Association of the dopamine transporter gene and ADHD symptoms in a Canadian population-based sample of same-age twins.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:1442 –1449 (2008) Association of the Dopamine Transporter Gene and ADHD Symptoms in a Canadian Population-Based Sample of Same-Age Twins Isabelle Ouellet-Morin,1,2 Karen G. Wigg,1 Yu Feng,1 Ginette Dionne,2 Philippe Robaey,3 Mara Brendgen,4 Frank Vitaro,5 Louise Simard,6 Russell Schachar,7 Richard E. Tremblay,8 Daniel Pérusse,9 Michel Boivin,2 and Cathy L. Barr1,7* 1 Genetics and Development Division, Toronto Western Research Institute, University Health Network, Toronto, Canada School of Psychology, Universite´ Laval, Que´bec, Canada 3 Department of Psychiatry, Universite´ de Montréal, Montréal, Canada 4 Department of Psychology, Universite´ du Que´bec à Montréal, Montréal, Canada 5 School of Psychoeducation, Universite´ de Montre´al, Montre´al, Canada 6 Faculty of Medicine, University of Manitoba, Winnipeg, Canada 7 Department of Psychiatry, Brain and Behaviour Program, The Hospital for Sick Children, Toronto, Canada 8 Department of Psychology, Universite´ de Montre´al, Montre´al, Canada 9 Departments of Anthropology and Psychiatry, Universite´ de Montréal, Montréal, Canada 2 Attention deficit hyperactivity disorder (ADHD) is the most prevalent psychiatric disorder emerging during childhood. Psychostimulant medications (e.g., methylphenidate) noticeably reduce ADHD symptoms in most children. Since methylphenidate inhibits dopamine transporter activity, the dopamine transporter gene (DAT1) was considered to be the prime candidate risk gene in ADHD. Several studies found evidence for an association between the 10-repeat allele of the variable number of tandem repeat (VNTR) located in the 30 untranslated region and ADHD and/or ADHD symptoms in clinical and population-based samples. However, this finding was not replicated in all samples. In this study, we investigated the association between the DAT1 gene and ADHD symptoms in a population-based twin sample from Québec (Canada). We used two polymorphisms, the VNTR and rs27072, the last providing the most significant results in a clinical sample from Toronto (Ontario, Canada). No association was noted between the VNTR and ADHD symptoms in children at 6 and 7 years of age, as reported by teachers. However, a significant association was found for the rs27072 polymorphism and symptoms of inattention and hyperactivity/ impulsivity. These findings indicate that the DAT1 gene contributes to ADHD symptoms in this sample and further suggest that the VNTR may not be the optimal polymorphism for study in all populations. ß 2007 Wiley-Liss, Inc. KEY WORDS: ADHD; genetics; dopamine transporter gene; association; twins Grant sponsor: Canadian Institutes of Health Research; Grant number: NET-54016. *Correspondence to: Cathy L. Barr, Ph.D., The Toronto Hospital Western Division, 399 Bathurst Street, MP14-302, Toronto, Ontario, Canada M5T 1S8. E-mail: email@example.com Received 25 October 2006; Accepted 23 October 2007 DOI 10.1002/ajmg.b.30677 Published online 28 December 2007 in Wiley InterScience (www.interscience.wiley.com) ß 2007 Wiley-Liss, Inc. Please cite this article as follows: Ouellet-Morin I, Wigg KG, Feng Y, Dionne G, Robaey P, Brendgen M, Vitaro F, Simard L, Schachar R, Tremblay RE, Pérusse D, Boivin M, Barr CL. 2008. Association of the Dopamine Transporter Gene and ADHD Symptoms in a Canadian Population-Based Sample of Same-Age Twins. Am J Med Genet Part B 147B:1442–1449. INTRODUCTION Attention deficit hyperactivity disorder (ADHD) is the most prevalent psychiatric disorder in childhood, affecting 8–12% of children worldwide [Faraone et al., 2005]. ADHD is defined as developmentally inappropriate levels of inattention, hyperactivity, and impulsivity emerging before 7 years of age and associated with a variety of negative outcomes, including academic impairments (e.g., poor performance or underachievement) and socio-emotional problems. Twin studies have repeatedly shown a strong genetic component to ADHD symptoms [Biederman and Faraone, 2005]. Willcutt [Willcutt, 2005] estimated the genetic contribution to be 0.73, based on twelve independent twin studies, while the remaining phenotypic variance (0.27) was attributed to non-shared environmental factors. Genetic and environmental contributions have been shown to be similar in continuous and dichotomized ADHD measures [Levy et al., 1997]. As a result, ADHD is considered to represent the extreme phenotypic manifestation of naturally occurring variation in inattentive and hyperactive/impulsive behaviors in the population. Furthermore, twin studies of DSM-IV symptom dimensions identified common genetic factors contributing to both the inattention and hyperactive/impulsive dimensions and specific genetic contributions, indicating that risk genes may contribute to both or either of the dimensions [Levy et al., 2001]. Psychostimulant medications (e.g., methylphenidate and dexamphetamine) have been shown to generate symptomatic improvement in up to 70% of ADHD children [Elia et al., 1999; Wigal et al., 1999; James et al., 2001]. The exact mechanism by which symptoms are improved is not clear; however, it is known from functional neuroimaging studies that methylphenidate reduces dopamine transporter availability and binding sites [Dresel et al., 2000; Krause et al., 2000], thereby increasing dopamine availability in synaptic areas [Cragg and Rice, 2004; Levy et al., 2006]. Since psychostimulant Association Between DAT1 and ADHD Symptoms medications have been shown to alter dopamine transporter regulation and contribute to ADHD symptom relief, the dopamine transporter gene (DAT1) was considered to be the primary candidate risk gene in ADHD [Cook et al., 1995]. The hypothesized role for DAT1 in ADHD also came from studies of DAT1 knock-out mice who displayed, in comparison to normal littermates, a wide range of phenotypic differences, including hyperactivity, cognitive and motor deficits, and calming responses to psychostimulant medication [Gainetdinov et al., 1999; Miller et al., 1999; Madras et al., 2005]. The DAT1 gene (SLC6A3), located on chromosome 5p15.3, contains an 40 bp variable number of tandem repeats (VNTR) polymorphism in the 30 -untranslated region (UTR) [Vandenbergh et al., 1992] that has been indicated to contribute to dopamine transporter transcription in some studies but not all [Fuke et al., 2001; Miller and Madras, 2002]. Cook et al.  first reported an association between the 10-repeat allele of the VNTR and ADHD and DSM-III-R attention deficit disorder. Subsequent studies have replicated this association [Gill et al., 1997; Waldman et al., 1998; Daly et al., 1999; Curran et al., 2001; Chen et al., 2003; Hawi et al., 2003; Galili-Weisstub et al., 2005; Todd et al., 2005; Mill et al., 2005b; Brookes et al., 2006; Lim et al., 2006], whereas others have not [Barr et al., 2001; Curran et al., 2001; Payton et al., 2001; Todd et al., 2001a; Muglia et al., 2002; Feng et al., 2005; Kim et al., 2005; Langley et al., 2005; Cheuk et al., 2006]. A meta-analysis conducted by Purper-Ouakil et al.  aggregated the data from 11 studies and found a non-significant association between the 10repeat allele of the VNTR and ADHD when the substantial sample heterogeneity was accounted for. The estimate of the contribution of this gene to ADHD (odds ratio of 1.13; 95% confidence interval, 0.94–1.30) is based on transmission of VNTR alleles and may be underestimated if this particular polymorphism is not the functional risk allele. Inconsistent results are also noted in studies investigating the role of the 10repeat allele of the VNTR to ADHD symptom improvement in children treated with psychostimulant medications. Some support of this role was observed in samples predominantly composed of American [Stein et al., 2005] and Irish Caucasian children [Kirley et al., 2003], whereas conflicting findings were reported in Brazilian [Roman et al., 2001], African-American [Winsberg and Comings, 1999] and Korean [Cheon et al., 2005] samples. Few studies have examined the association between the 10-repeat allele of the DAT1 VNTR and ADHD symptoms in population-based samples [Payton et al., 2001; Todd et al., 2001b; Mill et al., 2005b]. Conflicting results are again noted; one study reported a significant contribution of the VNTR when ADHD symptoms were measured as a continuum [Mill et al., 2005b]. A second study found a significantly higher frequency of the 10-repeat allele in 58 children selected from the population with ADHD scores in the 90th percentile as measured by the SWAN scale compared to 68 children who scored below the 10th percentile [Cornish et al., 2005]. Two other studies did not find evidence for biased transmission in dichotomized ADHD-related phenotypes with the 10-repeat allele [Payton et al., 2001; Todd et al., 2001a]. Altogether, conflicting findings have been reported between the DAT1 VNTR polymorphism and ADHD-related phenotypes. Some inconsistencies may have arisen because ADHD and/or ADHD symptoms were assessed differently across studies. ADHD has been evaluated using either DSM symptoms-based presence/absence of the diagnosis [Gill et al., 1997; Barr et al., 2001], continuous measures [Payton et al., 2001; Mill et al., 2005b], or dimensional approaches (e.g., latent class analyses, symptom severity, subtypes) [Waldman et al., 1998; Todd et al., 2005]. The inconsistent findings and large sample heterogeneity reported by Purper-Ouakil et al.  may also be due to the important variability regarding the age of 1443 participants both within and between the samples. Twin studies have shown that latent genetic factors contribute to both age-specific and stability of ADHD symptoms during childhood [Hay et al., 2004]. Data from postmortem studies in humans indicates age-related changes in expression of the dopamine transporter with peak expression occurring at 9–10 years of age [Meng et al., 1999; Haycock et al., 2003]; thus, the contribution of this gene to symptoms may also change over time. Furthermore, changes in DAT1 gene expression may also be involved in the observed decrease in ADHD symptoms during young adulthood. Investigating the association between the DAT1 VNTR polymorphisms and ADHD symptoms prospectively in an age-homogeneous sample could help to clarify the role of this gene during different periods of development. Finally, it is noteworthy that the majority of studies have focused on this single 30 -UTR VNTR because it was the first polymorphism identified for this gene and later because there was evidence that it was functional. However, other DAT1 polymorphisms or haplotypes have also shown evidence for association [Barr et al., 2001; Hawi et al., 2003; Galili-Weisstub et al., 2005]. Feng et al.  have shown that the rs27072 polymorphism, recognized by a MspI restriction site located 422 base pairs upstream of the VNTR, was significantly associated with ADHD in a clinical sample from Toronto while the VNTR itself was not. This study raised the possibility that conflicting results could arise if the VNTR is not the functional variant but, rather, in linkage disequilibrium (LD) with it. Inconsistent findings would occur if the degree of LD between the VNTR and the as yet unidentified functional variant differed across populations, resulting in significant associations in some samples and not in others. Consequently, the DAT1 VNTR may not be the optimal polymorphism in all samples. In this study, we sought to determine the relationship of the DAT1 gene to inattention and hyperactive/impulsive symptoms in a population-based sample of twins evaluated at 6 and again at 7 years of age. Investigating the association between the DAT1 gene and ADHD symptoms in this sample offered several advantages. First, ADHD symptoms were evaluated when children were 6 and 7 years of age. Second, teachers assessed ADHD symptoms independently when the twins were attending kindergarten and the first grade of primary school. Twin studies have shown that teachers are less prone than mothers to have contrast effect types of rater bias [Sherman et al., 1997; Simonoff et al., 1998], possibly because they may base their judgments on a larger reference group of children. Moreover, the propensity for teachers to confuse the twins was greatly diminished in this study since most twins were attending separate classes in kindergarten (70%) and first grade (76%). Finally, the age homogenous