selected because of its high
degree of sensitivity (97.2%) and specificity (100%). The testretest reliability and criterion validity was calculated as 0.89.
The inter-rater reliability kappa coefficient was 0.74 (P < 0.01).
On the ADHD subscale, the CDIS recognizes three types
of ADHD: ADHD inattentive type (ADHD-I), ADHD
hyperactive-impulsive type (ADHD-HI), and ADHD combined
type (ADHD-C). Two psychiatrists interviewed all parents
separately. Additionally, teachers completed the Rutter [1967]
202
Qian et al.
Scale to evaluate children’s behaviors in the school environment. Consensus diagnoses were assigned to all probands
based upon data collected from CDIS, parent interviews, and
teacher reports. Of the total sample, ADHD-I accounted for
51.9%, ADHD-C for 41.1%, and ADHD-HI for 7% of individuals.
In order to further characterize the association, some
alternative phenotypes were defined. ‘‘Pure ADHD’’ refers to
ADHD without any comorbidity, and ‘‘ADHD refined phenotype’’ refers to the ADHD patients fulfilling not only ICD-10
criteria, but also DSM-IV criteria.
Genotyping
DNA was extracted from venous blood from 340 children
with ADHD and 226 controls. DNA extraction was performed
using the protein-depositing method [Qian et al., 2003]. PCR
amplifications of the DRD4, DAT1, COMT, and DRD2 loci were
carried out on a PTC-200 thermal cycler (MJ Research,
Cambridge, MA). Methods for PCR amplification and primer
sequences of DRD4, DAT1, and COMT were described elsewhere [Qian et al., 2003; Qian et al., 2004]. Only 122 controls
were used for genotyping of DRD2.
PCR amplification of the 155 bp fragment of DRD2
241A > G was carried out using the following pair of primers:
upstream 50 -AAA CGA AAG ACT GGC GAG CA-30 and downstream 50 -TGG AAC GGG TAG GAG GGG-30 . Cycling consisted
of 35 cycles consisting of denaturation at 948C for 30 sec,
annealing at 628C for 45 sec, and extension at 728C for 90 sec.
The amplification process consisted of an initial denaturing
step at 958C for 3 min and a final extension step at 728C for
7 min. The 25 ml reaction mixture consisted of 100ng of genomic
DNA, 20 mM of each primer, 10 Mm of dNTPs, 2 U of Taq
polymerase, and 10Taq buffer (MBI Fermentas, Vilnius,
Lithuania). After digestion with MaeIII restriction enzyme
overnight at 558C, the PCR products were run on 4% agarose
gel. One common fragment (155 bp) and two variable
fragments (99 þ 56 bp) were available.
Genotypes were determined from Gel Doc 2000 (BIO-RAD,
Hercules, CA) readings by at least two researchers. Ambiguous
or unidentifiable results were reamplified and rescored.
Samples that continued to amplify poorly were eliminated
from the study.
Statistics
Repeat-length polymorphisms of DRD4 and DAT1 were
dichotomized into short- and long-repeat allele groups. The
dichotomization thresholds for these two polymorphisms were
selected as the boundaries that most closely divided each group
of alleles into approximately equal groups. For the DRD4
polymorphisms, the dichotomization threshold was between
3 and 4 repeats, while for the DAT1 polymorphism, the
threshold was between 10 and 11 repeats. The associations of
polymorphisms with ADHD and its subtypes were examined
by comparing allele and genotype frequencies between cases
and controls using the Chi-square test. We didn’t adjust the
Chi-square tests for multiple testing. To analyze potential
gene–gene interactions, binary logistic regression was performed with genotypes as predictor variables and a diagnosis
of ADHD as the criterion variable. All statistical tests were
performed using SPSS for Windows (Release 10.0).
RESULTS
DRD4
For DRD4, 307 ADHD cases and 165 controls were successfully genotyped. The frequencies and percentage of DRD4
genotypes and alleles are shown in Table I. There was no
evidence of differences in allele or genotype frequencies
between cases and controls when alleles and genotypes were
considered individually, or when these were divided into long
repeats and short repeats. When stratified by gender, the longrepeat genotypes and alleles were found more frequently in
male ADHD probands than in male controls (genotypes: 94.1
vs. 84.8%, P ¼ 0.004; alleles: 81.9 vs. 74.2%, P ¼ 0.021)
(Table II). The same results were found for males with the
ADHD-C subtype. In contrast, long-repeat alleles occurred less
frequently in female ADHD subjects than in female controls
(76.4 vs. 87.9%, P ¼ 0.033). However, the largest difference is
that between the male and female controls (e.g. for LRG: 84.8
vs. 97%).
