| Summary |
To test the dopaminergic hypothesis further, they have looked for association between ADHD and alleles of seven dopamine- related candidate genes using a family-based association approach in a sample of 150 children diagnosed with ADHD. They tested polymorphisms in genes encoding three dopamine receptors (DRD3, DRD4, and DRD5) and four dopamine-relevant enzymes: tyrosine hydroxylase [tyrosine hydroxylase (TH)], dopamine beta hydroxylase (DbH), catechol-O-methyltransferase (COMT), and monoamine oxidase A (MAOA). They were unable to detect a significant association with any of the polymorphisms genotyped, although there was a trend for preferential transmission of the DRD5 148 bp marker allele and the MAOA 122 bp marker allele. They conclude that none of the alleles they have tested makes a major contribution to ADHD, although much larger samples are required to exclude small effects. |
| Total Sample |
The sample includes 150 children of U.K. origin (parents and grandparents from U.K. or northern Europe) aged between 6 and 13 years (mean=9.1, SD=1.8 years). The children were derived from 145 families (including 5 affected siblings), with 144 mothers and 115 fathers also participating in the study. DNA samples were sent to the laboratory for 150 families. These comprised 122 complete trios and 28 duos. |
| Sample Collection |
Families of children with suspected or diagnosed ADHD were recruited from District Child and Adolescent Psychiatry Clinics in Greater Manchester, Cheshire and Wales, U.K. |
| Diagnosis Description |
Diagnoses were made according to information derived from the Child and Adolescent Psychiatric Assessment Parent Version (CAPA), which is a semi-structured, investigator-based research diagnostic interview [Angold et al., 1995], and a structured teacher telephone interview [Holmes et al., in preparation]. Using operationalized checklists, interviewers generated ICD-10, DSM-IV and DSM-III-R diagnoses. The following diagnoses were assigned to 150 probands: ICD-10 hyperkinetic disorder (63.7%), DSM-IV ADHD combined type (69.2%), DSM-IV ADHD hyperactive-impulsive type (13%), DSM-IV ADHD inattentive type (8.2%), and DSM-III-R ADHD (92.7%). |
| Technique |
DNA was obtained from venous blood for 138 children, and 12 cheek swabs for children where it was not possible to obtain a blood sample. DNA was also requested from both parents. The Bioline DNAce MaxiBlood Purification System was used to extract DNA from 5 ml of blood. DNA was resuspended in 200 ul of water (25-100 ng/ul) and stored in a 96-well format at 4oC. Buccal cells were harvested using three cytology brushes per child. Brushes were air-dried and stored at -20oC. To extract DNA, the brushes were incubated in 200 ul 50 mM NaOH for 5 min at 95oC and neutralized with 30 ul 1 M Tris pH 8.0. Samples were stored at -20oC. DOP-PCR [Telenius et al., 1992] was used to increase the number of priming sites of the DNA extracted from buccal cells prior to specific amplification. PCRs were carried out in 96-well microtiter plates with a final reaction volume of 20 ul containing NH4 buffer (Bioline), 1.5 mM MgCl2, 0.1 mM dNTP, 0.2 U Taq (Bioline, BioTaq), 0.6 uM primers, 2 ul DOP product or 2 u1 of genomic DNA and 1 M Betaine. RFLP conditions were performed as previously described (see Tables I and II in the original publication for references). |
| Analysis Method |
Association and linkage of candidate genes with ADHD was examined using the transmission disequilibrium test (TDT) [Spielman et al., 1993], which tests for distortion from the expected Mendelian pattern of inheritance from parent to affected offspring. Three candidate genes (dopamine receptor DRD5, tyrosine hydroxylase, and monoamine oxidase A) had several alleles. For these markers, an extension of the TDT for multiallelic markers was used, as implemented in the software ETDT [Sham and Curtis, 1995]. |
| Result Description |
They were unable to detect a significant association with any of the polymorphisms genotyped, although there was a trend for preferential transmission of the DRD5 148 bp marker allele and the MAOA 122 bp marker allele. They conclude that none of the alleles they have tested makes a major contribution to ADHD, although much larger samples are required to exclude small effects. |
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