sample allows us to investigate the contribution of this gene to symptoms at a distinct developmental stage, and thus remove one of the potential confounds of previous studies. MATERIALS AND METHODS Participants Participants of this study were part of the Quebec Newborn Twin Study (QNTS), a longitudinal population-based study of twins born in the greater Montreal area. Families were recruited between April 1995 and December 1998. Twins were first seen at 5 months of age and then prospectively assessed at 18, 30, 48, 60, 72, 84, and 100 months to gather data on a variety of child- and family-related characteristics. This study focuses on data collected at 72 and 84 months when children were 6 and 7 years old. Zygosity was assessed at 5 and 18 months using a shortened version the Zygosity Questionnaire for Young Twins [Goldsmith, 1991]. This questionnaire allows independent 1444 Ouellet-Morin et al. raters to aggregate their evaluation of the zygosity status through the assessment of the twins’ physical similarity. DNA was extracted for 31.3% of same-sex twin pairs selected randomly. Zygosity status was determined using 8–10 highly polymorphic micro-satellite markers. Both methods showed a high concordance rate at 5 months (91.9%) and at 18 months (93.8%) (reaching about 97%, when chorionicity was taken into account, as shown in Forget-Dubois et al. ). Genotypes generated in this study were in complete agreement with the zygosity status determined previously. The institutional review board at Sainte-Justine Hospital Research Centre approved the protocol. Written informed consent and assent from parents and children was obtained for DNA and data collections. ADHD symptoms were available for 789 twins at 6 years of age and 838 twins at 7 years of age (665 were assessed at both time collection). DNA was collected, genotyped, and analyzed for the DAT1 VNTR in 458 and 514 twins (at 6 and 7 years old, respectively) and rs27072 polymorphisms in 461 and 579 twins (at 6 and 7 years old, respectively). Note that only families for whom at least one parent and one child were genotyped were retained for the analyses. No phenotypic mean differences were noted between twins genotyped for the DAT1 VNTR [t(787) ¼ 0.71, P ¼ 0.48 and, t(836) ¼ 1.03, P ¼ .30 at 6 and 7 years old, respectively] and rs27072 polymorphisms [t(787) ¼ 0.54, P ¼ 0.59 and, t(836) ¼ 1.17, P ¼ .24 at 6 and 7 years old, respectively] and twins who were not. The total number of genotyped families and their distribution according to parent-child composition, zygosity status, and gender are summarized in Table I for each polymorphism and time of data collection. Of the twins that were genotyped for at least one marker, 88.3% were Caucasian, 1.9% were Asian, 1.1% were Black, 0.6% were of mixed ethnicity [Caucasian-Native North American (1 twin pair) and Caucasian-Black (1 twin pair)] and 2.2% were grouped in the ‘‘other ethnicity’’ category. The remaining families (5.9%) did not provide ethnicity information. Most of the participants (76.8%) had French-Canadian ancestors. grade teachers rated the children’s level of inattention and hyperactivity/impulsivity using eight items adapted from the Child Social Behavior Questionnaire (CBSQ) [Tremblay et al., 1987], itself derived from the Preschool Behavior Questionnaire (PSD) [Behar and Stringfield, 1974]. These items chosen to quantify ADHD symptoms in this population-based sample were not intended for use for a clinical diagnosis of ADHD. For instance, teachers reported to what extent a child ‘‘is easily distracted, has difficulty to pursue an activity,’’ ‘‘is impulsive, acts before thinking,’’ ‘‘has difficulty awaiting turn in games.’’ The same items were used at both collection times. All items were assessed on a three point Likert-type scale (0 ¼ never, 1 ¼ sometimes, and 2 ¼ often). This instrument has good criterion validity and high interrater and test–retest reliabilities in both population-based and clinical samples [Behar and Stringfield, 1974]. This scale yields high internal consistency in 6 and 7 year olds (a ¼ 0.85 and 0.91, respectively). ADHD symptoms were reasonably normally distributed at 6 (kurtosis ¼ 0.48, skewness ¼ 0.74) and 7 years of age (kurtosis ¼ 0.17, skewness ¼ 0.79) [Tabachnick and Fidell, 2001]. Isolation of DNA and Markers Typing Standard high salt extraction methods were used to isolate DNA from blood lymphocytes [Miller et al., 1988]. The DAT1 VNTR was genotyped as previously described [Vandenbergh et al., 1992]. The DAT1 rs27072 marker was genotyped according to Ueno et al. . Briefly, the 217-bp fragment was amplified at an annealing temperature of 608C using the primers 30 MspF: 50 -ccg tgt ctt gtg ttg ctg ta-30 and 30 MspR: 50 0 acg ggg att ctc agc agg tg 3 and PCR products subsequently digested with Msp1 for 2 hr. Restriction fragments (MspI polymorphism) and PCR products (VNTR) were electrophoresed on 3% agarose gels. Two investigators, blind to the zygosity status of the twins, scored the genotypes independently. Ambiguous results were reamplified. Samples that continued to amplify poorly were eliminated from the study, resulting in different numbers of twins genotyped for the VNTR and rs27072 polymorphisms. Procedure and Measures Teacher ratings of ADHD scores. To ensure that the teacher had sufficient time to know the children, data collections took place in the spring. Kindergarten and first Statistical Analyses The associations between DAT1 markers and the ADHD scores were tested using the quantitative transmission TABLE I. Characteristics of Children Rated for ADHD Symptoms and Genotyped for the DAT1 VNTR and rs27072 Polymorphisms Zygosity status (%) Gender (%) Data collection DAT1 polymorphisms Family composition Number of families MZ DZ Male Female 6 years of age VNTR 2 parents–2 children 2 parents–1 child 1 parent–2 children 1 parent–1 child Total 2 parents–2 children 2 parents–1 child 1 parent–2 children 1 parent–1 child Total 2 parents–2 children 2 parents–1 child 1 parent–2 children 1 parent–1 child Total 2 parents–2 children 2 parents–1 child 1 parent–2 children 1 parent–1 child Total 163 31 44 13 251 168 29 40 16 253 174 31 61 13 279 180 29 57 16 282 41 39 27 46 39 40 46 28 44 39 43 43 30 61 41 42 50 30 56 40 59 61 