| Summary |
Here, they describe linkage investigations of the 48-bp VNTR in exon 3 of DRD4 and ADHD in affected sibling pair (ASP) families and singleton families using both the transmission disequilibrium test (TDT) and a mean test of identity-by-descent (IBD) sharing. Using the TDT in the total sample, the 7 allele is differentially transmitted to ADHD children (P=0.03) while the mean test revealed no evidence of increased IBD sharing among ASPs. In the current sample, the 7 allele attributes a 1.5-fold risk for developing ADHD over non-carriers of the allele estimated under a model described by Risch and Merikangas. |
| Total Sample |
One hundred and thirty-three families with ADHD were available for study: 84 families contributed two or more affected ADHD siblings while 49 families contributed a single affected ADHD child. Parental blood for genotyping was available for 163 parents in the ASP families (five parents unavailable) and 87 parents of the single cases (11 parents unavailable). As expected based on epidemiologic studies of ADHD, comorbid psychiatric disorders were common with the following distributions observed in the 220 affecteds: conduct disorder (21%), oppositional defiant disorder (42%), mood disorders (18%), and two or more anxiety disorders (19%). The mean age of the 220 affected children is 10.9 (s.d.=3.6) and full scale IQ is 103.8 (s.d.=15.5) with IQ not assessed in 25 cases. Thirty unaffected siblings (mean age 11.4, s.d. 3.2) from 24 families were evaluated using the previously described diagnostic procedures (with the exception of cognitive testing) and blood was available for genotyping. The families are largely Caucasian (80%) and distributed across Hollingshead SES classes as follows: I (17%), II (34%), III (33%), IV (14%) and V (2%). |
| Sample Collection |
Families were identified through two studies. Fifty-four families were selected from a previous family study of ADHD in which ascertainment was through an affected child-of these families, 17 had at least one ASP available for linkage analyses. An additional sample of 79 families was identified in an ongoing molecular genetic of ADHD in which ascertainment is through two affected siblings. The families are largely Caucasian (80%). |
| Diagnosis Description |
Lifetime diagnoses of ADHD using DSM-III-R and DSM-IV were made using a semi-structured interview, KSADS-E modified for DSM-IV (used in the early study) or the KSADS-PL (used in the ongoing study). ADHD symptoms were generated from interviews with the child's mother followed by a direct interview with the child if 8 years of age or older. Teacher and parent rating scales were used to supplement the direct interview and determine ADHD diagnoses. Consensus diagnoses were obtained from a review of the clinical interview and parent-teacher questionnaire information by a diagnostic panel consisting of the clinical interviewers, child psychiatrists (DC, JM), and/or psychologists (JA). A definite ADHD diagnosis was made if all criteria were met as specified under DSM-III-R or DSM-IV. A probable diagnosis was made if a subject fell one symptom short (including age of onset) but impairment criteria were met. Full scale IQ was determined for affected offspring using age appropriate standardized tests, WISCIII or the WAIS-R. |
| Technique |
Blood samples were collected from each family member and DNA was isolated using the Puregene Kit following the manufacturer's recommendations (Gentra Systems, Minneapolis, MN, USA). A polymorphic region in the DRD4 gene consisting of a variable number of 48-bp repeats was scored by PCR with primers flanking the repeat sequence. For more detailed information about PCR amplification, please refer to the original publication. |
| Analysis Method |
The asymptotic McNemar test was used to test for linkage disequilibrium of the 7-repeat allele and ADHD in the sample of 133 families using the TDTEX program from the SAGE package. The asymptotic McNemar test available in TDTEX was used to obtain an extended TDT for multiple alleles. Multiple affected siblings per family were included in the TDT as independent affected-parent trios as described by Spielman and collegues. Attributable risk of the 7-repeat allele based on TDT results was determined following Risch and Merikangas. IBD sharing among ASPs was computed using ASPEX. Transmission of marker alleles from one informative parent to a pair of affected siblings was determined and the mean number of alleles IBD from all informative parental meioses computed. The mean test comparing the observed and expected IBD sharing rates and applying the normal approximation was used, a procedure which has been shown to provide the most powerful test of IBD sharing. Genotype inconsistencies were identified using LINKAGE version 5.1. Univariate analyses were conducted using programs in SAS version 6.12. |
| Result Description |
Using the TDT in the 133 families, the 7-repeat allele was transmitted significantly more often than an alternative allele, with significance levels slightly greater than the 0.01 level recommended for a replication by Lander and Kruglyak. The attributable risk for the 7-repeat allele is 1.5 based on the observed transmitted and non-transmitted proportions. The attributable risk of the 7-repeat allele was approximately 1.5 in TDT analyses across diagnostic classifications, probability levels, sex of the ADHD child, and in the subset of families of Caucasian ancestry. The results were similar for DSM-IV defined subtypes with and without hyperactive-impulsive behaviors. There were no other alleles at this VNTR that were more often transmitted to unaffected siblings than not (multiple allele McNemar TDT, P=0.21). Eighty-nine informative meioses were available to evaluate IBD status in the ASPs. The overall rate of IBD sharing, pi=0.57, was not significantly greater than that expected by chance alone. For more information, please refer to the original publication. |
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