DAT1
Probands (332 ADHD) and 216 controls were successfully
genotyped for DAT1. The frequencies of DAT1 genotypes and
alleles are shown in Table III. No individual allele or genotype
was differentially distributed between cases and controls. The
TABLE I. Frequencies of DRD4 Genotypes and Alleles for Controls, all ADHD Patients, and ADHD Subtypes
Genotype
1 (2/2)
2 (2/3)
3 (2/4)
4 (2/5)
5 (3/4)
6 (3/5)
7 (4/4)
8 (4/5)
9 (4/6)
Allele
2R
3R
4R
5R
6R
Control
ADHD
ADHD-C
ADHD-I
ADHD refined
phenotype
Pure ADHD
N ¼ 165 (%)
17 (10.3)
—
29 (17.6)
1 (0.6)
2 (1.2)
1 (0.6)
108 (65.5)
5 (3)
2 (1.2)
2N ¼ 330 (%)
64 (19.4)
3 (0.9)
254 (77)
7 (2.1)
2 (0.6)
N ¼ 307 (%)
15 (4.9)
4 (1.3)
60 (19.5)
3 (1)
14 (4.6)
—
196 (63.8)
13 (4.2)
2 (0.7)
2N ¼ 614 (%)
97 (15.8)
18 (2.9)
481 (78.3)
16 (2.6)
2 (0.3)
N ¼ 130 (%)
4 (3.1)
1 (0.8)
27 (20.8)
2 (1.5)
5 (3.8)
—
84 (64.6)
6 (4.6)
1 (0.8)
2N ¼ 260 (%)
38 (14.6)
6 (2.3)
207 (79.6)
8 (3.1)
1 (0.4)
N ¼ 157 (%)
9 (5.7)
2 (1.2)
30 (19.1)
1 (0.6)
8 (5.1)
—
99 (63.1)
7 (4.5)
9 (0.6)
2N ¼ 314 (%)
51 (16.2)
10 (3.2)
244 (77.7)
8 (2.5)
1 (0.3)
N ¼ 159 (%)
8 (5)
1 (0.6)
27 (17)
3 (1.9)
8 (5)
—
103 (64.8)
7 (4.4)
2 (1.3)
2N ¼ 318 (%)
47 (14.8)
9 (2.8)
250 (78.6)
10 (3.1)
2 (0.6)
N ¼ 86 (%)
7 (8.1)
1 (1.2)
14 (16.3)
1 (1.2)
3 (3.5)
—
55 (64)
5 (5.8)
—
2N ¼ 172 (%)
30 (17.4)
4 (2.3)
132 (76.7)
6 (3.5)
—
Evaluation of Potential Gene – Gene Interactions
203
TABLE II. Analysis of Long- and Short-Repeat DRD4 Genotypes/Alleles by Gender
Genotype
LRG
SRG
P
Allele
LRA
SRA
P
Male control
Male ADHD
Male ADHD-C
Male ADHD-I
Male ADHD
for refined
phenotype
N ¼ 99 (%)
84 (84.8)
15 (15.2)
—
2N ¼ 198 (%)
147 (74.2)
51 (25.8)
—
N ¼ 271 (%)
255 (94.1)
16 (5.9)
0.004**
2N ¼ 542 (%)
444 (81.9)
98 (18.1)
0.021*
N ¼ 116 (%)
111 (95.7)
5 (4.3)
0.006**
2N ¼ 232 (%)
192 (82.8)
40 (17.2)
0.031*
N ¼ 136 (%)
127 (93.4)
9 (6.6)
0.033*
2N ¼ 272 (%)
222 (81.6)
50 (18.4)
0.055
N ¼ 138 (%)
130 (94.2)
8 (5.8)
0.016*
2N ¼ 276 (%)
228 (82.6)
48 (17.4)
0.027*
Female
control
Female
ADHD
N ¼ 66 (%)
64 (97)
2 (3)
—
2N ¼ 132 (%)
116 (87.9)
16 (12.1)
—
N ¼ 36 (%)
33 (91.7)
3 (8.3)
0.480
2N ¼ 72 (%)
55 (76.4)
17 (23.6)
0.033*
LRG, long-repeat genotype, (2/4, 2/5, 3/4, 3/5, 4/4, 4/5, 4/6); SRG, short-repeat genotype, (2/2,2/3); LRA, long-repeat allele, (4–6) repeats; SRA, short-repeat
allele, (2–3) repeats.