73 54 61 60 54 72 56 61 57 57 70 39 59 58 50 70 44 60 52 39 61 46 51 51 41 65 44 52 51 32 64 61 52 51 35 65 56 52 48 61 39 54 49 49 59 35 56 48 49 68 36 39 48 49 65 35 44 48 rs27072 7 years of age VNTR rs27072 Association Between DAT1 and ADHD Symptoms 1445 TABLE II. Frequencies of the DAT1 VNTR and rs27072 Alleles disequilibrium test (QTDT) [Abecasis et al., 2000]. This extended version of the transmission disequilibrium test (TDT) allows conducting family-based association tests of quantitative phenotypes in nuclear families of any size, including monozygotic (MZ) and dizygotic (DZ) twin pairs. The variance-components approach allows for simultaneously modeling of the mean and variances enhancing the information gathered by the test [Abecasis et al., 2000]. The presence of spurious association due to population stratification, essentially relegated to the between familial component, could also be estimated (AP model). In the presence of population stratification, the association could be reliably tested by partitioning the genotype and phenotype scores into orthogonal within- and between-familial variance components (AO model). While exerting an adequate control for population stratification, the AO model constitutes a conservative test of allelic association. In the absence of population stratification, testing the total evidence for association (AT model) should otherwise be pursued [Fulker et al., 1999; Chen and Abecasis, 2007]. LD between markers was estimated using ldmax [Abecasis and Cookson, 2000]. We used one-sided tests because there was an a priori hypothesis for the biased transmission of the 10-repeat allele and the G allele of rs27072 in previous studies. Corrections for multiple testing were performed in the main analyses considering four tests [two markers and the two time points (6 and 7 years of age)] using the Bonferroni method with a critical level of a ¼ 0.05 (critical corrected value P ¼ 0.0125). This method is nevertheless known to be an overly conservative test because it does not account for the dependence structure existing between the DAT1 polymorphisms and phenotypes over time. Finally, because identical results are obtained for diallelic markers, only the G allele’s statistics of the rs27072 are presented. DAT1 polymorphisms VNTR rs27072 Alleles Allele frequencies 11 10 9 7 8 6 G A 0.011 0.700 0.279 0.002 0.004 0.004 0.834 0.166 sample of twins. Based on previous findings, we used two polymorphisms located in the 30 -UTR of the DAT1 gene: the VNTR and the rs27072 polymorphism positioned 422 bp upstream of the VNTR. High LD was observed between these polymorphisms (D0 ¼ 0.965, w2(2) ¼ 71.36, P ¼ 0.000). Table II shows the allelic distribution in this sample. The distribution of VNTR alleles is similar to those noted in mixed European and US populations [Vandenbergh et al., 1992; Kang et al., 1999]. In addition, VNTR and rs27072 allele frequencies were comparable to those observed for the parental chromosomes of a clinical sample composed of Canadian children with a diagnosis of DSM-IV ADHD [Feng et al., 2005]. ADHD scores for inattention and hyperactivity/impulsivity gathered prospectively from teachers when twins were attending kindergarten (6 year olds) and the first grade of primary school (7 year olds) were examined separately. The MZ and DZ intraclass correlations for the ADHD symptoms were high at both time points (6 year olds: MZ ¼ .65 and DZ ¼ .31; 7 year olds: MZ ¼ 0.63 and DZ ¼ 0.33), yielding to heritability estimates (95% confidence intervals) of similar amplitude to what is reported in other twin studies [h2 ¼ 0.651 (0.559–0.723) and h2 ¼ 0.647 (0.558–0.718) at 6 and 7 years of age, respectively]. Using QTDT, we did not observe any evidence of population stratification for the VNTR or rs27072 polymorphisms, as reported in Table III (AP model). Therefore, the total evidence for the association test (AT model) should be prioritized because it constitutes a reliable but less conservative test of allelic association when population stratification is unlikely [Abecasis et al., 2000]. No significant association was observed between the VNTR and teachers’ report of ADHD symptoms. However, we did observe a significant allelic association between the rs27072 polymorphism and ADHD scores when children were attending kindergarten [w2(1) ¼ 7.26, P ¼ 0.007] and a trend for a significant association when children were in the first grade of primary school [w2(1) ¼ 3.74, P ¼ 0.053]. To explore the possibility that the different findings between 6 and 7 years of age may have resulted from chance variation in the Power of the Sample The power of the sample to identify QTL associations with two-sided tests of significance was estimated using the Genetic Power Calculator for variance-component QTL association for sibships (http://pngu.mgh.harvard.edu/purcell/gpc/qtlassoc.html). For 266 twin pairs (averaged number of pairs included in statistical analyses) assuming the allele frequencies of the VNTR 10-repeat allele and that the risk alleles are in complete LD with a QTL for ADHD symptoms, this sample could detect QTLs contributing at least to 1.6% to trait variance (a ¼ 0.05 and 80% power). The power of the present sample is therefore slightly less powerful than the one investigated in Mills et al. [2005b] (1.6% vs. 1.3%). RESULTS The aim of this study was to examine the relationship between DAT1 and ADHD symptoms in a population-based TABLE III. QTDT Tests of Association for ADHD Symptoms Evaluated at 6 and 7 Years of Age by Teachers DAT1 polymorphisms VNTR 9 10 rs27072 G 6 years of age 7 years of age w2 fit statistic of the modelsa w2 fit statistic of the models AP AT AO AP AT AO 0.36 (P ¼ 0.551) 0.26 (P ¼ 0.614) 0.02 (P ¼ 0.897) 0.05 (P ¼ 0.822) 0.15 (P ¼ 0.694) 0.06 (P ¼ 0.801) 0.60 (P ¼ 0.437) 0.62 (P ¼ 0.431) 1.07 (P ¼ 0.300) 0.89 (P ¼ 0.345) 1.56 (P ¼ 0.221) 1.45 (P ¼ 0.228) 0.25 (P ¼ 0.616) 7.26 (P ¼ 0.007) 4.27 (P ¼ 0.039) 0.06 (P ¼ 0.808) 3.74 (P ¼ 0.053) 0.89 (P ¼ 0.345) The AP models showed no indication of population stratification. Thus, AT models could be prioritized over AO models. AP models, test for Population stratification; AT models, test for Total Association; AO model, test for Orthogonal Association. a TDT tests were only conducted for alleles with a frequency higher than 10%. 