Association Between Alpha-2a-adrenergic Receptor Gene and ADHD Inattentive Type

ORIGINAL ARTICLES

Association Between Alpha-2a-adrenergic Receptor Gene and ADHD Inattentive Type Marcelo Schmitz, Daniel Denardin, Tatiana Laufer Silva, Thiago Pianca, Tatiana Roman, Mara Helena Hutz, Stephen V. Faraone, and Luis Augusto Rohde Background: Previous investigations have demonstrated that an MspI polymorphism at the adrenergic ␣2A receptor gene (ADRA2A) is associated with severity of attention-deficit/hyperactivity disorder (ADHD) inattentive symptoms in clinical samples composed mainly of subjects with ADHD, combined type. This study aimed to investigate the association between this ADRA2A polymorphism and attention-deficit/hyperactivity disorder–inattentive type (ADHD-I) in a nonreferred sample. Methods: In a case– control study, we assessed a sample of 100 children and adolescents with ADHD-I and 100 non-ADHD controls. Cases and controls were matched by gender and age and were screened by using teacher reports in a revised version of the Swanson, Nolan, and Pelham rating scale at 12 schools. Psychiatric diagnoses were derived through structured diagnostic interviews. Results: Homozygous subjects for the G allele at the ADRA2A had significantly higher odds ratio (OR) for ADHD-I than did those with other genotypes (CC ⫹ CG genotypes), even after adjusting for potential confounders (p ⫽ .02; OR ⫽ 3.78; 95% confidence interval ⫽ 1.23–11.62). In family-based analyses, no significant associations were detected. Conclusions: Our results suggest that the ADRA2A may be associated with ADHD-I, replicating previous findings from clinical samples that have suggested the importance of this gene for the dimension of inattention. In addition, these results support the role of the noradrenergic system in ADHD. Key Words: ADRA2A gene, adrenergic receptors, attention-deficit/ hyperactivity disorder–inattentive type, candidate genes, children, molecular genetics

A

ttention-deficit/hyperactivity disorder (ADHD) is one of the most common mental disorders affecting children and adolescents, with an estimated prevalence ranging from 3% to 10% (American Psychiatric Association 1994; Faraone et al 2003; Rohde et al 1999). The causes of ADHD remain unclear, but investigations involving ADHD families, twin siblings, and adopted children have described ADHD as highly heritable, suggesting a strong genetic influence (Faraone et al 2005). Although dopaminergic genes are yet the most studied in ADHD (Biederman and Faraone 2005), genes related to the noradrenergic system also have been the focus of investigation in recent studies (Bobb et al 2005; Roman et al 2002, 2006). Among several noradrenergic genes, those encoding adrenergic receptors are good candidate genes for ADHD. Animal-based studies and clinical investigations suggest that noradrenergic projections to the prefrontal cortex improve cortical functions related to ADHD, such as working memory, basically through postsynaptic ␣2 receptors (Arnsten and Li 2005; Arnsten et al 1996; Biederman and Spencer 1999; Franowicz and Arnsten 1998; Jakala et al 1999). Among the several types of ␣2 receptors in the brain, ␣2A is very promising because of its presence in many cerebral regions. Moreover, it is the most prevalent noradrenergic receptor in the prefrontal cortex (Arnsten et al 1996), and it is the site From the ADHD Outpatient Clinic (MS, DD, TLS, TP, LAR) Child and Adolescent Psychiatric Division, Hospital de Clínicas de Porto Alegre; the Department of Genetics (MHH), Federal University of Rio Grande do Sul, Porto Alegre, Brazil; the Department of Morphological Sciences (TR), Federal School of Medical Sciences of Porto Alegre, Porto Alegre, Brazil; and the Department of Psychiatry (SVF), Upstate Medical University, Syracuse, New York. Address reprint requests to Luis Augusto Rohde, Sc.D., Serviço de Psiquiatria da Infância e Adolescência, Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Rua Ramiro Barcelos, 2350, Porto Alegre, Rio Grande do Sul, Brazil 90035-003; E-mail: [email protected]. Received October 31, 2005; accepted February 23, 2006.

