Association of Dopamine Transporter Genotype With Disruptive Behavior Disorders in an Eight-Year Longitudinal Study of Children and Adolescents

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1 American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B: (2007) Association of Dopamine Transporter Genotype With Disruptive Behavior Disorders in an Eight-Year Longitudinal Study of Children and Adolescents Steve S. Lee, 1 * Benjamin B. Lahey, 2 Irwin Waldman, 3 Carol A. Van Hulle, 2 Paul Rathouz, 2 William E. Pelham, 4 Jan Loney, 5 and Edwin H. Cook 6 1 Department of Psychology, University of California, Los Angeles, California 2 Department of Health Studies, University of Chicago, Chicago, Illinois 3 Department of Psychology, Emory University, Atlanta, Georgia 4 Department of Psychology, State University of New York at Buffalo, Buffalo, New York 5 Department of Pediatrics, University of Iowa, Iowa 6 Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois Associations between dopamine transporter (DAT1) variable number tandem repeats (VNTR), genotypes, and disruptive behavior were examined in an 8-year longitudinal study of children (n ¼ 183). Half of the children met criteria for attention-deficit/hyperactivity disorder (ADHD) at 4 6 years and half were non-referred comparison children. Consistent with several studies, the non-additive association for the 10-repeat allele was significant for hyperactivity-impulsivity (HI) symptoms. However, consistent with other studies, exploratory analyses of the non-additive association of the 9-repeat allele of DAT1 with HI and oppositional defiant disorder (ODD) symptoms also were significant. The inconsistent association between DAT1 and child behavior problems in this and other samples may reflect joint influence of the 10-repeat and 9-repeat alleles. ß 2006 Wiley-Liss, Inc. KEY WORDS: dopamine; genetics; attentiondeficit/hyperactivity disorder (ADHD); oppositional defiant disorder (ODD); conduct disorder (CD) Please cite this article as follows: Lee SS, Lahey BB, Waldman I, Van Hulle CA, Rathouz P, Pelham WE, Loney J, Cook EH Association of Dopamine Transporter Genotype With Disruptive Behavior Disorders in an Eight-Year Longitudinal Study of Children and Adolescents. Am J Med Genet Part B 144B: Attention-deficit/hyperactivity disorder (ADHD), oppositional defiant disorder (ODD), and conduct disorder (CD) are characterized by developmentally extreme and impairing Grant sponsor: National Institute of Mental Health Grant; Grant number: 2RO1 MH *Correspondence to: Steve S. Lee, Department of Psychology, University of California, Los Angeles, 1285 Franz Hall Box Los Angeles, CA stevelee@psych.ucla.edu. Received 3 February 2006; Accepted 23 August 2006 DOI /ajmg.b levels of distractibility, hyperactivity, hostility, and aggression [American Psychiatric Association, 1994]. These disruptive behavior disorders (DBDs) are the most common referral for mental health services for youth in the United States [Achenbach and Howell, 1993]. Much is known about the DBDs, but relatively little is known about their causes. This is a serious deficiency, as innovations in prevention and treatment can be expected from a better understanding of environmental and genetic influences. Early family aggregation studies established that ADHD was more prevalent among biological relatives of probands [Faraone and Doyle, 2001] and adoption studies found elevated rates of ADHD in biological siblings and parents of ADHD probands [Sprich et al., 2000]. A review of 20 twin studies from the US, Australia, and Europe yielded a pooled heritability estimate of 76% for ADHD [Faraone et al., 2005]. These studies suggest that ADHD is strongly heritable and is a compelling phenotype for molecular genetic studies. One promising set of candidate genes for ADHD are those regulating dopaminergic pathways, given their central role in regulating motor and reward activity. For example, the dopamine transporter (DAT1) gene regulates uptake of dopamine by presynaptic neurons, influencing the quantity of dopamine available in the synapses in the brain, including the striatum, prefrontal cortex, and hypothalamus. DAT1 is of interest partly because it is the site of action for pharmacologic agents that reduce ADHD symptoms. Transgenic murine models (e.g., knockout or knockdown of the DAT gene) show phenotypic transformations including learning deficits and hyperactivity [Madras et al., 2005] that may be analogous to ADHD in humans. The DAT1 polymorphism (SLC6A3) has been mapped to the short arm of chromosome 5 (5p15.3) and although many different polymorphisms in the DAT1 gene have been reported, the 40-bp VNTR in the 3 0 -untranslated region in exon 15 has been the most widely investigated. DAT1 genotypes show anywhere between 3 and 13 repeats of the 40-bp region, but the 9 and 10 repeats are the most common among Caucasian, Hispanic, and African American samples [Mitchell et al., 2000]. Several studies suggest that the 10-repeat allele of DAT1 is a risk allele for ADHD. Using family-based association methods, Cook et al. [1995] reported that the 10-repeat allele was associated with ADHD. This finding was replicated in a number of subsequent studies [Waldman et al., 1998; Barr et al., 2001], but recent reviews of this literature revealed variability across studies and modest pooled odds ratios for DAT1 and ADHD [Bobb et al., 2005; Faraone et al., 2005]. Both reviews suggest that further study of DAT1 and ADHD is needed. Differences in ascertainment procedures for ADHD, ß 2006 Wiley-Liss, Inc.