1446 Ouellet-Morin et al. TABLE IV. QTDT Tests of Association Between the rs27072 Polymorphism and ADHD Symptoms for Twins Evaluated at Both 6 and 7 Years Olds 6 years of age 7 years of age 2 a 2 w fit statistic of the models rs27072 G w fit statistic of the models AP AT AO AP AT AO 0.13 (P ¼ 0.714) 5.33 (P ¼ 0.021) 3.04 (P ¼ 0.081) 0.03 (P ¼ 0.861) 5.62 (P ¼ 0.018) 1.86 (P ¼ 0.172) The AP models showed no indication of population stratification. Thus, AT models could be prioritized over AO models. AP models, test for Population stratification; AT models, test for Total Association; AO model, test for Orthogonal Association. a TDT tests were only conducted for alleles with a frequency higher than 10%. twin families included in the analyses, we conducted an exploratory analysis on a subsample of twins that were rated for ADHD symptoms at both time points, as presented in Table IV. We found evidence for association between the rs27072 polymorphism and ADHD symptoms at 6 years of age [w2(1) ¼ 5.33, P ¼ 0.021] and at 7 years of age [w2(1) ¼ 5.62, P ¼ 0.018], indicating that random sample variation may explain, to some extent, divergent findings in the primary analyses. Including the sex of the twins as a covariate in the analyses did not alter the results (not shown). Because variance components models can be sensitive to the nonnormality of the phenotypic distribution [Iles, 2002], we recalculated the AP models using 10,000 Monte Carlo permutations to make sure that the results were not influenced by non-normality. Similar results were obtained for the VNTR [9 allele (w2(1) ¼ 0.36, P ¼ 0.58 and w2(1) ¼ 0.60, P ¼ 0.43 at 6 and 7 years of age, respectively) and 10 allele (w2(1) ¼ 0.26, P ¼ 0.60 and w2(1) ¼ 0.62, P ¼ 0.44 at 6 and 7 years of age, respectively)] and the rs27072 polymorphisms [w2(1) ¼ 0.25, P ¼ 0.65 and w2(1) ¼ 0.06, P ¼ 0.79 at 6 and 7 years of age, respectively]. These results suggest that the phenotypic distributions did not affect the test of population stratification. Finally, based on results from twin studies that indicate that there are shared as well as unique genetic influences contributing to symptom dimensions [Levy et al., 2001], we performed a secondary analysis, examining separately the relationship of the rs27072 marker and symptom dimensions of ADHD (inattention, and hyperactivity/impulsivity) using teacher-reported symptoms at 6 years of age. We found evidence for association with both dimensions and this marker (Table V). DISCUSSION The main objective of this study was to investigate the association between the DAT1 gene and ADHD symptoms in a TABLE V. QTDT Tests of Association Between the rs27072 Polymorphism and ADHD Dimensions at 6 Years of Age w2 fit statistic of the models ADHD dimensions AP Hyperactivity/impulsivity G 0.41 (P ¼ 0.52) Inattention G 0.04 (P ¼ 0.84) AT AO 6.78 (P ¼ 0.009) 4.48 (P ¼ 0.03) 5.43 (P ¼ 0.02) 2.59 (P ¼ 0.107) AP models, test for Population stratification; AT models, test for Total Association; AO model, test for Orthogonal Association. population-based sample. The polymorphism that has been examined most often in relation to ADHD symptoms is the 30 -UTR VNTR; however, inconsistent findings have been noted, suggesting that other DAT1 polymorphisms in LD with the VNTR could be involved. Accordingly, the rs27072 polymorphism, located 422 bp upstream of and in strong LD with the VNTR, was shown to be significantly associated with ADHD in a clinical sample of children aged between 6 and 16 years old [Feng et al., 2005]. In this study, we did not find a significant association between the DAT1 VNTR and ADHD symptoms in a population-based sample; however, a significant association was observed with the rs27072 polymorphism. Few studies have examined the DAT1 contribution to ADHD symptoms in population-based studies. Our DAT1 VNTR results contrasts with the conclusions of Cornish et al.  that found an association with the DAT1 VNTR in selected children with high ADHD scores. Our findings are in contrast to the main analyses reported by Mill et al. [2005a] that indicated an association between ADHD and the 10-repeat VNTR allele in a sample of 329 DZ male twins followed prospectively when a composite measure of symptoms was averaged across parental reports obtained at ages 2, 3, 4, and 7 years and the teacher assessment collected at 7 years of age. However, the results agree with the single point analysis of the measures reported by teachers at 7 years of age that were not significant. Nevertheless, our results are consistent with other population-based twin studies [Payton et al., 2001; Todd et al., 2001a] that did not find an association. Those two studies differ in the age of participants (5–17 years olds and 7–19 years olds, respectively) and phenotype analyzed. Payton et al.  compared selected MZ twins with extremely high and low symptoms whereas Todd et al. [2001a] targeted DSM-IV subtypes and latent classes. Because of the different phenotypes chosen for analyses (instruments, informant, age, selection criteria), conclusions based on the results of the different population based samples are difficult to interpret and potential age or informant related differences could be obscured. The present study provided evidence that the rs27072 polymorphism was significantly associated with ADHD symptoms even though it was in strong, but not complete, LD with the VNTR. This finding, based on a distinct sample with a more homogeneous ethnic background (mostly French ancestors), converges with results obtained by Feng et al.  using a clinical sample of Canadian school age children (ages 6–16) drawn from a much more heterogeneous ethnic background. Furthermore, this association was observed for both the inattention and hyperactive/impulsive symptom dimensions at 6 years of age, consistent with results from the abovementioned clinical sample (Crosbie et al., in preparation). Replication of these data is especially noteworthy not only because it further supports the previous report [Feng et al., 2005], but also because this convergent finding was obtained Association Between DAT1 and ADHD Symptoms from a population-based sample that was evaluated at the same age