**P < 0.05.
*P < 0.01.
long-repeat genotypes and alleles were seen more frequently in
ADHD probands than in controls (genotypes: 10.8 vs. 5.6%,
P ¼ 0.032; alleles: 5.4 vs. 2.8%, P ¼ 0.037). For ADHD subtypes,
similar results were found (Table IV).
COMT
For the COMT Val158Met polymorphism, 317 ADHD cases
and 194 controls were successfully genotyped. Results showed
no evidence for a difference in allele distribution or genotype
frequency between ADHD probands and controls (Table V). For
the three subtypes of ADHD (ADHD-C, ADHD-I, and ADHDHI), the ADHD refined phenotype, and pure ADHD, we
obtained the same results. These data are not shown.
DRD2
For the DRD2 241A > G polymorphism, 337 ADHD cases
and 115 controls were successfully genotyped. There was no
overall evidence for a difference in allele distribution and
genotype frequency between ADHD probands and controls
(Table VI). For the three subtypes of ADHD (ADHD-C, ADHDI, and ADHD-HI), the ADHD refined phenotype, and pure
ADHD, we obtained the same results. Additionally, the
stratification of results by gender showed no significant
difference (P > 0.05). These data are not shown.
Logistic Regression Analyses
Multivariate logistic regression was used to assess the
cumulative and interactive effects of the candidate genes on
the risk for ADHD. When comparing the 176 ADHD cases with
the ‘‘refined phenotype’’ to the 226 controls, Tables VII and VIII
show that the effects of the risk alleles of DAT1 and DRD4
found in the univariate analyses remained significant after
partialing out the effects of the other putative risk alleles. In
addition to these, male gender was an independent risk factor
for ADHD. We found no significant two-way interactions
among genes in the regression analyses.
DISCUSSION
Numerous investigations have supported a significant role of
genetic influences in the etiology of ADHD. To date, most
molecular genetic studies of ADHD have focused on genes
encoding proteins involved in the dopaminergic system,
including the dopamine transporter, dopamine receptors, and
dopamine-degrading enzymes such as monoamine-oxidase A
and COMT.
Findings from case-control association studies of ADHD and
dopamine candidate genes suggest allelic heterogeneity among
populations. This heterogeneity is clear from examining the
DRD4 7-repeat allele, which is not observed in Asian samples.
By contrast, research conducted in the Americas showed that
the 7-repeat allele is the second most prevalent allele, with a
frequency of 20.6% [Chang et al., 1996 #6315]. The 4-repeat
and 2-repeat alleles account for most of the alleles of the DRD4
48 bp repeat polymorphism in the Han Chinese population.
The difference in frequency for DRD4 between the male and
TABLE III. Frequencies of DAT1 Genotypes and Alleles for Controls, all ADHD Patients, and ADHD Subtypes
Genotype
1 (6/10)
2 (7/10)
3 (9/9)
4 (9/10)
5 (10/10)
6 (10/11)
7 (7/9)
8 (9/11)
9 (10/12)
Allele
6R
7R
9R
10R
11R
12R
Control
ADHD
ADHD-C
ADHD-I
ADHD refined
phenotype
Pure ADHD
N ¼ 216 (%)
3 (1.4)
9 (4.2)
1 (0.5)
14 (6.5)
177 (81.9)
12 (5.6)
—
—
—
2N ¼ 432 (%)
3 (0.7)
9 (2.1)
16 (3.7)
392 (90.7)
12 (2.8)
—
N ¼ 332 (%)
1 (0.3)
10 (3)
3 (0.9)
29 (8.7)
252 (75.9)
33 (9.9)
1 (0.3)
2 (0.6)
1 (0.3)
2N ¼ 664 (%)
1 (0.2)
11 (1.7)
38 (5.7)
578 (87)
35 (5.3)
1 (0.2)
N ¼ 139 (%)
1 (0.7)
3 (2.2)
3 (2.2)
12 (8.6)
102 (73.4)
17 (12.2)
—
1 (0.7)
—
2N ¼ 278 (%)
1 (0.4)
3 (1.1)
19 (6.8)
237 (85.3)
18 (6.5)
—
N ¼ 172 (%)
—
6 (3.5)
—
16 (9.3)
133 (77.3)
15 (8.7)
—
1 (0.6)
1 (0.6)
2N ¼ 344 (%)
—
6 (1.7)
17 (4.9)
304 (88.4)
16 (4.7)
1 (0.3)
N ¼ 171 (%)
1 (0.6)
4 (2.3)
2 (1.2)
18 (10.5)
123 (71.9)
21 (12.3)
—
2 (1.2)
—
2N ¼ 342 (%)
1 (0.3)
4 (1.2)
24 (7)
290 (84.8)
23 (6.7)
—
N ¼ 94 (%)
—
5 (5.3)
1 (1.1)
6 (6.4)
69 (73.4)
10 (10.6)
1 (1.1)
1 (1.1)
1 (1.1)
2N ¼ 188 (%)
—
6 (3.2)
10 (5.3)
160 (85.1)
11 (5.9)
1 (0.5)
204
Qian et al.
