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Large-scale studies

Data Summary

Published Variant
- SNP: 1391
- CNV: 398
- Others: 173
Published Gene: 359
Published Region: 128
Pathway by PBA: 8
Study: 361

Study Report

Basic Info

Reference	Tovo-Rodrigues L, 201121403674
Citation	Tovo-Rodrigues L., Rohde L. A., Roman T., Schmitz M., Polanczyk G., Zeni C., Marques F. Z., Contini V., Grevet E. H., Belmonte-de-Abreu P., Bau C. H. and Hutz M. H. (2011) "Is there a role for rare variants in DRD4 gene in the susceptibility for ADHD? Searching for an effect of allelic heterogeneity." Mol Psychiatry.
Study Design	case-control
Study Type	Candidate-gene association study and Mutational study
Sample Size	786 cases and 330 controls
Predominant Ethnicity	Caucasian
Population	Brazil
Age Group	Children/Adolescents and Adults

Detail Info

Summary	In this study, a total of 786 individuals with ADHD were genotyped for DRD4 exon 3 VNTR. All 7R homozygous subjects were selected for VNTR re-sequencing. Subjects homozygous for the 4R allele were selected paired by age, ancestry and disorder subtypes in order to have a sample as homogeneous as possible with 7R/7R individuals. Using these criteria, 103 individuals (66 with ADHD and 37 control individuals) were further investigated. An excess of rare variants were observed in the 7R alleles of ADHD patient when compared with controls (P=0.031). This difference was not observed in 4R allele. Furthermore, nucleotide changes that predict synonymous and non-synonymous substitutions were more common in the 7R sample (P=0.008 for total substitutions and P=0.043 for non-synonymous substitutions). In silico prediction of structural/functional alterations caused by these variants have also been observed. These findings suggest that not only repeat length but also DNA sequence should be assessed to better understand the role of DRD4 exon 3 VNTR in ADHD genetic susceptibility.
Total Sample	The total sample included 786 ADHD individuals and 330 non-ADHD controls.
Sample Collection	The total sample included 786 ADHD individuals. The sample was composed by 378 referred children with ADHD recruited at the Child and Adolescent Psychiatric Division of the Hospital de Clinicas de Porto Alegre (HCPA), and 308 adults with ADHD recruited at the adult clinic in the same hospital. The third source was a community sample including 100 children with ADHD inattentive type ascertained from 12 public schools. A total of 330 non-ADHD controls (230 adult blood donors and 100 children) were also included. All 7R homozygous subjects were selected for VNTR sequencing. The 4R homozygotes were selected paired by age, ancestry and disorder subtypes, in order to have a sample as homogeneous as possible with the one including the 7R/7R individuals. Using these criteria, 103 individuals (66 ADHD and 37 control individuals) were further investigated. This sample comes from a population of essentially European ancestry with little evidence of African or Native USAn admixture.
Diagnosis Description	For the first two samples, an ADHD consensus diagnosis based on Diagnostic and Statistical Manual of Mental Disorders-IV criteria was achieved through an extensive approach including both clinical interviews and application of semi-structured instruments as described in detail previously. For the third sample, an extensive diagnostic process based in Diagnostic and Statistical Manual of Mental Disorders-IV criteria and similar to the one used in the samples described above was also implemented in this sample and was fully detailed elsewhere.
Technique	DNA was extracted from whole blood by a salting out procedure. The 48-bp VNTR in DRD4 exon III was genotyped as previously described. The PCR product was purified with exonuclease I (Exo) and shrimp alkaline phosphatase treatment before sequencing as reported. The primers used for sequencing were D4-3 (5'-GCGACTACGTGGTCTACTCG-3') and D4-J3 (5'-CCTTCCCCCACGCCA-3') described by Lichter et al. When new mutations were observed or when sequences were dubious, the samples were resequenced through independent PCR products.
Analysis Method	Sequence analyses were accomplished using CodonCode Aligner v3.0.1 software (Codon Code Corporation, Dedham, MA, USA). Haplotypes were estimated using a Bayesian method implemented in PHASE2.1 software. In order to verify possible alterations in secondary structure of the loop caused by mutations, inferred variant amino acids sequences were submitted to Psipred and Phyre web servers. Detection of functional sites in the sequences was accomplished with Scansite 2.0, SH3-Hunter and ELM web servers. Allele distributions were compared using Fisher's exact test, and the comparisons of molecular sub-stitutions were performed using Kruskal-Wallis test. All tests were two-tailed and significance level was set at 0.05. The analyses were performed with the SPSS software, version 16.
Result Description	DNA sequence analyses of the 206 alleles obtained from Brazilian ADHD probands and controls identified 17 different haplotypes. At the 4R allele 6 variable sites were observed whereas 31 different sites were found at the 7R. Three new VNTR motifs were observed, all with new mutations. These repeats were called 36, 37 and 38 in accordance to the nomenclature proposed by Ding et al. The divergence among them and motif 2 consists of five nucleotide changes. Moreover, five haplotypes not previously described were also observed. New repeats were identified in three haplotypes being one in a 4R allele and two in the 7R. All of them were observed in ADHD patients. All new haplotypes were in heterozygosis with the most common haplotype. The worldwide most frequent haplotypes 4R (1-2-3-4) and 7R (1-2-6-5-2-5-4) were also the most frequent in Brazilians. These haplotypes accounted for 91 and 85% of the chromosomes investigated, respectively. Rare variants were more frequent in ADHD subjects than in controls. Neither controls nor ADHD group showed private rare variants. The 4R distribution did not differ between groups (P=0.086) while an excess of 7R rare haplotypes were observed in ADHD subjects (P=0.031). The 4R variability did not differ between ADHD cases and controls (P=0.149), but the 7R haplotype distribution was more variable in ADHD patients (P=0.01). When nucleotide changes that predict synonymous and non-synonymous substitutions were compared, it was observed that the 7R ADHD group presented more variability for both total substitutions (P=0.008) and non-synonymous substitutions (P=0.043). These differences were not observed for 4R alleles.

Other variant reported by this study (count: 1)

Variant Name	Allele Change	Risk Allele	Statistical Values	Author Comments	Result of Statistical Analysis
DRD4 exon3 VNTR	4, 7 repeat		Haplotypic distribution analysis by Fisher's exact test: P-v...... Haplotypic distribution analysis by Fisher's exact test: P-value=0.031 for 7R rare haplotypes, P-value=0.086 for 4R; variability test, P-value=0.01 for 7R haplotype distribution, P-value=0.149 for 4R; nucleotide changes test, P-value=0.008 for total substitutions of 7R ADHD group, P-value=0.043 for non-synonymous substitutions of 7R ADHD group. More...	An excess of rare variants were observed in the 7R alleles of ADHD patient when compared with controls. This difference was not observed in 4R allele. Furthermore, nucleotide changes that predict synonymous and non-synonymous substitutions were more common in the 7R sample.	Significant

Genes reported by this study (count: 1)