for all twins. It is currently unknown if the DAT1 gene influences ADHD risk differently across age groups. However, evidence from postmortem studies indicating temporal changes in DAT1 expression during development [Meng et al., 1999; Haycock et al., 2003] suggests this possibility. Continued study of the twins over additional time points will allow us to determine if there is in fact a developmental change. Prospective molecular studies conducted with age-homogenous participants are now needed to replicate the rs27072 polymorphism association with ADHD symptoms and to determine if this association is specific to a given period of development or is stable over time. As stated previously, the 30 VNTR polymorphism has been the focus of the majority of molecular genetic studies involving the DAT1 gene because there is some evidence suggesting that the VNTR alleles may influence transcription [Fuke et al., 2001; Miller and Madras, 2002]. Using in vitro expression assays, several groups have provided experimental evidence that variability in the repeat number of the VNTR and the 30 UTR sequence may influence dopamine transporter protein levels. The first study of this polymorphism found that luciferase expression was significantly higher when the 30 UTR containing the 10-repeat allele was transfected in the COS7 (African Green Monkey kidney) cell line compared to constructs harboring either the 7- or 9-repeat alleles [Fuke et al., 2001]. However, another study found the opposite result as greater expression was observed when constructs containing the 9-repeat were transfected into a human embryonic kidney (HEK-293) cells [Miller and Madras, 2002]. These authors also identified a DNA variant (T/C), located 134 bp downstream of the VNTR recognized by the restriction enzyme DraI. This DNA variant was reported to influence levels of protein expression but in a promoter-dependent manner. This DNA variant was not observed in 102 individuals with ADHD, which suggests that this DNA change is unlikely to be relevant to ADHD symptoms [Feng et al., 2005]. Two additional studies, using either a mouse dopaminergic cell line [Greenwood and Kelsoe, 2003] or human neuroblastoma and human embryonic kidney cell lines [Mill et al., 2005a], found no effect on transcription by the 10- or 9-repeat alleles. The varying results from the in vitro studies may be a function of the different constructs used including the specificity of the promoter, different cell types used for transfection studies or possibly from the sequence around the VNTR included in the construct. Evidence in support of the latter can be gleamed from a study that found that the inclusion of the 800 bp sequence from the stop codon to just 50 of the VNTR influences DAT1 transcription, mRNA stability or translation as evident from the lower DAT1 protein density in an in vitro assay [VanNess et al., 2005]. Notably, the 800 bp sequence includes the rs27072 polymorphism but excluded the VNTR. The position of rs27072 within a functional region may thus explain the significant result for this marker in our sample. In conclusion, this study gives some support to the idea that the DAT1 VNTR may not be the best polymorphism to investigate the association between DAT1 and ADHD related phenotypes in all samples. While the role of the VNTR sequence on gene expression remains unclear, other polymorphisms have now been identified that also appear to be functional. These include a VNTR located in intron 8 [Brookes et al., 2006; Guindalini et al., 2006], several variants that change DAT1 amino acid sequence [Dar et al., 2006], and several promoter polymorphisms [Kelada et al., 2005]. Further molecular studies are now needed to ascertain the relationship between these as well as the rs27072 polymorphism and ADHD symptoms, particularly in the studies that previously reported no association between the DAT1-VNTR and ADHD symptoms. 1447 ACKNOWLEDGMENTS This work was supported by a New Emerging Teams Grant from the Canadian Institutes of Health Research (NET-54016), and by NHRDP and FQRSC grants. Isabelle Ouellet-Morin is supported by fellowships from the Canadian Institutes of Health Research (CIHR) and the Behavioral, Gene and Environment Training Grant Program (CIHR). We thank all families that have generously accepted to participate in this study. REFERENCES Abecasis GR, Cookson WO. 2000. GOLD–graphical overview of linkage disequilibrium. Bioinformatics 16(2):182–183. Abecasis GR, Cardon LR, Cookson WO. 2000. A general test of association for quantitative traits in nuclear families. Am J Hum Genet 66(1):279–292. Barr CL, Xu C, Kroft J, Feng Y, Wigg K, Zai G, Tannock R, Schachar R, Malone M, Roberts W, et al. 2001. Haplotype study of three polymorphisms at the dopamine transporter locus confirm linkage to attention-deficit/hyperactivity disorder. Biol Psychiatry 49(4):333–339. Behar L, Stringfield S. 1974. A behavior rating scale for the preschool child. Dev Psychol 10:601–610. Biederman J, Faraone SV. 2005. Attention-deficit hyperactivity disorder. Lancet 366(9481):237–248. Brookes KJ, Mill J, Guindalini C, Curran S, Xu X, Knight J, Chen CK, Huang YS, Sethna V, Taylor E, et al. 2006. A common haplotype of the dopamine transporter gene associated with attention-deficit/hyperactivity disorder and interacting with maternal use of alcohol during pregnancy. Arch Gen Psychiatry 63(1):74–81. Chen W-M, Abecasis GR. 2007. Family-based association tests for genomewide associated scans. Am J Hum Genet 81:913–925. Chen CK, Chen SL, Mill J, Huang YS, Lin SK, Curran S, Purcell S, Sham P, Asherson P. 2003. The dopamine transporter gene is associated with attention deficit hyperactivity disorder in a Taiwanese sample. Mol Psychiatry 8(4):393–396. Cheon KA, Ryu YH, Kim JW, Cho DY. 2005. The homozygosity for 10-repeat allele at dopamine transporter gene and dopamine transporter density in Korean children with attention deficit hyperactivity disorder: Relating to treatment response to methylphenidate. Eur Neuropsychopharmacol 15(1):95–101. Cheuk DK, Li SY, Wong V. 2006. No association between VNTR polymorphisms of dopamine transporter gene and attention deficit hyperactivity disorder in Chinese children. Am J Med Genet Part B 141B(2): 123–125. Cook EH Jr, Stein