0006-3223/06/$32.00 doi:10.1016/j.biopsych.2006.02.035

of action of guanfacine and clonidine, drugs that are used to treat ADHD (Biederman and Faraone 2005). The ␣2A adrenoreceptor gene (ADRA2A) is located in chromosome 10q24 –26. A ⫺1291 C¡G single-nucleotide polymorphism (SNP), creating an MspI site in the promoter region of the gene, was identified by Lario et al (1997). Seven studies evaluated the association between this ADRA2A polymorphism and ADHD (Comings et al 1999; Park et al 2004; Roman et al 2003, 2006; Stevenson et al 2005; Wang et al 2006; Xu et al 2001). In our first study, we found an association between the GG genotype at ADRA2A gene and inattentive scores in a sample of 92 subjects with ADHD (Roman et al 2003). In a subsequent independent sample of children with the disorder, the association between inattentive symptoms and the GG genotype again was detected (Roman et al 2006). Similar findings also were obtained by Park et al (2004). Those investigators investigated a possible role of ADRA2A gene in ADHD by assessing three different SNPs, including the ⫺1291 C¡G SNP. A significant effect of this polymorphism was detected through quantitative TDT (QTDT) in both inattentive and hyperactive–impulsive symptom dimensions, particularly through the G allele, replicating our findings. Moreover, haplotype analyses showed significant effects of this polymorphism by either TDT or QTDT. In both cases, the G allele of ⫺1291 C¡G SNP appeared to contribute to an increased risk, especially when inattentive symptoms were considered. However, Xu et al (2001) did not find a role for this polymorphism in a family association study with ADHD probands. None of these studies investigated specifically the association of the ADRA2A gene and ADHD–inattentive type, and all used clinically referred samples. Previous studies on other psychiatric diseases consistently have suggested that clinical heterogeneity may obscure a positive finding in molecular genetic studies because it frequently is associated with etiological heterogeneity (State et al 2000). Therefore, the reduction of ADHD’s clinical heterogeneity through the selection of specific ADHD types appears to be a rationale strategy, because groups with specific biological and environmental components may be identified (Faraone et al 1998; Woo and Rey 2005) In addition, several studies have indicated differences in social, academic, and behavioral funcBIOL PSYCHIATRY 2006;60:1028 –1033 © 2006 Society of Biological Psychiatry

M. Schmitz et al tioning among the three DSM-IV types of ADHD (Eiraldi et al 1997; Gaub and Carlson 1997; Paternite et al 1996), and many investigators agree that the greatest differences can be found between the inattentive type and the other types (Brito et al 1999; Gansler et al 1998). Moreover, Woo and Rey (2005) recently reported that the inattentive type was the most common subtype of ADHD in community samples, accounting for about half of the cases. The main objective of this study was to investigate the association between ADRA2A and ADHD predominantly inattentive type (ADHD-I) in a nonclinically referred sample. On the basis of previous studies from our group that used different samples (see Roman et al 2003, 2006), we hypothesized that ADRA2A would be associated with ADHD-I.

