| Summary |
To detect micro-deletions and micro-duplications that may have a role in the pathogenesis of ADHD, they carried out a genome-wide screen for copy number variations (CNVs) in a cohort of 99 children and adolescents with severe ADHD. Using high-resolution array comparative genomic hybridization (aCGH), a total of 17 potentially syndrome-oassociated CNVs were identified. The aberrations comprise 4 deletions and 13 duplications with approximate sizes ranging from 110 kb to 3 Mb. Two CNVs occurred de novo and nine were inherited from a parent with ADHD, whereas five are transmitted by an unaffected parent. Candidates include genes expressing acetylcholine-metabolizing butyrylcholinesterase (BCHE), contained in a de novo chromosome 3q26.1 deletion, and a brain-specific pleckstrin homology domain-containing protein (PLEKHB1), with an established function in primary sensory neurons, in two siblings carrying a 11q13.4 duplication inherited from their affected mother. Other genes potentially influencing ADHD-related psychopathology and involved in aberrations inherited from affected parents are the genes for the mitochondrial NADH dehydrogenase 1 alpha subcomplex assembly factor 2 (NDUFAF2), the brain-specific phosphodiesterase 4D isoform 6 (PDE4D6) and the neuronal glucose transporter 3 (SLC2A3). The gene encoding neuropeptide Y (NPY) was included in a ~3 Mb duplication on chromosome 7p15.2-15.3, and investigation of additional family members showed a nominally significant association of this 7p15 duplication with increased NPY plasma concentrations (empirical family-based association test, P=0.023). Lower activation of the left ventral striatum and left posterior insula during anticipation of large rewards or losses elicited by functional magnetic resonance imaging links gene dose-dependent increases in NPY to reward and emotion processing in duplication carriers. These findings implicate CNVs of behaviour-related genes in the pathogenesis of ADHD and are consistent with the notion that both frequent and rare variants influence the development of this common multifactorial syndrome. |
| Total Sample |
A cohort of children and adolescents with ADHD (n=99; 78 male, 21 female) were included in the CNV scan. Sixty-seven patients were from nuclear families with at least 2 members affected with ADHD, 8 patients were from extended multigenerational families with high density of ADHD and 24 patients had sporadic ADHD. As reference DNA for the aCGH experiments, a sex-matched pool of unscreened blood donors (n = 100, 50 females) of European ancestry and originating from the same catchment area as the patients was used. |
| Sample Collection |
Patients and their families were recruited and phenotypically characterized by a team of experienced psychiatrists in the outpatient units of the Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy and the Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, Germany, according to Diagnostic and Statistical Manual of Mental Disorders , 4th Edition (DSM-IV-TR) criteria. The study was approved by the ethics committee of the University of Wuerzburg. |
| Diagnosis Description |
Nuclear families, if they had one or more children affected with ADHD, were recruited for family-based segregation and association studies. The index patient was required to be older than 8 years and to fulfil DSM-IV criteria for the ADHD combined subtype, and other affected siblings in a family had to be older than 6 years. Exclusion criteria were the following: (1) general IQ<=80; (2) potentially confounding psychiatric diagnoses such as schizophrenia, any pervasive developmental disorder, Tourette's disorder, or primary affective or anxiety disorder; (3) neurological disorders such as epilepsy; (4) history of any acquired brain damage or evidence of fetal alcohol syndrome; (5) premature deliveries and/or (6) maternal reports of severe prenatal, perinatal or postnatal complications. Psychiatric classification was based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children Present and Lifetime version (K-SADS-PL). In addition, the Child Behaviour Checklist and a German Teachers' Report were used on ADHD symptoms according to DSM-IV. For more information, please refer to the original publication. |
| Technique |
aCGH was performed for array comparative genomic hybridization as described previously. In brief, samples of sonicated patient and pooled male or female reference DNA were labelled by random priming (BioPrime Array CGH; Invitrogen, Carlsbad, CA, USA) with Cy3 and Cy5 (Amersham Biosciences, Piscataway, NJ, USA), respectively, and hybridized to a tiling path BAC array consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center), a 1 Mb resolution BAC set (Wellcome Trust Sanger Centre) and a set of sub-telomeric clones (assembled by members of the COSTB19 Action: molecular cytogenetics of solid tumours). For the analysis and visualization of aCGH data, the software package CGHPRO was used. No background subtraction was applied, and raw data were normalized by 'Subgrid LOWESS'. For more information, please refer to the original publication. |
| Analysis Method |
The family-based association test (FBAT) was used to investigate whether the 7p15.2-15.3 duplication is associated with ADHD, sex, BMI (kg m-2), binge eating (no/yes) and NPY plasma concentrations (pmol l-) within a multigenerational pedigree comprising 20 individuals. By means of 10,000 simulations, empirical two-sided P-values were obtained. The offset parameter was set to null for residuals of BMI and NPY that were adjusted for sex and age, whereas for binge eating, an offset minimizing variance of the test statistic was chosen. The reported P-values are nominal, that is, not adjusted for multiple testing, at the significance level of 0.05. |
| Result Description |
Array comparative genomic hybridization: Using stringent criteria, a total of 11 duplications and 2 deletions were identified. For additional comparison, they indicate the number of times a similar CNV has been described in the DoGV. Finally, they detected an additional two duplications and two deletions that the authors consider potentially syndrome-associated despite the fact that they did not reach the high-stringency threshold scores because they were also observed at low frequency in one or both of the reference data sets. Among apparent candidates is the gene encoding neuropeptide Y (NPY) contained in a duplication on chromosome 7p15.2-15.3. Using aCGH, the 7p15.2-15.3 duplication was detected in several additional family members across three generations. NPY plasma concentrations were significantly higher in offsprings having inherited the 7p15 duplication than in non-carriers (empirical FBAT, P=0.023). There was a trend towards a preferable transmission of the 7p15 duplication to affected family members (empirical FBAT, P=0.138, 8 transmissions versus 3 non-transmissions) and binge eating (empirical FBAT, P=0.117, 6 transmissions versus 1 non-transmissions). However, these results did not reach an overall significance level if corrected by Bonferroni's approach. Finally, the empirical FBAT for BMI indicated no association with this trait (P=0.192). |
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