| Summary |
To explore potential gene-gene interactions among candidate genes for ADHD, they studied the dopamine D2 receptor (DRD2), dopamine D4 receptor (DRD4), dopamine transporter (DAT1), and catechol-O-methyltransferase (COMT) genes in the Han Chinese population. A sample of 340 children with ADHD was diagnosed according to the DSM-IV criteria. 226 unrelated controls were also recruited. Identified polymorphisms included a 48-base-pair-repeat in Exon 3 of DRD4, a 40-base-pair-repeat in the 3' 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. They 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. |
| Total Sample |
The sample consisted of 340 Chinese children. 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). A control group (n=226) was also recruited, which was drawn from the Han population, and was comprised of healthy blood donors and individuals from their institute. |
| Sample Collection |
Subjects with ADHD were recruited from the child psychiatric clinics at Peking University Institute of Mental Health between September 1999 and August 2001. Children in this study originate from the Han population. |
| Diagnosis Description |
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) [USAn Psychiatric Association, 1994 #7949]; (2) have a full scale IQ above 70 (mean=100.6, standard deviation (SD)=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 study was approved by the Ethics Committee of the Health Science Center, Peking University. For more details about diagnoses, please refer to the original publication. |
| Technique |
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. 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. |
| Analysis Method |
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. They 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). |
| Result Description |
For DRD4, 307 ADHD cases and 165 controls were successfully genotyped. 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 long-repeat genotypes and alleles were found more frequently in male ADHD probands than in male controls. 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. However, the largest difference is that between the male and female controls (e.g. for LRG: 84.8 vs. 97%). For DAT1, 332 ADHD cases and 216 controls were successfully genotyped. No individual allele or genotype was differentially distributed between cases and controls. The long-repeat genotypes and alleles were seen more frequently in ADHD probands than in controls. For ADHD subtypes, similar results were found. 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. For the three subtypes of ADHD, the ADHD refined phenotype, and pure ADHD, the same results were obtained. 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. For the three subtypes of ADHD, the ADHD refined phenotype, and pure ADHD, the same results were obtained. Additionally, the stratification of results by gender showed no significant difference. 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, results 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. No significant two-way interactions among genes were found in the regression analyses. |
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