Methods and Materials Subjects The sample for this study was obtained from 12 public schools in Porto Alegre, Brazil. These schools were selected as a result of their location near our University Hospital. The inclusion criteria for cases were as follows: (1) age between 6 and 18 years, (2) contact with the biological mother, and (3) presence of at least four inattentive symptoms and at most three hyperactivity and impulsivity symptoms as detected by use of a screening scale (SNAP-IV, a revision of the Swanson, Nolan, and Pelham [SNAP] Questionnaire; Swanson et al 2001) by the teacher who best knew the student. Each positive case selected was matched with a control (same gender and age) meeting criteria 1 and 2 just mentioned and who had at most three inattentive symptoms and three hyperactivity and impulsivity symptoms in the SNAP-IV scale that was completed by the teacher. The exclusion criteria for cases and controls were an estimated IQ lower than 70 and the diagnosis of psychosis. Screening Procedure First, a child and adolescent psychiatrist gave a lecture for teachers at each school providing information about the research project and ADHD. Research assistants were trained to approach teachers and to guide them to complete the screening instrument. These assistants contacted the teachers of the 12 schools, requesting that they identify all students with inattentive problems (potential cases) and some students without inattentive problems (potential controls). The teachers completed the screening instrument for ADHD (the SNAP-IV) for all identified students. Students with positive screening and controls (and their families) were invited to take part in the diagnostic phase of the study. The project was approved by the ethical committee of our university hospital, which is an approved institutional review board by the U.S. Office for Human Research Protections. Written informed consent was obtained from parents for the assessment of children. Children or adolescents provided verbal assent to participate in the study. Diagnostic Process The diagnoses of ADHD-I and its comorbidities were performed in our outpatient clinic through a three-stage process that was described extensively in previous investigations (Rohde 2002; Rohde et al 2005). First, an initial evaluation was performed with a semistructured interview (Schedule for Affective Disorders and Schizophrenia for School-Age Children, Epidemiological Version [K-SADS-E]; Orvaschel 1985) that was modified to assess DSM-IV criteria and was administered to the parents by trained

BIOL PSYCHIATRY 2006;60:1028 –1033 1029 research assistants. The interrater reliability for the ADHD diagnosis had been assessed elsewhere (kappa coefficient ⫽ .94; p ⬍ .001; Polanczyk et al 2003). Second, each diagnostic derived from K-SADS-E was discussed in a clinical committee that was chaired by an experienced child and adolescent psychiatrist (LAR). And third, clinical evaluation of ADHD-I and comorbid conditions was performed according to DSM-IV criteria by a child and adolescent psychiatrist who previously had received the results of the K-SADS-E and who conducted interviews with the parents and the child or adolescent. When a diagnostic disagreement occurs in the three-stage process, priority is given to diagnoses derived from clinical interviews (Rohde 2002). In addition, subjects‘ overall functioning was assessed by the CGAS (Clinical Global Assessment Scale; Shaffer et al 1983). The CGAS is a widely used measure of child and adolescent global functioning and has adequate psychometric properties (test– retest and interrater reliability and concurrent and discriminative validity; Shaffer et al 1983). Confirmed ADHD-I cases presenting, or not, comorbidity with other disorders and non-ADHD controls were included in the study. To ensure that we would be dealing with a relatively pure ADHD–inattentive type, we only included cases fulfilling DSM-IV criteria for ADHD–inattentive type, but that presented at most three symptoms of hyperactivity and impulsivity after this extensive evaluation. The inattention score of the SNAP-IV scale was used in the dimensional analyses of inattention. The SNAP-IV scale (Swanson et al 2001) has four subscales (Total Scores: 26 items, Inattention: 9 items, Hyperactivity/Impulsivity: 9 items, and Oppositional: 8 items). The SNAP-IV items are rated on a scale from 0 to 3. This measure frequently has been used in ADHD investigations, including those designed to assess clinical interventions (Swanson et al 2001). The internal consistency of the SNAP-IV varies from good to excellent (Stevens et al 1998). In a study published elsewhere, we obtained a Cronbach’s alpha coefficient of .74 for the complete scale (26 items) in a different sample (Corrêa-Filho et al 2005). The scale was completed by the subjects’ parents. The estimated IQ score was obtained from the Vocabulary and Block Design subtests of the Wechsler Intelligence Scale– Third Edition (WISC III; Wechsler 1991) administered by trained psychologists. The flow chart of the patients’ participation in the study is shown in Figure 1. Assessment of Demographic Variables and Confounders Some demographic variables (age, gender, ethnicity, and schooling) and possible confounders (alcohol, nicotine, and other drug use during pregnancy; maternal age; and infant birth weight) were assessed with the caretakers or the biological mothers through a questionnaire designed for this study. Socioeconomic status was defined by the socioeconomic scale developed by the Brazilian Association of Market Research Institutes (Associação Brasileira de Empresas de Pesquisa 2003). Comorbidity was evaluated through the extensive clinical assessments described in Diagnostic Process. Parental ADHD was assessed by a child and adolescent psychiatrist by using the ADHD module of the K-SADS-E, modified to assess DSM-IV criteria. This strategy has been used in several previous studies (Biederman et al 2004; Roman et al 2003). Genotyping High-molecular-weight genomic deoxyribonucleic acid (DNA) was extracted from whole blood by a salting-out procedure (Lahiri www.sobp.org/journal

