| Summary |
In this report, we describe results of analysis of the DRD4 120-bp repeat promoter polymorphism in a sample of 371 children with ADHD and their parents, using the transmission disequilibrium test (TDT). Results showed a significant preferential transmission of the 240-bp (long) allele with ADHD. Exploratory analyses of the Inattentive phenotypic subtype of ADHD strengthened the evidence for linkage. These data add further support for the role of DRD4 variants conferring increased risk for ADHD, and imply that additional studies of DRD4 and other related genes are needed. |
| Total Sample |
The sample consisted of 371 children with ADHD from 197 families, including 46 families of a single child with ADHD (23%), 132 families with two children with ADHD (67%), 15 families with three affected children with ADHD (8%), and four families with four affected children with ADHD (2%). Genotyping data were available for 308 parents (156 mothers and 152 fathers). |
| Sample Collection |
The study was approved by the UCLA Human Subject Protection Committee. All children and parents provided written informed consent for their participation. As a portion of ongoing molecular-genetic studies of ADHD, 197 families including 371 affecteds and their parents underwent phenotype assessments to assess lifetime psychopathology and to identify families with both single and multiple children affected with ADHD. Families were accepted into the multiplex ADHD study if at least two children were affected with ADHD (any subtype) based on DSM-IV criteria, and at least one member met definite criteria with the other member definite or probable ADHD. Probable ADHD was defined as falling one symptom short of diagnostic criteria (including number of behavioral symptoms, age of onset, duration, or presence in two settings), but the child still had demonstrated impairment (cf sample description). |
| Diagnosis Description |
All probands and first-degree relatives were directly interviewed and assessed using the K-SADS-PL for children and adolescents, and the SADS-LA-IV for adults. Both parent report of child symptoms and child report (for subjects 8 years or older) were obtained. The structured interview data were supplemented by teacher and parent rating scales (SNAP-IV, CBCL, TRF). All interviews were performed by experienced, research trained clinical psychologists familiar with ADHD who had completed reliability training on the instruments. Interrater reliability (kappa) exceeded 0.98 for all ADHD diagnoses, 0.95 for conduct disorder, and 0.84 for 10 other major psychiatric diagnoses (ie major depression, dysthymic disorder, etc) with greater than 5% frequency in the sample. A consensus diagnostic panel led by senior child psychiatrists (JJM, JTM) generated 'best estimate' summary DSM-IV and DSM-III-R diagnoses from structured interview and questionnaire data reviewed blind to family status. In addition, full scale IQ estimates were determined for all probands using either the WISC-III or the WAIS-R, with all children required to have an IQ estimate of 70 or greater. All children evaluated were screened by history to rule out the presence of any potentially confounding comorbid medical or genetic disorders. The mean IQ was 105 ¡À 15. The families were predominantly Caucasian (81%) and represented a range of SES classes according to the Hollingshead ranks of: I (21%), II (38%), III (27%), IV (12%), and V (2%). |
| Technique |
Blood samples were collected from family members and genomic DNA was extracted from whole blood with the Qiagen Kit using the manufacturer's protocol (Qiagen, Valencia, CA, USA). The DRD4 120-bp tandem repeat polymorphism was assayed according to methods described elsewhere with minor modifications. PCR amplication of 100 ng of genomic DNA was performed for genotyping. Ten microliters of PCR products were electrophoresed in 1.5% agarose gels in 1xTBE buffer for 1 h at 120 V. The gels were stained with ethidium bromide and alleles were determined by comparison with known molecular weight standards. The PCR reaction yields distinct bands at 429 bp (short allele) and 549 bp (long allele). All gels were scored by two readers and discrepancies clarified. |
| Analysis Method |
Using the TDTEX program from the SAGE package, the asymptotic McNemar test was used to test for linkage disequilibrium of the 120-bp repeat gene in the 197 families. Multiple affected siblings per family were included in the TDT as independent affected-parent trios. Genotype relative risk of the 120-bp repeat gene was calculated according to the method of Risch and Merikangas. IBD sharing among the subset of affected sib pairs was determined using SIBPAL. Genotype and phenotype data were entered and double-checked using doubleentry verification procedures. Linkage disequilibrium of the 120-bp and 48-bp VNTR loci was tested using the Associate program in LINKAGE. Genotype inconsistencies were identified using LOD-LINK. Univariate analyses were conducted using SAS Version 6.12. |
| Result Description |
The frequency of the allele in the overall sample is similar to that reported from analysis of a small number of subjects of mixed European origin, with the long allele (240 bp) appearing more common (81%) than the short allele (19%). As expected, the 120-bp repeat gene was in linkage disequilibrium (allelic association) with the exon 3 VNTR from the same sample. Results from the transmission disequilibrium test (TDT) in families selecting only one affected child per family revealed a significant association of the 240 allele and ADHD. Results from the TDT showed preferential transmission of the 240-bp (long) allele in the trios for probable or definite ADHD by DSM-IV criteria. The results of the TDT test are also marginally significant when applied to the sample diagnosed by the earlier DSM-III-R criteria for probable or definite ADHD. The most robust evidence for linkage with the 240-bp allele was observed when the TDT analysis was restricted to those trios formed by probands meeting DSM-IV criteria for ADHD, Inattentive subtype The results of analyses, in spite of reducing the sample, continued to show significant linkage of the 240-bp allele with both ADHD, all subtypes as well as ADHD, Inattentive subtype only. The DRD4 7-repeat continued to show greater transmission than not 7-repeats, the difference did not reach a 0.05 significance level in the larger sample. Haplotypes were analyzed by TDT, with evidence found for preferential non-transmission of the 120-bp short allele/not 7 allele haplotype. |
Hot Results
Basic Info
Detail Info