TABLE IV. Case-Control Analysis of DAT1 Long- and Short-Repeat Genotypes/Alleles
Genotype
LRG
SRG
P
Allele
LRA
SRA
P
Control
ADHD
ADHD-C
ADHD refined
phenotype
Pure ADHD
N ¼ 216 (%)
12 (5.6)
204 (94.4)
—
2N ¼ 432 (%)
12 (2.8)
420 (97.2)
—
N ¼ 332 (%)
36 (10.8)
296 (89.2)
0.032*
2N ¼ 664 (%)
36 (5.4)
628 (94.6)
0.037*
N ¼ 139 (%)
18 (12.9)
121 (87.1)
0.014*
2N ¼ 27 (%)
18 (6.5)
260 (93.5)
0.017*
N ¼ 171 (%)
23 (13.5)
148 (86.5)
0.007**
2N ¼ 342 (%)
23 (6.7)
319 (93.3)
0.009**
N ¼ 94 (%)
12 (12.8)
82 (87.2)
0.029*
2N ¼ 188 (%)
12 (6.4)
176 (93.6)
0.032*
LRG, long-repeat genotype, (10/11, 9/11, 10/12); SRG, short-repeat genotype, (6/10, 7/9, 7/10, 9/9, 9/10, 10/10); LRA, long-repeat allele, (11, 12) repeats; SRA,
short-repeat allele, (6, 10) repeats.
*P < 0.05.
**P < 0.01.
TABLE V. COMT Val158Met Allele and Genotype Frequencies in all Cases and Controls
Genotype
ADHD (N ¼ 317)
Control (N ¼ 194)
P
Allele
Val/Val
Val/Met
Met/Met
Val
Met
153 (48.3%)
99 (51.0%)
0.656
140 (44.2%)
78 (40.2%)
24 (7.6%)
17 (8.8%)
446 (70.3%)
276 (71.1%)
0.789
188 (29.7%)
112 (28.9%)
TABLE VI. DRD2 241A > G Allele and Genotype Frequencies in all Cases and Controls
Genotype
ADHD (N ¼ 337)
Control (N ¼ 115)
P
Allele
A1A1
A1A2
A2A2
A1
A2
232 (68.8%)
80 (69.6%)
0.873
96 (28.5%)
31 (27%)
9 (2.7%)
4 (3.5%)
560 (83.1%)
190 (82.6%)
0.868
114 (16.9%)
40 (17.4%)
A1, (241A); A2, (241G).
female controls tells us some gender differences in the
dopamine system. Such difference could also be observed in
some animal studies. Andersen and Teicher [2000] found that
male rats exhibited a much greater degree of striatal overproduction and elimination of dopamine D1 and D2 family
receptors compared to females across periadolescence; the
female rats sustained a stable density of dopamine receptors
throughout the lifetime.
The 40 bp VNTR of DAT1 also displays a high degree of
variability among populations. The frequency of the 10-repeat
allele is significantly higher in Asia, especially China and
Japan (above 90%) than it is in Caucasians, Hispanics,
and African-Americans (71.9, 70.9, and 72.9%, respectively)
[Doucette-Stamm et al., 1995 #10870; Mitchell et al., 2000
#10869]. The Chinese population shows a reduced frequency of
TABLE VII. Logistic Regression Analysis of 176 ADHD
‘‘Refined Phenotype’’ Cases and 226 Controls
DRD4 LRG
DAT1 LRG
COMT A1A1
DRD2 A1A1
Male gender
Constant
OR
95% CI for OR
P value
2.805
3.066
0.979
1.091
3.545
—
1.056–7.452
1.064–8.835
0.566–1.696
0.600–1.985
1.833–6.856
0.039
0.038
0.941
0.774
0.000
—
CI, confidence interval; LRG, long-repeat genotype.
the 9-repeat allele and an increased frequency of the 10-repeat
allele when compared with Caucasians [Li et al., 1994 #10871].
