Evaluation of potential geneЦgene interactions for attention deficit hyperactivity disorder in the Han Chinese population.код для вставкиСкачать
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: firstname.lastname@example.org 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  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  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. REFERENCES Faraone SV, Sergeant J, Gillberg C, Biederman J. 2003. The worldwide prevalence of ADHD: Is it an American condition? World Psychiatry 2:104–113. American Psychiatric Association. 1994. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). Washington, DC: American Psychiatric Association. Andersen SL, Teicher MH. 2000. Sex differences in dopamine receptors and their relevance to ADHD. Neurosci Biobehav Rev 24:137–141. Asghari V, Sanyal S, Buchwaldt S, Paterson A, Jovanovic V, Van Tol HH. 1995. Modulation of intracellular cyclic AMP levels by different human dopamine D4 receptor variants. J Neurochem 65:1157–1165. Barkley RA. 1998. Attention deficit hyperactivity disorder: A handbook for diagnosis and treatment. New York: Guilford. Barr CL, Wigg K, Malone M, Schachar R, Tannock R, Roberts W, Kennedy JL. 1999. Linkage study of catechol-O-methyltransferase and attentiondeficit hyperactivity disorder. Am J Med Genet Neuropsychiatr Genet 88:710–713. Blum K, Sheridan PJ, Wood RC, Braverman ER, Chen TJH, Comings DE. 1995. Dopamine D2 receptor gene variants: Association and linkage studies in impulsive-addictive-compulsive behaviour. Pharmacogenetics 5:121–141. Chang FM, Kidd JR, Livak KJ, Pakstis AJ, Kidd KK. 1996. The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus. Hum Genet 98:91–101. Chen CH, Lee YR, Wei FC, Koong FJ, Hwu HG, Hsiao KJ. 1997. Association study of NlaIII and MspI genetic polymorphisms of catecholO-methyltransferase gene and susceptibility to schizophrenia. Biol Psychiatry 41:985–987. Civelli O, Bunzow JR, Grandy DK, Zhou Q-Y, Van Tol HHM. 1991. Molecular Biology of the dopamine receptors. Eur J Pharmacol (Mol Pharmacol Section). 207:277–286. Comings DE, Wu H, Chiu C, Ring RH, Gade R, Ahn C, Mac Murray JP, Dietz G, Muhleman D. 1996. Polygenic inheritance of Tourette syndrome, stuttering, attention deficit hyperactivity, conduct and oppositional defiant disorder: The additive and subtractive effect of the three dopaminergic genes—DRD2, DbH and DAT1. Am J Med Genet Neuropsychiatr Genet 67:264–288. Doucette-Stamm LA, Blakely DJ, Tian J, Mockus S, Mao JI. 1995. Population genetic study of the human dopamine transporter gene (DAT1). Genet Epidemiol 12:303–308. Eisenberg J, Mei-Tal G, Steinberg A, Tartakovsky E, Zohar A, Gritsenko I, Nemanov L, Ebstein RP. 1999. Haplotype relative risk study of catecholO-methyltransferase (COMT) and attention deficit hyperactivity disorder (ADHD): Association of the high-enzyme activity Val allele with ADHD impulsive-hyperactive phenotype. Am J Med Genet Neuropsychiatr Genet 88:497–502. Faraone SV, Biederman J. 1998. Neurobiology of attention deficit hyperactivity disorder. Biol Psychiatry 44:951–958. Faraone SV, Biederman J. 2004. Neurobiology of Attention Deficit Hyperactivity Disorder. In: Charney DS, Nestler EJ, editors. Neurobiology of Mental Illness, Second Edition. New York, NY: Oxford University Press. Faraone SV, Doyle AE, Mick E, Biederman J. 2001. Meta-analysis of the association between the 7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyperactivity disorder. Am J Psychiatry 158:1052–1057. Faraone SV, Perlis RH, Doyle AE, Smoller JW, Goralnick J, Holmgren MA, Sklar P. 2005. Molecular genetics of attention deficit hyperactivity disorder. Biol Psychiatry 57:1313–1323. Gelernter J, Kennedy JL, Van Tol HHM, Civelli O, Kidd KK. 1992. The D4 dopamine receptor (DRD4) maps to distal 11p close to HRAS. Genomics 13:208–210. Giros B, Jaber M, Jones SR, Wightman RM, Caron MG. 1996. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379:606–612. Grossman MH, Szumlanski C, Littrell JB, Weinstein R, Weinshilboum RM. 1992. Electrophoretic analysis of low and high activity forms of catecholO-methyltransferase in human erythrocytes. Life Sciences 50:473–480. Hawi Z, Millar N, Daly G, Fitzgerald M, Gill M. 2000. No association between catechol-O-methyltransferase (COMT) gene polymorphism and attention deficit hyperactivity disorder (ADHD) in an Irish sample. Am J Med Genet Neuropsychiatr Genet 96:282–284. 