1030 BIOL PSYCHIATRY 2006;60:1028 –1033 CASES

CONTROLS

486 STUDENTS WERE SELECTED AT THE SCHOOLS BASED ON TEACHERS’ SCORES IN THE SNAP-IV

245 STUDENTS WERE SELECTED AT THE SCHOOLS BASED ON TEACHERS’ SCORES IN THE SNAP-IV

ALL INVITED TO DIAGNOSTIC PHASE: 1) K-SADS-E APPLIED BY RESEARCH ASSISTANTS 2) ESTIMATED IQ BY TRAINED PSYCHOLOGISTS

M. Schmitz et al

151 INVITED TO DIAGNOSTIC PHASE: 13 REFUSES 331 NEGATIVE FOR ADHD-I 18 MENTAL RETARDATION 24 BIOLOGICAL MOTHER NOT AVAILABLE

1) K-SADS-E APPLIED BY RESEARCH ASSISTANTS 2) ESTIMATED IQ BY TRAINED PSYCHOLOGISTS

3) FULL CLINICAL ASSESSMENT BY A CHILD PSYCHIATRIST

3) FULL CLINICAL ASSESSMENT BY A CHILD PSYCHIATRIST

100 CASES

100 CONTROLS

37 REFUSES 12 HAD ADHD 02 HAD MENTAL RETARDATION

Figure 1. Flow chart of the patients’ participation.

and Nurnberger 1991). Primers and protocols for polymerase chain reaction amplifications of genomic DNA samples and genotyping were used and performed as previously described (Lario et al 1997; Roman et al 2003). Data Analyses Allele frequencies were estimated by counting. For familybased association analysis, we used the original TDT (i.e., a McNemar’s ␹2 test of biased transmission alleles from heterozygous parents to their affected offspring in trios only; Ewens and Spielman 1995) and the haplotype relative-risk (HRR) statistics (Terwilliger and Ott 1992). For the HRR analysis, both trios composed of father, mother, and affected child and parent– proband pairs were included. Heterozygous parent–proband pairs with the same genotype were excluded because the transmission status of parental alleles could not be determined (Curtis and Sham 1995). In the case– control approach, we used conditional logistic regression analysis (Hosmer and Lemeshow 2000) to compare genotype frequencies between probands and controls in the context of potential confounders. They were defined on the basis of conceptual analyses of the literature or by using a broad statistical definition (association with both the study factor and outcome for a p ⱕ 0.20). This approach assured very conservative analyses. Cases and controls were matched by gender and age. A significance level of 5% was accepted in all other analyses.

Results The sample consisted of 100 subjects with ADHD-I and 100 non-ADHD controls. We also genotyped 173 parents who were assessed from 73 mother, father, and affected child or adolescent trios, and 27 mother–affected child or –adolescent pairs. Subjects’ demographic and clinical characteristics are shown in Table 1. Consistent with their diagnosis, the ADHD group had more maternal diagnoses of ADHD (p ⬍ .001), lower IQ estimates (p ⫽ .002), and more oppositional defiant disorder (p ⫽ .001), generalized anxiety disorder (p ⫽ .05), and social phobia (p ⫽ .004; see Table 1). The frequencies for the ⫺1291C allele and for the ⫺1291G allele in patients, their parents, and controls were, respectively, www.sobp.org/journal

