| Summary |
They performed a genome-wide linkage scan for ADHD in nine patients from a genetically isolated population in the Netherlands, who were linked to each other within 10 generations through multiple lines of descent. The genome-wide scan was performed with a set of 400 microsatellite markers with an average spacing of 10-12cM. They performed multipoint parametric linkage analyses using both recessive and dominant models. This genome scan pointed to several chromosomal regions that may harbour ADHD susceptibility genes. None exceeded the empirical genome-wide significance threshold, but the Log of odds (LOD) scores were >1.5 for regions 6p22 (Heterogenetic log of odds (HLOD)=1.67) and 18q21-22 (HLOD=2.13) under a recessive model. They followed up these two regions in a larger sample of ADHD patients (n=21, 9 initial and 12 extra patients). The LOD scores did not increase after increasing the sample size (6p22 (HLOD=1.51), 18q21-22 (HLOD=1.83)). However, the LOD score on 6p22 increased to 2 when a separate analysis was performed for the inattentive type ADHD children. The linkage region on chromosome 18q overlaps with the findings of association of rs2311120 (P-value=E-05) and rs4149601 (P-value=E-04) in the genome-wide association analysis for ADHD performed by the Genetic Association Information Network consortium. Furthermore, there was an excess of regions harbouring serotonin receptors (HTR1B, HTR1E, HTR4, HTR1D, and HTR6) that showed a LOD score >1 in this genome-wide scan. |
| Total Sample |
A total of 26 patients were involved in this study. Of these 26 patients, 21 were inbred, of whom only 9 patients, who could be linked to each other within no more than 10 generations, were used in the initial analysis, and all 21 inbred patients were used in the follow-up analysis. |
| Replication Size |
21 patients including 9 initial and 12 extra patients |
| Sample Collection |
This study was conducted within the framework of the Genetic Research in Isolated populations (GRIP) programme. Approximately 150 individuals founded this population, located in the South West of the Netherlands, in the middle of the eighteenth century. The population expanded from 700 inhabitants in 1848 to more than 20 000 inhabitants at present. For this population, a genealogical database including records for more than 100 000 individuals is available. For this study, two paediatric neurologists, to whom ADHD patients are referred in GRIP, asked all of their patients diagnosed with ADHD to participate in this study . Thirty-three (67%) patients and their parents agreed to participate. Of these 33 patients, 2 were excluded from analysis because their genealogy could not be worked out, and 5 children were excluded because they did not fulfil the criteria used for the diagnosis of ADHD in this study. |
| Diagnosis Description |
The Dutch version of the National Institute of Mental Health Diagnostic Interview Schedule for Children (NIMH DISC or DISC)-IV was used to assess DSM-IV diagnoses. Psychologists and psychology students trained by the authors of the Dutch DISC-IV administered the DISCs. Parent DISCs (DISC-P) were administered during face-to-face contacts, at a community general health centre or in a children's hospital. Furthermore, lifetime ADHD symptoms were also assessed with the DISC-P. Teachers were interviewed with the ADHD section (current, not lifetime) of the teacher DISC (DISC-T) through telephone. The child version of the DISC (DISC-C) was not applied, as most of the children included in our sample were too young (<11 years of age). Children receiving treatment were withdrawn from medication for this study before the interview. |
| Technique |
Blood was drawn for all patients and their parents. DNA was extracted from peripheral leucocytes using standard procedures. They performed the genome-wide linkage scan on nine patients and their parents with a set of 400 fluorescently labelled, highly polymorphic microsatellite markers (distance between markers 10-12cM) covering the whole genome. The remaining, distantly related, 17 patients and their parents were only typed for the markers in the regions of interest on chromosomes 6 (n=12) and 18 (n=7). The genotyping experiments were carried out following the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). |
| Analysis Method |
For the nine patients used in the initial genome-wide scan, the large pedigree was divided into two smaller, analysable subpedigrees using PEDCUT software. All the markers were checked for Mendelian inconsistencies using PEDCHECK, and a second round of laboratory quality control was performed. For the genome-wide linkage analysis, they performed affected-only analyses using both dominant and the recessive models. The genome-wide significance thresholds were determined empirically by performing 1000 genome-wide simulations of their data under the null hypothesis of no linkage. Their simulations showed that an HLOD (the highest heterogenetic log of odds) score of 2.65 corresponds to a genome-wide type I error rate of 5% and that an HLOD of 1.78 corresponds to a genome-wide type I error of 50%. For detailed information of statistical analysis, please refer to the original publication. |
| Result Description |
There was no genome-wide significant evidence for linkage under either model. The highest LOD score under the dominant model was observed at 6q16 (HLOD=0.91). Homozygosity mapping yielded five genomic regions with HLOD>=1. The strongest evidence of linkage was observed at 18q21-22 (HLOD=2.13). Other regions with HLOD >=1 include 6p23 (HLOD=1.68), 6p12 (HLOD=1.07), 1p36 (HLOD=1.09), 18p11 (HLOD=1.15), and 15q25 (HLOD=1.19). The patients' haplotypes at chromosome 18 show excess of homozygosity but not at a single marker. The LOD scores did not increase by increasing the sample size, but rather decreased due to adding noninformative individuals. However, the LOD score on 6p22 increased to 2 when a separate analysis was performed for the inattentive type ADHD children. The linkage region on chromosome 18q overlaps with the findings of association of rs2311120 (P-value=10-5) and rs4149601 (P-value=10-4) in the genome-wide association analysis for ADHD performed by GAIN. |
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