| Summary |
The aim of this study was to test for association between the DBH gene and ADHD in a sample of 88 Brazilian nuclear families. Haplotype relative risk (HRR) analysis of the DBH TaqI restriction site polymorphism showed a preferential transmission of the TaqI A2 allele in our whole ADHD sample (P=0.03). The significant effect of the A2 allele was stronger when only families with no ADHD parental diagnosis were considered (P=0.01). The results suggest a contribution of this gene to ADHD susceptibility, partially replicating previous findings that have demonstrated an association between the DBH TaqI A2 allele and ADHD. |
| Total Sample |
They genotyped 88 ADHD probands and 155 parents assessed from 67 mother, father, and affected child trios, and 21 parent/child pairs. Whereas most of the probands presented DSM-IV ADHD combined type (77%), 16% of the probands were of the inattentive type, and 7% of the hyperactive/impulsive type of the disorder. Eighty-six percent of the subjects were from European descent. Only 25% of the probands did not meet diagnostic criteria for other psychiatric disorders. The most common comorbid condition was oppositional defiant disorder (41%), but anxiety disorders (15%), conduct disorder (14%), and bipolar disorder (8%) also co-occurred with ADHD. The mean IQ in the subjects was 91. Forty-one percent of the families had ADHD parental diagnosis. |
| Sample Collection |
Patients were drawn from the ADHD outpatient clinic at the Child and Adolescent Psychiatric Division of Hospital de Clinicas de Porto Alegre (HCPA). This investigation was approved by the Ethical Committee of HCPA and by the Coordinating Committee of the Graduate Program in Genetics and Molecular Biology of the Federal University of Rio Grande do Sul. Parents provided written informed consent and probands provided verbal assent to participate. |
| Diagnosis Description |
A consensus diagnosis of ADHD, based on the fourth edition of the Diagnostic and Statistical Manual, revised (DSM-IV) [USAn Psychiatric Association, 1994] criteria, was achieved through a three-stage process, described in detail previously [Roman et al., 2001]. Briefly, it comprised: a) evaluation with a semistructured interview (Schedule for Affective Disorders and Schizophrenia for School-Age Children, Epidemiological Version-K-SADS-E) modified to assess DSM-IV criteria and applied to the parents by trained research assistants; b) discussion of each diagnosis derived through the K-SADS-E in a clinical committee chaired by SL.A.R.; and c) clinical evaluation of ADHD and comorbid conditions using DSM-IV criteria by a child psychiatrist. In addition to this diagnostic procedure, parents completed the Child Behavior Checklist (CBCL), a behavior symptom checklist that assesses children's behavioral problems and social competencies. For probands who were attending school, teachers completed the Attention Problems scale of the CBCL-Teacher Report Form (TRF), which includes items related to ADHD in the classroom. Cognitive evaluation based on vocabulary and block design subtests of the Weschler Intelligence Scale-third edition (WISC-III) was applied by a trained psychologist to estimate the probands overall IQ. Information about socio-demographic data was systematically collected from the parents. The parents were also evaluated for past (childhood) or present diagnosis of ADHD by a child psychiatrist, using the ADHD module from the K-SADS-E, modified to assess DSM-IV criteria. Familiality was defined as the presence of at least one parent with a positive diagnosis of ADHD. Parents who fulfilled all criteria except age-of-onset of impairment criterion were included in the parental ADHD group to increase its sample size. |
| Technique |
High molecular weight genomic DNA was extracted from whole blood by a salting out procedure [Miller et al., 1988]. The TaqI polymorphism in intron 5 of the DBH gene was amplified by the polymerase chain reaction (PCR) using the primers and conditions described by Daly et al. [1999]. The 464 base pair (bp) PCR fragments were digested with TaqI, and the digested products were separated using 2% agarose gels. TaqI identified a bi-allelic polymorphism with an undigested band of 464 bp (allele A1) and two bands of 300 bp and 164 bp (allele A2). A 100 bp ladder was used to score the alleles. The allele terminology 'A1' (TaqI absent) and 'A2' (TaqI present) were used. |
| Analysis Method |
Allele frequencies were estimated by counting, and the Hardy-Weinberg equilibrium was calculated based on these allele frequencies by the goodness-of-fit Chi-square test. The haplotype relative risk (HRR) statistics was performed for family-based association analysis. This method was chosen to avoid any potential effect of population stratification. Both trios composed by father, mother, and affected child and parent/proband pairs were included in the HRR analyses. Heterozygote parent/proband pairs with the same genotype were excluded because the transmission status of parental alleles could not be determined. A one-tailed significance level of 5% was used for each test. |
| Result Description |
The transmissions for the HRR tests could be determined unambiguously in 82 families. The HRR analysis in the whole sample confirmed the association between the DBH gene and ADHD. The A2 allele was significantly more transmitted than not transmitted(P=0.03). Preferential transmission was also observed when the inattentive and the hyperactive/impulsive type probands were excluded from this analysis (P=0.03). In an attempt to further replicate the findings from Daly et al. [1999], sub-sets of probands defined by the presence and the absence of familial history of ADHD were also evaluated. No preferential transmission was observed in the families with ADHD parental diagnosis (P=0.42). However, the significant association with the A2 allele was maintained in families where there was no familial history of the disorder (P=0.01). |
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