.64 and .36, .63 and .37, .69 and .31. These frequencies were similar to those reported in our previous studies for this ADRA2A polymorphism (see Roman et al 2003, 2006). The genotype frequencies (CC homozygous, CG heterozygous, and GG hoTable 1. Demographic and Clinical Characteristics of Cases With ADHD-I and Controls Characteristic Age in mo, mean (SD) Gender (Male) Ethnicity European-Brazilian African-Brazilian Schooling in y, mean (SD) SES (Middle Class) Maternal ADHD Estimate IQ in y, mean (SD) Alcohol Use in Pregnancy Birth Weight (kg) in kg, mean (SD) Maternal Age at Delivery in y, mean (SD) Comorbidities Mood Disorders Major Depression Dysthymia Anxiety Disorders Simple Phobia GAD SAD Social phobia Agoraphobia Disruptive behavior disorders ODD CD

ADHD-I (n ⫽ 100)

Controls (n ⫽ 100)

142 (39.5) 68

140 (38.5) 68

p Value .72 1 .13

62 38 4.4 (2.7) 49 28 94 (11) 9 3.32 (0.6)

73 27 4.7 (3) 56 3 99.3 (11.4) 4 3.29 (0.6)

.12 .13 ⬍.001 .002 .2 .74

27 (6.5)

27.6 (6.7)

.53

4 4

1 1

.25 .22

21 14 8 21 11

19 5 3 5 5

.69 .05 .18 .004 .1

38 2

14 1

.001 .57

Data are percentages unless otherwise noted. ADHD-I, attention-deficit/hyperactivity disorder–inattentive type; CD, conduct disorder; GAD, generalized anxiety disorder; IQ, intelligence quotient; ODD, oppositional defiant disorder; SAD, separation anxiety disorder; SES, socioeconomic status.

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M. Schmitz et al Table 2. Odds Ratios (OR) for ADHD-I According to ADRA-2A Genotype, Adjusted for Potential Confounders

Alcohol Use During Pregnancy Social Phobia Maternal ADHD G-Allele Homozygosity

Wald ␹2

p Value

OR

95% CI

.16 8.97 11.52 5.39

.687 .003 .001 .02

1.32 10.3 17 3.78

.34–5.16 2.24–47.38 3.31–87.32 1.23–11.62

ADHD-I, attention-deficit/hyperactivity disorder–predominantly inattentive subtype; CI, confidence interval.

mozygous) were respectively .45, .37, and .18 in probands; .42, .41, and .17 in their parents; and .47, .45, and .08 in controls. These frequencies were under Hardy-Weinberg equilibrium. Only unrelated European-Brazilian subjects were included in these analyses. In the conditional logistic regression analysis, children homozygous for the G allele at the ADRA2A had significantly higher odds ratios (ORs) for ADHD-I than did those with other genotypes (CC ⫹ CG genotypes; p ⫽ .02; OR ⫽ 3.78; 95% confidence interval [CI] ⫽ 1.23–11.62), even after adjusting for potential confounders (maternal ADHD, comorbidity with social phobia, and maternal use of alcohol during pregnancy; these were variables associated with both study factor and outcome for a p ⱕ 0.20; Table 2). Because population stratification might bias case– control approach, we also assessed the effects of the GG genotype controlling for ethnicity (p ⫽ .046; OR ⫽ 3.15; 95% CI ⫽ 1.02–9.74). Because the simultaneous inclusion in the model of both maternal ADHD and genotype at ADRA2A might be interpreted as overcontrolling because of the fact that both variables have the potential to measure the same condition (genetic effect), we also performed analyses that excluded maternal ADHD. The effect of the GG genotype still was significant (p ⫽ .02; OR ⫽ 2.92; 95% CI ⫽ 1.17–7.31). Finally, because the conceptual analyses of the literature documented effects of both smoking during pregnancy and birth weight as potential risk factors for ADHD (Ben Amor et al 2005; Huizink and Mulder 2006), we decided to include these variables in the first model of the conditional logistic regression analysis. Findings in this new model were similar, and homozygosity for the G allele also was associated with a significant OR for ADHD-I (p ⫽ .042; OR ⫽ 3.23; 95% CI ⫽ 1.04 –9.98). We obtained data on allele transmission for HRR analysis from 97 families (74 trios and 23 dyads). No significant association between the ADRA2A ⫺1291 C¡G variant and ADHD-I was detected, because no allele was preferentially transmitted (G allele: transmitted ⫽ 71, nontransmitted ⫽ 73; C allele: transmitted ⫽ 99; nontransmitted ⫽ 97; ␹2 ⫽ .01; p ⫽ .91). As expected, similar findings were obtained through TDT analyses of the ADHD diagnosis or symptom dimensions (available upon request).

