| Summary |
Molecular genetic investigations of ADHD have found positive associations with the 480-bp allele of a VNTR situated in the 3' untranslated region of DAT1 and allele 7 of a VNTR in exon 3 of DRD4. A number of independent studies have attempted to replicate these findings but the results have been inconsistent. In this study, they used both family-based and case control approaches to examine these polymorphisms in a sample of 137 children diagnosed with ICD-10, DSM-IV or DSM-III-R ADHD. No evidence of association with the DAT1 polymorphism was found, despite a sample size that has up to 80% power to detect a previously reported effect size. A significant increase in the DRD4 7 repeat allele amongst ADHD probands (21.7%) and their parents (18.9% in mothers, 22.3% in fathers) was observed, compared to ethnically matched controls (12.8%). However TDT analysis showed no preferential transmission of allele 7 to ADHD probands. |
| Total Sample |
The sample included 137 British Caucasian children. These children were derived from 133 families (included four affected sibling pairs), with 132 mothers and 107 fathers also participating in the study. In total, 442 controls were used in case-control analysis. On the basis of parent and teacher reports, the following diagnoses were assigned to 137 probands: ICD-10 hyperkinetic disorder (65.0%), DSM-IV ADHD combined type (71.5%), DSM-IV ADHD hyperactive-impulsive type (10.9%), DSM-IV ADHD inattentive type (7.3%), and DSM-III-R ADHD (94.9%). High co-morbidity rates were observed amongst the sample: specifically 58.4% of the sample also met criteria for oppositional defiant disorder, 12.4% conduct disorder, 13.1% tic disorders, 2.9% anxiety disorders and 1.5% depressive disorders. |
| Sample Collection |
Families with children who had suspected ADHD were recruited from Child and Adolescent Psychiatry Clinics throughout Greater Manchester and Cheshire, United Kingdom. A control group was also incorporated to allow for comparisons with previous case-control findings. The control group (n=295) was comprised of British Caucasian healthy blood donors and healthy individuals selected at random from general practice registers in East Anglia, United Kingdom. An additional random sample of controls (n=147) was taken from St Mary's Hospital in Manchester, United Kingdom and genotyped for the DRD4 polymorphism. Socio-demographic data for the control sample were not available. |
| Diagnosis Description |
Those families who gave consent to participate in the study were approached by the research team and underwent the assessment process. Potential subjects were first assessed using the Wechsler Intelligence Scale for Children, version III (WISC-III). Only those children with a full scale IQ score above 70 (mean=91.2, SD=13.1) were included in the study. Other exclusion criteria were: any evidence of major medical or neurological conditions, Tourette's syndrome, pervasive developmental disorders or laboratory evidence of fragile X syndrome. Mothers were interviewed using the Child and Adolescent Psychiatric Assessment (CAPA), a semi-structured, interviewer-administered, diagnostic interview. Information on ADHD symptoms in school (which was required to determine whether children fulfilled the diagnostic criterion of symptom pervasiveness) was obtained from teachers using a semi-structured teacher telephone interview that has been shown to have good test-retest reliability and criterion validity. This telephone interview was typically conducted with the child's class teacher, as the majority of children who participated in the study were in primary school. However for those children in secondary school, the teacher interview was conducted with the teacher who had the most extensive knowledge of the child's behaviour. Hence, one teacher was interviewed for each child participating in the study. Diagnoses were made according to ICD-10, DSM-IV and DSM-III-R criteria. Interviews were conducted by a psychologist (JH) and two child psychiatrists (ALT, HF) who were trained to use the CAPA by attending a 3-day intensive training workshop (conducted by AT and RCH). Furthermore, interviews were audiotaped and supervised on a weekly basis by an experienced child and adolescent psychiatrist (AT). Interrater reliability kappa coefficients were calculated for ICD-10 Hyperkinetic Disorder (0.83), DSM-IV ADHD (0.88), DSM-III-R ADHD (0.88) and for an overall diagnosis of ADHD (ie fulfilling ICD-10, DSM-IV or DSM-III-R criteria) (kappa=1.0). |
| Technique |