2 DAT1 and Disruptive Behavior Disorders 311 experimental design (e.g., population vs. family-based association), and sample characteristics (e.g., age and comorbidity) may have contributed to these inconsistent results. One important aspect of the inconsistent findings regarding DAT1 is that not all positive findings implicate the 10-repeat allele. A large research collaboration across four sites found preferential transmission of the 9-repeat allele for severe combined symptoms of ADHD (hyperactivity and inattention) [Todd et al., 2005]. Thus, further study of DAT1 is needed to clarify how it is associated with ADHD, if it is associated at all. Because there is evidence that the covariation between ADHD, ODD, and CD is largely due to shared genetic influences [Nadder et al., 2002; Dick et al., 2005], it is likely that at least some genes are related to general susceptibility to the DBDs [Dick et al., 2005]. Although there are fewer studies of relations between dopamine genes and ODD and CD than ADHD [Comings et al., 1996], individual differences in dopaminergic genes may influence all of the externalizing disorders. To date, links between dopamine and aggression appear more consistent with dopamine receptor genes than DAT1, however. Among a sample of parents of ADHD children, fathers with the 7-repeat allele of the DRD4 gene reported higher ratings of their childhood symptoms of CD [Rowe et al., 2001]. The 7-repeat allele also was preferentially transmitted among children with ADHD and comorbid ODD [Kirley et al., 2004]. However, Young et al. [2002] used a sibling-based method to assess the association between DAT1 and a broad measure of externalizing behavior in a sample of twins and singletons (adopted and control children). In this sample, children with at least one DAT1 9-repeat allele received higher parent ratings on the CBCL externalizing scale [Achenbach, 1991] at ages 4 and 7 (and marginally at age 9). Although several studies have suggested that the 10-repeat VNTR of DAT1 is a risk allele for disruptive behavior, two studies raise the possibility that the 9-repeat VNTR could be a risk allele. In this study, we examine whether polymorphisms in the 3 0 untranslated region VNTR in DAT1 are associated with differences in DBD symptoms in an ethnically diverse sample [Lahey et al., 2004]. This study uses measures of the DBD phenotypes that are identical to those used in the DSM-IV field trials to test the DSM-IV diagnostic criteria [Lahey et al., 1994]. Unlike previous studies, the present test of the association of DAT1 with disruptive behavior is conducted using data from a longitudinal study. The use of data gathered in seven assessments conducted over an 8-year period both enhances statistical power and allows tests of interactions with age. MATERIALS AND METHODS Participants Two cohorts of 3.8- to 7.0-year-old children were recruited in consecutive years in Chicago and Pittsburgh. In Chicago, all probands were recruited from a university child psychiatry clinic where they presented with parent and/or teacher complaints of ADHD. In Pittsburgh, 42% of ADHD probands were recruited from a university child psychiatry clinic and 58% were recruited through advertisements. Probands recruited through advertisements did not differ significantly on numbers of ADHD symptoms, demographic characteristics, or impairment measures from those who presented at the psychiatry clinics [Lahey et al., 1998]. Participants were required to be enrolled in structured educational programs: 36% preschool, 43% kindergarten, 21% first grade, and 1% second grade. All participants were required to live with their biological mothers, regardless of whether other parent figures were also present in the home. Five potential probands (two in Pittsburgh; three in Chicago) were excluded because they received diagnoses of pervasive developmental disorder, mental retardation, or seizure disorder. Comparison children had never been referred for mental health problems but were not excluded if they met criteria for a mental disorder other than ADHD in the initial assessment. They were recruited from the same schools as probands or from schools in similar neighborhoods and were selected to approximately match probands in terms of sex, ethnicity, and age. Of the 310 eligible participants, 259 parents (Chicago ¼ 120; Pittsburgh ¼ 139) gave written informed consent after the study was explained; all children gave oral assent. Four of these children were excluded due to below average intelligence, yielding a final sample of 255 eligible children (125 probands and 130 comparison children). The two groups were matched for age and sex and did not significantly differ on family income