Discussion In a nonreferred sample of Brazilian children and their families, we found that homozygosity for the G allele at the ADRA2A ⫺1291C¡G polymorphism increased the risk for ADHD-I compared with other genotypes, even after adjusting for potential confounders in a case– control approach. To the best of our knowledge, this is the first study showing such an association. No association was observed through family-based methods.

Our findings indicating an association between ADHD-I and ADRA2A concur with other studies from our group using dimensional analysis that have suggested a significant role for this SNP in symptoms of inattention (Roman et al 2003, 2006). In our first attempt to investigate the ADRA2A gene in a Brazilian sample of ADHD patients, the ⫺1291 C¡G SNP was analyzed in 92 subjects and their biological parents (Roman et al 2003). Mean scores at both inattentive and combined (inattentive ⫹ hyperactive and impulsive) symptoms were significantly higher in the probands with the GG genotype than in probands with other genotypes. In another sample of 128 probands from our ADHD outpatient clinic, the association with inattentive symptoms again was detected, because the mean SNAP-IV scores in this dimension were significantly higher in probands with GG genotype than in probands with other genotypes (p ⫽ .017; Roman et al 2006). Overall, our findings appear to indicate that at least in our population, this gene is related to inattentive scores of ADHD. In addition, our results corroborate to studies showing that inattention and hyperactivity–impulsivity are related, but separated, symptom domains (Hudziak et al 1998; Rohde et al 2001). Therefore, it is possible to speculate that different genetic components might be related to each of these symptom dimensions. Considering these findings, it is fundamental to understand the potential functional significance of the ADRA2A ⫺1291C¡G polymorphism. Belfer et al (2005) recently reported that a single haplotype block spanned ADRA2A gene. This haplotype block is composed by 9 different SNPs that distribute from the 5= end to the 3= end of ADRA2A locus, including the ⫺1291C¡G and a nonsynonymous amino acid change in position 251, known to be of functional relevance for ␣2A adrenoreceptor. As noted by those investigators, this ADRA2A haplotype block was sufficient to capture the information content even when the functional locus was not included. Thus, the association of the ADRA2A gene with ADHD detected herein could be a result of another functional SNP either in the coding or regulatory regions, in linkage disequilibrium with the ⫺1291C¡G SNP. We found no association between the ␣2A gene and ADHD, inattentive type, by using family-based approaches. No preferential transmission of the G allele was detected in the HRR or TDT analyses. However, the negative family-based results could be explained by the probable small effect of this gene in the disorder, an effect that would be difficult to detect in association studies with small to moderate samples (Crowe 1993). Therefore, the use of different approaches, such as the case– control study, would be more effective in detecting small genetic effects. Moreover, Bobb et al (2005) observed in a review on molecular genetic studies that 46% of the 26 studies using family-based and case– control approaches on the same population and polymorphism found divergent results, indicating that both methods should be used to prevent the possibility of type II error. The strengths of this study include the following. The sample is comprised of nonreferred subjects, making it more representative of the population. In addition, the sample includes only subjects with ADHD–predominantly inattentive subtype. This is a step forward in the ADHD research field because of the shortage of studies specifically focused on this subtype. Furthermore, it is important to highlight that the diagnoses in our study were obtained through an extensive clinic evaluation that was performed by a child and adolescent psychiatrist, instead of being derived from scores of self-reported scales of symptoms, which is a limitation in some studies. Also, only subjects with at most three symptoms of hyperactivity were included, so the sample includes subjects with relatively pure ADHD–inattentive subtype (e.g., www.sobp.org/journal