DNA was obtained from venous blood for 125 children and from cheek swabs for 12 children. DNA was also requested from both parents. For families where parental DNA was not available, DNA was obtained from unaffected full biological siblings (n=5) aged between 5 and 15 years who were screened for ADHD using the CAPA. Additional control DNA was extracted from 442 United Kingdom controls. All genotypes for DAT1 and DRD4 for ADHD cases, parents and controls were checked for Mendelian inheritance. For details about DNA extraction from blood and cheek scrapes, please refer to the original paper. DOP-PCR 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 microtitre plates with a final reaction volume of 100 ¦Ìl containing. PCR amplification was performed on the PTC-225 Peltier Thermal Cycler (MJ Research, Essex, United Kingdom). For details about DRD4 and DAT1 specific amplification precedure, please refer to the original paper. |
| Analysis Method |
Initial statistical analysis was carried out on the total sample of ADHD probands. A further subgroup was defined: namely 'Swanson's refined phenotype' which refers to DSM-IV diagnosed ADHD, without serious comorbidity, with the exception of oppositional defiant disorder. An additional sub-group of 'methylphenidate responders' was also defined. This group was classified as those children on methylphenidate who were rated as 'very improved' (by parent and referring psychiatrist) on the global improvement item of the Clinical Global Impression Scale. Seventy-seven children were classified as definite methylphenidate responders. Association of DRD4 and DAT1 with ADHD was examined by: (i) comparing cases and controls; and (ii) using family controls in an extension of the transmission disequilibrium test. In this paper, the abbreviated term TDT was used to refer to the logistic regression TDT described below. Allele frequencies in different groups of subjects were compared using Fisher's exact test. Odds ratios (and 95% confidence intervals) were calculated to estimate the risk of disease associated with possession of the candidate allele (allele 10 (480 bp) for DAT1 and allele 7 for DRD4). For the family-based analysis, a conditional logistic regression framework was used to combine data on ADHD children and either their parents or siblings. Three types of case-control sets were constructed depending on the availability of family members: (1) Parent-child trios, (2) Parent-child pairs, (3) Sibships. The data from the three types of case-control sets defined above were combined in one conditional logistic regression analysis using the statistical software package Stata. Departures from Hardy-Weinberg equilibrium were tested using exact tests as implemented in the software Genepop. This program estimates the P-value using a Markov chain method when the number of alleles makes complete enumeration infeasible. |
| Result Description |
Case-control analysis of DAT1: There was no evidence of an overall difference in distribution in allele frequencies (P=0.82). Possession of allele 10 (480 bp) confers no additional risk of ADHD (odds ratio 0.96 (95% confidence interval 0.7, 1.3)). There is no evidence against Hardy-Weinberg equilibrium for DAT1 in the controls (P=0.41), ADHD cases (P=0.72) or parents (P=1.00). TDT analysis of DAT1: There was no overall evidence of association between DAT1 and ADHD (likelihood ratio chi-squared statistic 0.56, 2 df, P=0.75). An additional analysis was carried out grouping together alleles 9 and 11. This gave an estimated odds ratio of 0.89 (95% confidence interval 0.6, 1.4) for allele 10 (480 bp). Thus we detected no evidence of association with DAT1 using either TDT or case control analysis. Case-control analysis of DRD4: There was strong evidence of a difference in distribution (P=0.01, using Fisher's exact test). This difference was observed even more strongly between parents of cases and the controls (P=0.001). (There was no significant difference between mothers and fathers (P=0.1).) Possession of the candidate allele 7 is significantly more common in ADHD cases than in the controls (odds ratio=1.9 (1.3, 2.7), P=0.001). It is also similarly more common in mothers (odds ratio=1.6 (1.1, 2.5), P=0.02) and fathers (odds ratio=2.5 (1.6, 3.8), P<0.0001) of ADHD cases than in controls. There was some evidence against Hardy-Weinberg equilibrium in controls (P=0.02), weaker evidence in cases (P=0.08) and some evidence in parents (P=0.03), although the differences between the observed and expected distribution of genotypes was small. TDT analysis of DRD4: From the combined regression analysis, there was no overall evidence of association between DRD4 and ADHD (likelihood ratio chi-squared statistic 5.97, 5 df, P=0.31). a further analysis grouping together all alleles other than allele 7 was carried out. This gave an estimated odds ratio of 0.95 (95% confidence interval 0.6, 1.5) for allele 7. This allele was transmitted 38 times and not transmitted 39 times by a heterozygous parent. |
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