| Summary |
Previous research found an association between single nucleotide polymorphisms (SNPs) in the promoter region of DRD4 and statistically derived phenotypes generated from ADHD symptoms. This study sought to replicate this finding by using the same methodology in an independent sample of ADHD individuals. Four SNPs were genotyped in and around DRD4 in 2631 individuals in 642 families. They developed a quantitative phenotype at each SNP by weighting nine inattentive and nine hyperactive-impulsive symptoms. The weights were selected tomaximize the heritability at each SNP. Once a quantitative phenotype was generated at each SNP, the screening procedure implemented in PBAT was used to select and test the five SNPs/genetic model combinations with the greatest power to detect an association for DRD4. One of the four SNPs was associated with the quantitative phenotypes generated from the ADHD symptoms. A rank ordering of the correlation between each of the ADHD symptoms and the quantitative phenotype suggested that hyperactive-impulsive symptoms were more strongly correlated with the phenotype; however, including inattentive symptoms was necessary to achieve a significant result. This study partially replicated a previous finding by identifying an association between rs7124601 and a quantitative trait generated from ADHD symptoms. The rs7124601 is in linkage disequilibrium (LD) with the SNPs identified previously. In contrast to the previous study, this finding suggests that both hyperactive-impulsive and inattentive symptoms are important in the association. |
| Total Sample |
The final IMAGE dataset had 776 DSM-IV combined type affected individuals, of which 674 were probands. DNA was available for both parents in 614 families (90%) and from one parent in 49 families (7%). To be informative in this analysis, families must have had at least two genotyped people and one individual with ADHD symptom information. |
| Sample Collection |
The sample used in this research is a part of the International Multi-Center ADHD Genetics Project (IMAGE). Briefly, European Caucasian subjects were recruited from 12 specialist clinics in 8 countries: Belgium, Germany, the Netherlands, Ireland, Israel, Spain, Switzerland, and United Kingdom. |
| Diagnosis Description |
Probands were required to have a clinical diagnosis of DSM-IV combined ADHD subtype and have at least one full sibling available for ascertainment of clinical information and DNA collection. Exclusion criteria for either the probands or their siblings included autism, epilepsy, low intelligence quotient (IQ) (<70), brain disorders, and any genetic or medical disorders associated with externalizing behaviors that might mimic ADHD. Probands were also excluded if their last medication-free period was more than 2 years ago. The clinical assessments for children were made during medication-free periods or recall back to medication-free periods. To ensure that the recall was accurate, affected individuals were required to have had a medication-free period within the last 2 years. For more details about clinical measures, please refer to the original publication. |
| Technique |
The DNA sample used in this research is a part of a larger genetic sample from the IMAGE study. Details of the DNA and SNP collection can be found elsewhere (Brookes K. et.al., 2006a). Geneservice Ltd., Cambridge, United Kingdom, performed whole genome amplification on all samples with less than 100 ug stock DNA, using the REPLI-g Kit (Quiagen Ltd., Crawley, United Kingdom). DNA samples were arrayed into 96-well plates at a concentration of 50 ng/uL and delivered to Illumina Inc. (San Diego, California) under dry ice. Four SNPs were genotyped throughout DRD4, including rs3758653, rs916457, rs752306, and rs7124601. These SNPs were selected as tag SNPs to explain the linkage disequilibrium (LD) structure throughout the gene. Each SNP was evaluated to ensure Hardy-Weinberg equilibrium. The initial study was genotyped prior to the HapMap and used Celera (Rockville, Maryland and Alameda, California) SNPs. The attempt to replicate this finding using the IMAGE sample was performed using genotypes completed on an existing Illumina array (Illumina Golden Gate Assay, Illumina Inc., San Diego, California) and therefore the replication sample was one of convenience. For these reasons, the SNPs selected for replication were not the same SNPs that were used in the initial study, which limits the extent that the initial finding can be replicated directly; however, the SNPs do cover the same haplotype block structure as the previous sample. |
| Analysis Method |
They developed a quantitative phenotype at each SNP by weighting nine inattentive and nine hyperactive-impulsive symptoms. The weights were selected tomaximize the heritability at each SNP. Once a quantitative phenotype was generated at each SNP, the screening procedure implemented in PBAT was used to select and test the five SNPs/genetic model combinations with the greatest power to detect an association for DRD4. |
| Result Description |
FBAT-PC Analysis: SNP rs7124601 achieves overall significance after adjusting for the five tests. The overtransmitted allele for rs7124601 was A. The average correlation of the hyperactive-impulsive symptoms was 0.16, while the average correlation for the inattentive symptoms was 0.02. The top three most influential variables on the phenotype were all hyperactive-impulsive variables. The most influential inattentive symptom was often forgetful in daily activities. When the FBAT-PC analysis was repeated using only the hyperactive-impulsive symptoms, the finding remained nominally significant (unadjusted P-value=0.028), whereas the finding was not significant when only the inattentive symptoms were used (unadjusted P-value=0.44). After adjusting for multiple comparisons in these cases, neither finding remained significant. |
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