206 Qian et al. Leung PWL, Luk SL, How TP, Taylor E, Mak FL, Bacon-Shone J. 1996. The diagnosis and prevalence of hyperactivity in Chinese schoolboys. Br J Psychiatry 168:486–496. Li T, Yang L, Wiese C, Xu CT, Zeng Z, Giros B, Caron MG, Moises HW, Liu X. 1994. No association between alleles or genotypes at the dopamine transporter gene and schizophrenia. Psychiatr Res 52:17–23. Li T, Sham PC, Vallada H, Xie T, Tang X, Murray RM, Liu X, Collier DA. 1996. Preferential transmission of the high activity allele of COMT in schizophrenia. Psychiatr Genet 6:131–133. Lotta T, Vidgren J, Tilgmann C, Ulmanen I, Melen K, Julkunen I, Taskinen J. 1995. Kinetics of human soluble and membrane-bound catechol Omethyltransferase: A revised mechanism and description of the thermolabile variant of the enzyme. Biochemistry 34:4202–4210. Maher BS, Marazita ML, Ferrell RE, Vanyukov MM. 2002. Dopamine system genes and attention deficit hyperactivity disorder: A metaanalysis. Psychiatr Genet 12:207–215. Milberger S, Biederman J, Faraone SV, Guite J, Tsuang MT. 1997. Pregnancy, delivery and infancy complications and attention deficit hyperactivity disorder: Issues of gene-environment interaction. Biol Psychiatry 41:65–75. repeats contribute to genetic risk for the disorder. Am J Med Genet Part B Neuropsychiatr Genet 128B:84–89. Roman T, Schmitz M, Polanczyk G, Eizirik M, Rohde Luis A, Hutz Mara H. 2001. Attention-deficit hyperactivity disorder: A study of association with both the Dopamine transporter gene and the Dopamine D4 receptor gene. Am J Med Genet Neuropsychiatr Genet 105:471–478. Rowe DC, Almeida DM, Jacobson KC. 1999. School context and genetic influences on aggression in adolescence. Psychol Sci 10:277–280. Rubinstein M, Phillips TJ, Bunzow JR, Falzone TL, Dziewczapolski G, Zhang G, Fang Y, Larson JL, McDougall JA, Chester JA, Saez C, Pugsley TA, Gershanik O, Low MJ, Grandy DK. 1997. Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine. Cell 90:991–1001. Rutter M. 1967. A children’s behaviour questionnaire for completion by teacher: preliminary finding. J Child Psychol Psychiatry 8:1–11. Szobot C, Roman T, Cunha R, Acton P Hutz M, Rohde Luis A, 2005. Brain perfusion and dopaminergic genes in boys with attention-deficit/ hyperactivity disorder. Am J Med Genet Part B Neuropsychiatr Genet 132B:53–58. Mitchell RJ, Howlett S, Earl L, White NG, McComb J, Schanfield MS, Briceno I, Papiha SS, Osipova L, Livshits G, Leonard WR, Crawford MH. 2000. Distribution of the 30 VNTR polymorphism in the human dopamine transporter gene in world populations. Hum Biol 72:295–304. Tahir E, Curran S, Yazgan Y, Ozbay F, Cirakoglu B, Asherson PJ. 2000. No association between low- and high-activity catecholamine-methyltransferase (COMT) and attention deficit hyperactivity disorder (ADHD) in a sample of Turkish children. Am J Med Genet Neuropsychiatr Genet 96:285–288. Palmatier MA, Kang AM, Kidd KK. 1999. Global variation in the frequencies of functionally different catechol-O-methyltransferase alleles. Biol Psychiatry 46:557–567. Vandenbergh DJ, Persico AM, Hawkins AL, Griffin CA, Li X, Jabs EW, Uhl GR. 1992. Human dopamine transporter gene (DAT1) maps to chromosome 5p15.3 and displays a VNTR. Genomics 14:1104–1106. Qian Q, Wang Y, Zhou R, Li J, Wang B, Glatt S, Faraone SV. 2003. Familybased and case-control association studies of catechol-O-methyltransferase in attention deficit hyperactivity disorder suggest genetic sexual dimorphism. Am J Med Genet Part B Neuropsychiatr Genet 118B:103–109. Van Tol HH, Wu CM, Guan HC, Ohara K, Bunzow JR, Civelli O, Kennedy J, Seeman P, Niznik HB, Jovanovic V. 1992. Multiple dopamine D4 receptor variants in the human population. Nature 358:149–152. Qian Q, Wang Y, Zhou R, Yang L, Faraone SV. 2004. Family-based,Casecontrol Association. Studies of DRD4 and DAT1 polymorphisms in Chinese attention deficit hyperactivity disorder patients suggest long Wang E, Ding YC, Flodman P, Kidd JR, Kidd KK, Grady DL, Ryder OA, Spence MA, Swanson JM, Moyzis RK. 2004. The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus. Am J Hum Genet 74:931–944.