| Summary |
A genome scan was performed on 164 Dutch affected sib pairs (ASPs) with ADHD. Initially, a narrow phenotype was defined, in which all the sib pairs met the full ADHD criteria (117 ASPs). In a broad phenotype, additional sib pairs were included, in which one child had an autistic-spectrum disorder but also met the full ADHD criteria (164 ASPs). A set of 402 polymorphic microsatellite markers with an average intermarker distance of 10 cM was genotyped and analyzed using the Mapmaker/sibs program. Regions with multipoint maximum likelihood scores (MLSs) >1.5 in both phenotypes were fine mapped with additional markers. This genome scan indicated several regions of interest, two of which showed suggestive evidence for linkage. The most promising chromosome region was located at 15q, with an MLS of 3.54 under the broad phenotype definition. This region was previously implicated in reading disability and autism. In addition, MLSs of 3.04 and 2.05 were found for chromosome regions 7p and 9q in the narrow phenotype. Except for a region on chromosome 5, no overlap was found with regions mentioned in the only other independent genome scan in ADHD reported to date. |
| Total Sample |
238 children from 106 families were incluede. The sample comprised 117 ASPs with narrow phenotype, and additional 47 ASPs with a broad phenotype. |
| Sample Collection |
The sample in the present study consists of 238 children in 106 families, whose diagnoses were assigned according to DSM-IV criteria. The families were recruited from three academic child psychiatric outpatient clinics, in Utrecht (n=24), Groningen (n=25), and Amsterdam (n=3). Other families (n=54) were recruited through a patient organization and by placing advertisements in journals and on the Internet. All subjects were white and of Dutch descent. |
| Diagnosis Description |
All subjects were phenotyped according to criteria set out in the Diagnostic and Statistical Manual Of Mental Disorders, 4th edition. The children and their parents were invited to participate in extensive diagnostic evaluations that lasted ~1d. After evaluation, ASPs were included in the study if they met the following five criteria: (1) at least one member of each ASP met all criteria for the DSM-IV-defined combined, inattentive, or hyperactive/impulsive type of ADHD; (2a) the other member of the ASP met the same criteria, (2b) the other member met at least five of nine DSM-IV criteria for inattention and/or five of nine criteria for hyperactivity/impulsivity (subthreshold ADHD), or (2c) the other member met all DSM-IV cri-teria for ADHD but had been found to have an autistic-spectrum disorder, which excludes an ADHD diagnosis; (3) both members were at least 3 years old but not >18 years old; (4) for those with a history of educational problems, only children with an estimated full-scale IQ >80 on the Wechsler Intelligence Scale for Children-Revised or the Wechsler Preschool and Primary Scale of Intelligence were included in the analysis; and (5) all four grandparents were white and of Dutch descent, with the exception of two families, each with two affected siblings, in which one of the parents was not white. Relationships between siblings were verified using the program GRR. The diagnosis was verified in clinical interviews with the parents and the children. For all the patients the DSM-IV version of the Diagnostic Interview Schedule for Children (DISC) was conducted with both parents by specially trained graduate students in medicine or child psychology. The Conners and Childhood Behavior Checklist/Teacher Report Form were collected from teachers and parents. |
| Technique |
DNA was extracted from peripheral blood lymphocytes or buccal mucosa, using established procedures. Samples obtained from buccal mucosa were purified with an additional phenol extraction step. DNA concentration was measured with a spectrophotometer (Tecan), and samples were diluted with distilled water to a concentration of 15ng/ul. The Mammalian Genotyping Service of the Marshfield Medical Research Foundation performed the genotyping for the initial screen. In the second stage of the screen, chromosomal regions were fine mapped with additional microsatellite markers from the Marshfield database. Marker positions in these regions were verified using the Ensembl, Celera, and Decode human sequence databases. Either the forward or the reverse oligonucleotide primer was labeled with 6-FAM, HEX, or NED fluorescent dyes (Biolegio and Applied Biosystems). PCRs were performed. Data were analyzed using Genescan 3.6 and Genotyper 3.5 for Windows NT (Applied Biosystems). |
| Analysis Method |
Inheritance within families was verified using the Pedcheck program (O'Connell and Weeks 1998). Data were analyzed using the Mapmaker/sibs program. Maximum-likelihood-scores (MLSs) were determined in single-point and multipoint analyses and were calculated using the possible-triangle method. In multipoint analysis, MLSs were calculated at 10 intervals between two adjacent markers, with off-end ranges of 10 cM at both ends of the chromosome. In those regions with an MLS >2, data were also analyzed by means of a weighted procedure. In the analysis of the X chromosome, the separate LOD scores for sister-sister, sister-brother, and brother-brother pairs were summed. CIs (95%) under linkage peaks were determined by calculating, and determining the map position of the point obtained by subtracting 1 from the maximum. Locus-specific lambdasib values were calculated by dividing the observed ZO value at the point of the maximal MLS by the expected value of 0.25. Overall information content of the markers was obtained by calculating the average multipoint information content for all markers, including the 10-cM offb-end scales. Exclusion mapping was performed for different relative risks. |
| Result Description |
Average genotyping completeness was 97.3%, and the average marker information content across the genome was 0.72. The most promising regions on chromosomes 7, 9, 10, and 15 were fine mapped with additional markers, and genotyping in these areas was repeated for markers from the initial screen. On chromosome region 7p, the MLS was 3.04, with a 95% CI of 16 cM (26.6 Mb). The estimated sibling relative risk of this locus would be 1.19. The relative risk of a locus on chromosome 15 was estimated to be 1.60 (with 1.32 being the lower limit of of the 95% CI). Here, the MLS was 3.54, and the 95% CI was 18 cM (16 Mb). Because one-fifth of the families in sample had more than two affected siblings, the effect of weighting multiple sibships in the regions with MLS >2 were investigated. When this procedure was followed, the MLS on chromosomes 15q and 7p decreased to 2.49 and 2.27, respectively, and the MLS on chromosome 9q increased to 2.34. Exclusion mapping showed that the existence of a locus with a relative risk of 1.5 could be excluded only for 8.3% of the total genome. However, 53.9% and 87% of the genome could be excluded for loci with relative risk values of 2 and 3, respectively. |
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