In our Han Chinese controls, the repeat numbers at the 40bp
locus ranged from 6 to 7 and 9 to 11. As expected, the 10-repeat
allele was the most frequent (90.7%).
As for the COMT Val158Met locus, the frequency of the lowenzyme activity allele varies significantly from 1 to 62% (e.g.,
Europe and Southwest Asia, 47%; East Asia, 24%; North and
Central America, 34%; South America, 18%; and Africa, 23%).
For Chinese, this allele occurred at a frequency of 25.5%
[Palmatier et al., 1999 #10872]. The frequency of the COMT
Met158 allele in our study (28.9%) was similar to that of the
previous studies in China [e.g., 32%, Li et al., 1996 #10873]
and [e.g., 27%, Chen et al., 1997 #8832]. Thus the allele and
genotype frequencies (Val/Val, 51.0%; Val/Met, 40.2%; and
Met/Met, 8.8%) in the control group may reflect essential
features of the Han Chinese population.
Our exploratory binary logistic regression analysis with
the sample having a refined ADHD phenotype showed
that male gender and long-repeat genotypes of DRD4 and
DAT1 were risk factors for ADHD. We found no evidence
for gene–gene interactions among the candidate genes we
studied. There were several previous studies addressing gene–
gene interaction in the candidate dopaminergic genes for
ADHD. The interaction effect of both DRD4 7-repeat allele and
DAT1 10-repeat allele genes on ADHD was suggested [Roman
et al., 2001; Szobot et al., 2005; Carrasco et al., 2006]. However,
the DRD4 7-repeat allele was not found in our present samples.
The important facet towards understanding the ADHD is the
Evaluation of Potential Gene – Gene Interactions
205
TABLE VIII. The Sequence Analysis of OR Values in Logistic Regression Model
Male gender
4.021*
4.134*
4.577*
4.860*
3.545*
LRG for DAT1
LRG for DRD4
A1A1 of COMT gene
A1A1 of DRD2
2.890**
2.655**
2.381***
3.066**
2.485**
2.883**
2.805**
1.010
0.808
0.861
A1 of DRD2 ¼ (241A); A1 of COMT ¼ Val158; LRG, long-repeat genotype.
*P < 0.01.
**P < 0.05.
***P ¼ 0.053.
marked sex differences in its prevalence. ADHD is more often
diagnosed in males than females (four- to ninefold more
prevalent in males [American Psychiatric Association,
1994]). Our analysis showed that male gender was an
independent risk factor for the disorder.
However, our study has following limitations: First and
foremost, the division of DRD4 and DAT1 alleles into long and
short is an exploratory one. Without taking into account the
functionality of the alleles assessed in the specific population,
our attempt to put several of them in the ‘‘same basket’’ might
obscure their effects. The study suggested that most 2R alleles
are 7R derivatives [Wang et al., 2004]. The 7R variant exhibits
a blunted ability to reduce camp levels, in comparison with that
of the common 4R variant [Asghari et al., 1995]. Interestingly,
the 2R variant also has a blunted cAMP response, but one that
is midway between those of the 4R allele and 7R allele. The 2R
and 7R alleles are genetically and functionally related, each
exhibiting suboptimal dopamine-signaling, in comparison
with that of 4R alleles [Wang et al., 2004]. The 7R allele has
been identified as a risk allele for ADHD in Caucasian children.
However, no 7R allele has been found in the Chinese children
with ADHD in our study. Second, the study’s case-control
design may suffer from population stratification. Given the
exploratory nature of this analysis, the positive finding must be
interpreted cautiously.
Some recent evidence suggests that genetic predisposition
may be only one piece of the etiologic puzzle, while interactions
between genes and environmental factors may give rise to the
expression of the illness. For example, a study by Milberger
and colleagues investigated the role of pregnancy, delivery,
and infancy complications (PDICs) in relation to the etiology of
ADHD, and found a positive association between ADHD and
PDICs [Milberger et al., 1997 #5568]. Therefore, future
research must continue to examine the role and influence of
gene–gene and gene–environment interactions in the etiology
of this disorder.
Carrasco X, Rothhammer P, Moraga M, Henrı’quez H, Chakraborty R,
Aboitiz F, Rothhammer F. 2006. Genotypic interaction between DRD4
and DAT1 loci is a high risk factor for attention-deficit/hyperactivity
disorder in Chilean families. Am J Med Genet Part B Neuropsychiatr
Genet 141B:51–54.
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