| Summary |
They analyzed three single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of the TPH2 gene in 103 families with 225 affected children. Allelic association in families with more than one affected child was assessed using the pedigree disequilibrium test. Preferential transmissions were detected for the two SNPs in TPH2's regulatory region, but not for the third SNP in intron 2. Haplotype analysis revealed a strong trend of association between the regulatory region SNPs and ADHD. Their results link potentially functional TPH2 variations to the pathophysiology of ADHD, and further support the relevance of 5-HT in disorders related to altered motor activity and cognitive processes. |
| Total Sample |
A total of 225 children with ADHD from 103 families were included in this study. In all 103 families, both parents were ascertained and no half-siblings were included. The vast majority of parents were of German origin, and only 12 parents came from other European countries. |
| Sample Collection |
A total of 225 children with ADHD from 103 families were ascertained and phenotypically characterized by physicians in the Departments of Child and Adolescent Psychiatry of the Universities of Aachen, Lubeck, Marburg, Wurzburg, and in the Psychiatric Clinic for Children and Adolescents of the District Oberpfalz, Regensburg. |
| Diagnosis Description |
Families were included if they had one or more children affected with ADHD to perform family-based association studies and a genome-wide scan (Hebebrand et al, submitted). The index patient was required to be older than 8 years, and other affected siblings in a family had to be older than 6 years. All children fulfilled diagnostic criteria for ADHD according to DSM-IV1 index patients were required to fulfill lifetime criteria for the combined type. Psychiatric classification was based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children Present and Lifetime version (K-SADS-PL). |
| Technique |
For genotyping of SNPs, pairs of flanking primers and allele-specific probes were designed as previously described by Mossner et al. Polymerase chain reactions (PCR) were carried out in a 25 ul volume containing 20 ng genomic DNA, 0.4 uM primers, 75mM Tris-HCl (pH 9.0), 20mM ammonium sulfate, 0.01% Tween 20, 1.5mM magnesium chloride, 0.25mM dNTP and 1U Taq polymerase. The amplification protocol consisted of 95oC for 5min, followed by 36 cycles of 95ofor 45 s, 51.9-56.3oC for 45 s and 72oC for 45 s. Genotype-based allele frequencies were determined on 116 chromosomes of a sample of unrelated subjects and were found to be 22.4% for the T allele of SNP rs4570625, 5% for the A allele of SNP rs11178997 and 31.0% for the T allele of SNP rs4565946. For detection of a rare loss-of-function Pro441Arg substitution in TPH2, a 492-bp fragment containing the exon 11 G1463A SNP was amplified by PCR using a modified protocol, and more details in the original publication. |
| Analysis Method |
They used the pedigree disequilibrium test (PDT) to test for allelic association in these families with more than one affected child and report here P-values for the avePDT statistic, which gives equal weight to all families. Analysis of LD between the investigated TPH2 polymorphisms and of association of haplotypes in these families was made with the program FAMHAP. As a correction for multiple testing when analyzing all combinations of the three SNP markers, we used the simulation approach implemented in FAMHAP,50 which results in a global P-value. |
| Result Description |
Preferential transmissions were detected for the two SNPs in TPH2's regulatory region (rs4570625, P=0.049; rs11178997, P=0.034), but not for the third SNP in intron 2 (rs4565946, P=0.3517). Haplotype analysis revealed a strong trend of association between the regulatory region SNPs (rs4570625, rs11178997) and ADHD (P=0.064). Their results link potentially functional TPH2 variations to the pathophysiology of ADHD, and further support the relevance of 5-HT in disorders related to altered motor activity and cognitive processes. |
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