1032 BIOL PSYCHIATRY 2006;60:1028 –1033 samples including subjects with six or more inattentive symptoms and four or five hyperactive and impulsive symptoms might have higher chances of including subthreshold ADHD-combined cases instead of real inattentive cases). Finally, the results of this study were presented in the context of conservative statistical analyses, because a broader criterion for the inclusion of variables as potential confounders was considered, and the main confounders described in the literature were included in the analyses. The limitations of this study also should be considered. First, case– control analyses always have the potential for populational stratification. However, the results still are significant, even adjusting for ethnicity in conditional logistic regression analyses (see Results). Second, we assessed a relatively small sample size, especially for family-based approaches. However, as far as we are aware, this study included the largest sample of clinically assessed nonreferred subjects with specific ADHD-I. In summary, our findings in a nonreferred sample provide evidence that the ADRA2A gene is associated with ADHD– inattentive type, supporting the relevance of the noradrenergic system in the pathophysiology of ADHD, especially in the dimension of inattention. As pointed out by Talmud and Humphries (2001), it is important to note that the best way to reduce the possibility of spurious associations is to replicate findings in independent samples, restricted to only those traits found initially with statistical significance. In this regard, this was the third replication of the association between this SNP at ADRA2A and inattention in independent samples from our population. The search for functionality of this SNP, as well as of other polymorphisms at the ADRA2A, is warranted to further clarify the putative effect of this gene on ADHD and its inattentive dimension. This work was partially supported by research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil; Grant No. 307780/2004-0), Programa de Apoio a Núcleos de Excelência (PRONEX, Brazil), and Hospital de Clínicas de Porto Alegre and by an unrestricted grant from Eli Lilly. The ADHD outpatient program receives research support from the following pharmaceutical companies: Bristol-Myers Squibb, Eli-Lilly, Janssen-Cilag, and Novartis. LAR is on the speakers’ bureau or is a consultant for the same companies. MS is on the speakers’ bureau of Novartis and Janssen-Cilag. SF receives research support from the following sources: McNeil Consumer & Specialty Pharmaceuticals, Shire Laboratories, Eli Lilly & Company, NIMIH, NICHD, and the National Institute of Neurological Diseases and Stroke. SVF is a speaker on the following speakers’ bureaus: Eli Lilly & Company, McNeil Consumer & Specialty Pharmaceuticals, and Shire Laboratories. Also, he has had an advisory or consulting relationship with the following pharmaceutical companies: McNeil Consumer & Specialty Pharmaceuticals, Noven Pharmaceuticals, Shire Laboratories, and Eli Lilly & Company. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, 4th ed. Washington, DC: American Psychiatric Press, 1994. Arnsten AF, Li BM (2005): Neurobiology of executive functions: Catecholamine influences on prefrontal cortical functions. Biol Psychiatry 1;57: 1377–1384. Arnsten AF, Steere JC, Hunt RD (1996): The contribution of alpha 2-noradrenergic mechanisms of prefrontal cortical cognitive function. Potential significance for attention-deficit hyperactivity disorder. Arch Gen Psychiatry 53:448 – 455. Associação Brasileira de Empresas de Pesquisa (2003): Critério de classificação econômica Brasil. Available at: http://www.anep.org.br/ codigosguias/CCEB.pdf. Accessed April 18, 2005.

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