MA, Krasowski MD, Cox NJ, Olkon DM, Kieffer JE, Leventhal BL. 1995. Association of attention-deficit disorder and the dopamine transporter gene. Am J Hum Genet 56(4):993–998. Cornish KM, Manly T, Savage R, Swanson J, Morisano D, Butler N, Grant C, Cross G, Bentley L, Hollis CP. 2005. Association of the dopamine transporter (DAT1) 10/10-repeat genotype with ADHD symptoms and response inhibition in a general population sample. Mol Psychiatry 10(7):686–698. Cragg SJ, Rice ME. 2004. DAncing past the DAT at a DA synapse. Trends Neurosci 27(5):270–277. Curran S, Mill J, Tahir E, Kent L, Richards S, Gould A, Huckett L, Sharp J, Batten C, Fernando S, et al. 2001. Association study of a dopamine transporter polymorphism and attention deficit hyperactivity disorder in UK and Turkish samples. Mol Psychiatry 6(4):425–428. Daly G, Hawi Z, Fitzgerald M, Gill M. 1999. Mapping susceptibility loci in attention deficit hyperactivity disorder: Preferential transmission of parental alleles at DAT1, DBH and DRD5 to affected children. Mol Psychiatry 4(2):192–196. Dar DE, Metzger TG, Vandenbergh DJ, Uhl GR. 2006. Dopamine uptake and cocaine binding mechanisms: The involvement of charged amino acids from the transmembrane domains of the human dopamine transporter. Eur J Pharmacol 538(1–3):43–47. Epub 2006 Mar 28. Dresel S, Krause J, Krause KH, LaFougere C, Brinkbaumer K, Kung HF, Hahn K, Tatsch K. 2000. Attention deficit hyperactivity disorder: Binding of [99mTc]TRODAT-1 to the dopamine transporter before and after methylphenidate treatment. Eur J Nucl Med 27(10):1518–1524. Elia J, Ambrosini PJ, Rapoport JL. 1999. Treatment of attention-deficithyperactivity disorder. N Engl J Med 340(10):780–788. 1448 Ouellet-Morin et al. Faraone SV, Perlis RH, Doyle AE, Smoller JW, Goralnick JJ, Holmgren MA, Sklar P. 2005. Molecular genetics of attention-deficit/hyperactivity disorder. Biol Psychiatry 57(11):1313–1323. Epub 2005 Jan 21. Feng Y, Wigg KG, Makkar R, Ickowicz A, Pathare T, Tannock R, Roberts W, Malone M, Kennedy JL, Schachar R, et al. 2005. Sequence variation in the 30 -untranslated region of the dopamine transporter gene and attention-deficit hyperactivity disorder (ADHD). Am J Med Genet B Neuropsychiatr Genet 4:4. Forget-Dubois N, Perusse D, Turecki G, Girard A, Billette JM, Rouleau G, Boivin M, Malo J, Tremblay RE. 2003. Diagnosing zygosity in infant twins: Physical similarity, genotyping, and chorionicity. Twin Res 6(6):479–485. Fuke S, Suo S, Takahashi N, Koike H, Sasagawa N, Ishiura S. 2001. The VNTR polymorphism of the human dopamine transporter (DAT1) gene affects gene expression. Pharmacogenomics J 1(2):152–156. Fulker DW, Cherny SS, Sham PC, Hewitt JK. 1999. Combined linkage and association sib-pair analysis for quantitative traits. Am J Hum Genet 64(1):259–267. Gainetdinov RR, Wetsel WC, Jones SR, Levin ED, Jaber M, Caron MG. 1999. Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactivity. Science 283(5400):397–401. Galili-Weisstub E, Levy S, Frisch A, Gross-Tsur V, Michaelovsky E, Kosov A, Meltzer A, Goltser T, Serretti A, Cusin C, et al. 2005. Dopamine transporter haplotype and attention-deficit hyperactivity disorder. Mol Psychiatry 10(7):617–618. Gill M, Daly G, Heron S, Hawi Z, Fitzgerald M. 1997. Confirmation of association between attention deficit hyperactivity disorder and a dopamine transporter polymorphism. Mol Psychiatry 2(4):311–313. Goldsmith HH. 1991. A zygosity questionnaire for young twins: A research note. Behav Genet 21(3):257–269. Greenwood TA, Kelsoe JR. 2003. Promoter and intronic variants affect the transcriptional regulation of the human dopamine transporter gene. Genomics 82(5):511–520. Guindalini C, Howard M, Haddley K, Laranjeira R, Collier D, Ammar N, Craig I, O’Gara C, Bubb VJ, Greenwood T, et al. 2006. A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample. Proc Natl Acad Sci USA 103(12):4552–4557. Epub 2006 Mar 14. Hawi Z, Lowe N, Kirley A, Gruenhage F, Nothen M, Greenwood T, Kelsoe J, Fitzgerald M, Gill M. 2003. Linkage disequilibrium mapping at DAT1, DRD5 and DBH narrows the search for ADHD susceptibility alleles at these loci. Mol Psychiatry 8(3):299–308. Hay DA, Bennett KS, McStephen M, Rooney R, Levy F. 2004. Attention deficit-hyperactivity disorder in twins: A developmental genetic analysis. Aust J Psychol 56:99–107. Haycock JW, Becker L, Ang L, Furukawa Y, Hornykiewicz O, Kish SJ. 2003. Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum. J Neurochem 87(3):574–585. Krause KH, Dresel SH, Krause J, Kung HF, Tatsch K. 2000. Increased striatal dopamine transporter in adult patients with attention deficit hyperactivity disorder: Effects of methylphenidate as measured by single photon emission computed tomography. Neurosci Lett 285(2): 107–110. Langley K, Turic D, Peirce TR, Mills S, Van Den Bree MB, Owen MJ, O’Donovan MC, Thapar A. 2005. No support for association between the dopamine transporter (DAT1) gene and ADHD. Am J Med Genet Part B 139B(1):7–10. Levy F, Hay DA, McStephen M, Wood C, Waldman I. 1997. Attention-deficit hyperactivity disorder: a category or a continuum? Genetic analysis of a large-scale twin study. J Am Acad Child Adolesc Psychiatry 36(6):737– 744. Levy F, McStephen M, Hay DA. 2001. The diagnostic genetics of ADHD symptoms and subtypes. In: Levy F, Hay D, editors. Attention-Genes and ADHD. Hove, UK: Brunner-Routledge. Levy F, Hay DA, Bennett KS. 2006. Genetics of attention deficit hyperactivity disorder: A current review and future prospects. Int J Disabil Dev Educ 53(1):5–20. Lim MH, Kim HW, Paik KC, Cho SC, Yoon do Y, Lee HJ. 2006. Association of the DAT1 polymorphism with attention deficit hyperactivity disorder (ADHD): A family-based approach. Am J Med Genet Part B 141B(3): 309–311. Madras BK, Miller GM, Fischman AJ. 2005. The dopamine transporter and attention-deficit/hyperactivity disorder. Biol Psychiatry 57(11):1397– 1409. Meng SZ, Ozawa Y, Itoh M, Takashima S. 1999. Developmental and agerelated changes of dopamine transporter, and dopamine D1 and D2 receptors in human basal ganglia. Brain Res 843(1–2):136–144. Mill J, Asherson P, Craig I, D’Souza UM. 2005a. Transient expression analysis of allelic variants of a VNTR in the dopamine transporter gene (DAT1). BMC Genet 6(1):3. Mill J, Xu X, Ronald A, Curran S, Price T, Knight J, Craig I, Sham P, Plomin R, Asherson P. 2005b. Quantitative trait locus analysis of candidate gene alleles associated with attention deficit hyperactivity disorder (ADHD) in five genes: DRD4, DAT1, DRD5, SNAP-25, and 5HT1B. Am J Med Genet Part B 133B(1):68–73. Miller GM, Madras BK. 2002. Polymorphisms in the 30 -untranslated region of human and monkey dopamine transporter genes affect reporter gene expression. Mol Psychiatry 7(1):44–55. Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3): 1215. Miller GW, Gainetdinov RR, Levey AI, Caron MG. 1999. Dopamine transporters and neuronal injury. Trends Pharmacol Sci 20(10):424– 429. Muglia P, Jain U, Inkster B, Kennedy JL. 2002. A quantitative trait locus analysis of the dopamine transporter gene in adults with ADHD. Neuropsychopharmacology 27(4):655–6562. Iles MM. 2002. Linkage and association: The transmission/disequilibrium test for QTLs. In: Camp NJ, Cox A, editors. Methods in molecular biology. Quantitative trait loci: Methods and protocols, vol 195. Totowa, NJ: Humana Press. pp 101–138. Payton A, Holmes J, Barrett JH, Sham P, Harrington R, McGuffin P, Owen M, Ollier W, Worthington J, Thapar A. 2001. Susceptibility genes for a trait measure of attention deficit hyperactivity disorder: A pilot study in a non-clinical sample of twins. Psychiatry Res 105(3):273–278. James RS, Sharp WS, Bastain TM, Lee PP, Walter JM, Czarnolewski M, Castellanos FX. 2001. Double-blind, placebo-controlled study of singledose amphetamine formulations in ADHD. J Am Acad Child Adolesc Psychiatry 40(11):1268–1276. Purper-Ouakil D, Wohl M, Mouren MC, Verpillat P, Ades J, Gorwood P. 2005. Meta-analysis of family-based association studies between the dopamine transporter gene and attention deficit hyperactivity disorder. Psychiatr Genet 15(1):53–59. Kang AM, Palmatier MA, Kidd KK. 1999. Global variation of a 40-bp VNTR in the 30 -untranslated region of the dopamine transporter gene (SLC6A3). Biol Psychiatry 46(2):151–160. Roman T, Schmitz M, Polanczyk G, Eizirik M, Rohde LA, Hutz MH. 2001. Attention-deficit hyperactivity disorder: A study of association with both the dopamine transporter gene and the dopamine D4 receptor gene. Am J Med Genet 105(5):471–478. Kelada SN, Costa-Mallen P, Checkoway H, Carlson CS, Weller TS, Swanson PD, Franklin GM, Longstreth WT Jr, Afsharinejad Z, Costa LG. 2005. Dopamine transporter (SLC6A3) 50 region haplotypes significantly affect transcriptional activity in vitro but are not associated with Parkinson’s disease. Pharmacogenet Genomics 15(9):659– 668. Kim YS, Leventhal BL, Kim SJ, Kim BN, Cheon KA, Yoo HJ, Badner J, Cook EH. 2005. Family-based association study of DAT1 and DRD4 polymorphism in Korean children with ADHD. Neurosci Lett 390(3):176– 181. Kirley A, Lowe N, Hawi Z, Mullins C, Daly G, Waldman I, McCarron M, O’Donnell D, Fitzgerald M, Gill M. 2003. Association of the 480 bp DAT1 allele with methylphenidate response in a sample of Irish children with ADHD. Am J Med Genet 121(1):50–54. Sherman DK, Iacono WG, McGue MK. 1997. Attention-deficit hyperactivity disorder dimensions: A twin study of inattention and impulsivityhyperactivity. J Am Acad Child Adolesc Psychiatry 36(6):745–753. Simonoff E, Pickles A, Hervas A, Silberg JL, Rutter M, Eaves L. 1998. Genetic influences on childhood hyperactivity: Contrast effects imply parental rating bias, not sibling interaction. Psychol Med 28(4):825– 837. Stein MA, Waldman ID, Sarampote CS, Seymour KE, Robb AS, Conlon C, Kim SJ, Cook EH. 2005. Dopamine transporter genotype and methylphenidate dose response in children with ADHD. Neuropsychopharmacology 30(7):1374–1382. Tabachnick BG, Fidell LS. 2001. Using Multivariate Statistics. 4th edition. Boston, USA: Ally and Bacon. Association Between DAT1 and ADHD Symptoms Todd RD, Jong YJ, Lobos EA, Reich W, Heath AC, Neuman RJ. 2001a. No association of the dopamine transporter gene 30 VNTR polymorphism with ADHD subtypes in a population sample of twins. Am J Med Genet 105(8):745–748. Todd RD, Rasmussen ER, Neuman RJ, Reich W, Hudziak JJ, Bucholz KK, Madden PA, Heath A. 2001b. Familiality and heritability of subtypes of attention deficit hyperactivity disorder in a population sample of adolescent female twins. Am J Psychiatry 158(11):1891–1898. Todd RD, Huang H, Smalley SL, Nelson SF, Willcutt EG, Pennington BF, Smith SD, Faraone SV, Neuman RJ. 2005. Collaborative analysis of DRD4 and DAT genotypes in population-defined ADHD subtypes. J Child Psychol Psychiatry 46(10):1067–1073. Tremblay RE, Desmarais-Gervais L, Gagnon C, Charlebois P. 1987. The preschool behaviour questionnaire: Stability of its factor structure between cultures, sexes, ages and socioeconomic classes. Int J Behav Dev 10:467–484. Ueno S, Nakamura M, Mikami M, Kondoh K, Ishiguro H, Arinami T, Komiyama T, Mitsushio H, Sano A, Tanabe H. 1999. Identification of a novel polymorphism of the human dopamine transporter (DAT1) gene and the significant association with alcoholism. Mol Psychiatry 4(6): 552–557. 1449 Vandenbergh DJ, Persico AM, Hawkins AL, Griffin CA, Li X, Jabs EW, Uhl GR. 1992. Human dopamine transporter gene (DAT1) maps to chromosome 5p15.3 and displays a VNTR. Genomics 14(4):1104–1106. VanNess SH, Owens MJ, Kilts CD. 2005. The variable number of tandem repeats element in DAT1 regulates in vitro dopamine transporter density. BMC Genet 6:55. Waldman ID, Rowe DC, Abramowitz A, Kozel ST, Mohr JH, Sherman SL, Cleveland HH, Sanders ML, Gard JM, Stever C. 1998. Association and linkage of the dopamine transporter gene and attention- deficit hyperactivity disorder in children: Heterogeneity owing to diagnostic subtype and severity. Am J Hum Genet 63(6):1767–1776. Wigal T, Swanson JM, Regino R, Lerner MA, Soliman I, Steinhoff K, Gurbani S, Wigal SB. 1999. Stimulant medications for the treatment of ADHD: Efficacy and limitations. Ment Retard Dev Disabil Res Rev 5(3):215–224. Willcutt EG. 2005. The etiology of ADHD: Behavioral and molecular genetic approaches. In: Barch D, editor. Cognitive and affective neuroscience of psychopathology. Oxford: Oxford University Press. Winsberg BG, Comings DE. 1999. Association of the dopamine transporter gene (DAT1) with poor methylphenidate response. J Am Acad Child Adolesc Psychiatry 38(12):1474–1477.