| Summary |
Several studies have suggested a possible association of a polymorphism at the dopamine D4 receptor gene and attention-deficit hyperactivity disorder. The allele reported to be associated with attention-deficit hyperactivity disorder (ADHD) is the allele with seven copies of the 48 bp repeat in the third exon. In this study, they extended the study of the dopamine D4 gene and ADHD by testing for linkage using two additional polymorphisms in the dopamine D4 receptor gene and a polymorphism in the closely linked gene, tyrosine hydroxylase. They also searched for two previously reported deletions, a 13 bp and a 21 bp deletion in the first exon. They examined the haplotypes of three polymorphisms of the D4 receptor gene and observed biased transmission of two of these haplotypes. These findings further support the role of the dopamine D4 gene in ADHD. |
| Total Sample |
In this study, they genotyped 82 families consisting of 72 with a proband and both parental DNAs genotyped and 10 families with a proband with DNA available and genotyped for a single parent. There were 15 affected siblings genotyped in the sample. Haplotypes could be determined unambiguously in 60 families consisting of 55 trios, 5 parent-child pairs, and 10 siblings. |
| Sample Collection |
A sample of 82 families with an ADHD proband collected in Toronto was used in this study. This protocol was approved by The Hospital for Sick Children Research Ethics Board and informed consent was obtained for all participants. |
| Diagnosis Description |
To be included in this study, children met the Diagnostic and Statistical Manual of Mental Disorders, fourth edition, [DSM¨CIV; USAn Psychiatric Association, 1994] criteria for one of the three ADHD subtypes (inattentive, hyperactive-impulsive, combined) and were between 7 and 16 years of age. Subjects were excluded if they scored below 80 on both the Performance and Verbal Scales of the Weschler Intelligence Scale for Children [WISC-III, Weschler, 1991] had evidence of neurological or chronic medical illness, bipolar affective disorder, psychotic symptoms, Tourette syndrome, chronic multiple tics, or had a comorbid anxiety, depressive, or developmental disorder that could better account for the behaviors (as specified by DSM-IV). Subjects with comorbid oppositional defiant disorder or conduct disorder were not excluded from this study. Children were free of medication 24 hr before their assessment. Subjects were referrals for clinical assessment of attention, behavior, and learning problems to one of two clinics at The Hospital for Sick Children. For every case, diagnosis was based on information obtained from a semistructured interview of parents [Parent Interview for Child Symptoms, PICS-IV; Schachar and Ickowicz, unpublished] and teacher [Teacher Telephone Interview-IV, TTI; Tannock and Schachar, unpublished]. This information was supplemented by evidence about behavior, development, and medical history derived from standardized parent and teacher questionnaires; Conners Parent and Teacher Rating Scales-Revised [Conners, 1997] and the Ontario Child Health Survey Scales-Revised [Boyle et al., 1993]. Academic achievement in reading, arithmetic, and spelling were measured using the Wide Range Achievement Test-III, [Wilkinson, 1993]. Intelligence was measured using the WISC-III [Wechsler, 1991]. Receptive and oral language skills were tested with the Clinical Evaluation of Language Fundamentals, third edition [Semel et al., 1995]. Children were assessed using self-report for depression [Children's Depression Inventory, Kovacs, 1995] and anxiety [Children's Manifest Anxiety Scale, Reynolds and Richmond, 1985]. All interviews [Schachar et al., 1995] and measures have established reliability and validity. |
| Technique |
DNA was extracted directly from blood lymphocytes using a high salt method [Miller et al., 1988]. The (G)n mononucleotide repeat polymorphism located in the first intron of DRD4 was typed according to Petronis et al. [1994]. The 48 pb repeat polymorphism in the third exon was typed as previously described [Lichter et al., 1993]. The 12 bp insertion/deletion polymorphism, and the 13 bp and 21 bp deletions in the first exon of DRD4 were assessed using one PCR reaction [Catalano et al., 1993] followed by restriction enzyme digestion of the PCR product with PstI to separate the polymorphisms into two fragments. The 12 bp repeat polymorphism is located in the larger of the two fragments. The size of the fragment was of either 202 bp for the 2-repeat allele or 190 bp for a single 12 bp repeat. The 21 bp deletion, if present, reduces these two bands to 181 bp (two repeats of the 12 bp repeat and the deletion) or 169 bp (1 repeat of the 12 bp repeat and the deletion). The 13 bp deletion separates into the smaller of the PstI fragments (96 bp without the deletion). The PstI fragments were electrophoresed on 6% polyacrylamide mini gels (Novex, San Diego, CA) followed by silver staining to detect the alleles. The tetranucleotide repeat polymorphism at TH was typed as described [Edwards et al., 1992]. |
| Analysis Method |
Statistical analysis was performed using the ETDT program [Sham and Curtis, 1995]. The ETDT program can be used to examine the pattern of preferential transmission of certain alleles across genotypes (allele-wise TDT) and also the genotype-wise analysis (considers every heterozygous parental genotype separately and examines whether each allele of the genotype is transmitted to affected offspring on 50% of occasions). Linkage disequilibrium between the markers was estimated with the EH program [Ott, 1991]. |
| Result Description |
For the trios, the 2-repeat allele of the 12 bp repeat polymorphism was transmitted 9 times compared to 12 times that it was not transmitted (allele-wise TDT P=0.513). For the entire sample, 15 transmissions of the 2-repeat allele compared to 13 transmissions of the 1 repeat allele (allele-wise TDT P=0.706) were observed. For the (G)n mononucleotide repeat in the first intron, A trend for the 142 bp allele was observed not to be transmitted. The 142 bp allele was transmitted 32 times compared to 49 times that it was not transmitted (allele-wise TDT P=0.059). This polymorphism is in strong linkage disequilibrium with the exon 3 repeat polymorphism. One point is that in this sample the linkage to the exon II polymorphism is not robust. It is significant using the one-sided P value but not the two-sided P value. The allele-wise TDT X2 for this sample for the transmission of the 7-repeat allele was 2.882 (one-sided P=0.045, two-sided P=0.090). For the tyrosine hydroxylase (TH) polymorphism, no significant difference in the transmission of any of the alleles was observed. |
Hot Results
Basic Info
Detail Info