or Full Scale IQ. Tests of Bias Due to Longitudinal Attrition The percent of children whose parent was interviewed was 100%, 95%, 94%, 94%, 89%, 85%, and 86% for baseline, wave 2, wave 3, wave 4, wave 6, and wave 7, respectively (wave 5 was not assessed due to limited funding). Families that did not participate in 1 year typically participated in the next assessment. As a result, 96.6% were assessed in year 7 or 8. The percent with completed teacher assessments in each year was 100%, 88%, 83%, 84%, 76%, 70%, and 71%, respectively. Of the original sample of 255 children, we obtained DAT1 genotypes on 72% of the sample (n ¼ 183) (see Table I for distribution of genotypes). To determine if this subsample was biased, we tested whether missing DNA data were associated with ADHD, ODD, and CD. Longitudinal log linear regressions, controlling for wave, revealed that missing DNA was not significantly associated with CD, hyperactivity, inattention, or ODD (b ¼ 0.12, z ¼ 0.53, P ¼ 0.60, b ¼ 0.11, z ¼ 0.98, P ¼ 0.33, b ¼ 0.10, z ¼ 0.93, P ¼ 0.36, and b ¼ 0.16, z ¼ 1.02, P ¼ 0.28, respectively). Thus, children with DNA are representative of the overall sample with respect to the core DBD outcome measures. Measures DNA collection and genotyping procedures. Saliva was collected from all children (probands and controls) and mothers. Because paternal DNA was collected in very few families, Mendelian errors could not be fully discerned. However, in all cases when mothers were homozygous (e.g., 10/10), the child s genotype reflected Mendelian patterns (e.g., the child had at least one 10-allele). For each locus, PCR was TABLE I. Distribution of DAT1 Genotypes by Race-Ethnicity 9/9 (n ¼ 13), 9/10 (n ¼ 56), 10/10 (n ¼ 105), 10/11 (n ¼ 3), 7/10 (n ¼ 2), 9/11 (n ¼ 3) 8/9 (n ¼ 1), n(%) Caucasian 10 (5.5%) 38 (20.8%) 68 (57.4%) 3 (1.6%) 0 3 (1.6%) 0 African American 2 (15.4%) 15 (26.8%) 30 (28.6%) 0 2 (1.1%) 0 1 (1.1%) Other 1 (1.1%) 3 (1.6%) 7 (3.8%) Group differences not statistically significant.

3 312 Lee et al. carried out in a 10 ml volume containing 50 ng of genomic template, 0.5 mm of each primer, one of which was 5 0 fluorescently labeled, 200 mm of each dntp, 1 PCR buffer, 1.5 mm MgCl 2, and 0.3 units of DyNAzyme TM EXT DNA polymerase (Finnzymes Oy, Espoo, Finland), with 0.5 M GC-melt (Clontech, Palo Alto, CA). The DAT1 primer sequence was 5 0 -NED-TGTGGTGTAGGGAACGGCCTGAG-3 0 and 5 0 CTTCCTGGAGGTCACGGCTCAAGG-3 0. Samples were amplified on an Applied Biosystems 9700 thermal cycler (Foster City, CA) with an initial 12 min step to heat-activate the enzyme at 968C, followed by 45 cycles consisting of a denaturation step of 968C for 30 sec, an annealing step for 45 sec at 688C for DAT1 and an extension step of 728C for 3 min, with final extension step at 728C for 10 min. Post-PCR products were purified with Sephadex G-50 gel filtration system, and then added to 10 ml of deionized formamide and 0.5 ml of ROX labeled size standard. PCR products were injected and detected by laser-induced fluorescence on an ABI PRISM 3730 Genetic Analyzer at the University of Chicago DNA Sequencing and Genotyping Core. Electropherograms were processed with Genescan software and alleles were called with Genotyper software, blind to all but a consecutively assigned number that is unrelated to whether the subject is a child, father, or mother, and without indication of pedigree relationship to adjacent numbers. Diagnostic measures. Independent diagnostic interviews of the mother and child were conducted concurrently by two lay interviewers. The Diagnostic Interview Schedule for Children [DISC; Shaffer et al., 1993] was administered to the parent during each annual assessment. Information was obtained from the parent on DSM-III-R diagnostic criteria for ADHD, ODD, CD, anxiety disorders (i.e., simple phobia, social phobia, agoraphobia, separation anxiety, panic, and overanxious disorders), major depression, and dysthymia during the last 6 months. In addition, questions used in the DSM-IV field trials version of the DISC [Lahey et al., 1994] to assess DSM-IV and ICD-10 symptoms of DBD, not used in DSM-III-R, were asked. As a part of each assessment, teachers were sent the DSM-IV version of the DBD Rating Scale [Pelham et al., 1992] by mail. Following standard procedures [Pelham et al., 1992], symptoms rated pretty much or very much were scored as present. As in the DSM-IV field trials, symptoms were considered present if reported by either parent or teacher [Piacentini et al., 1992]. At baseline, we assessed impairment in two ways to address the DSM-IV requirement that impairment be cross-situational to diagnose ADHD. First, the parent was asked in the DISC if the child s ADHD symptoms had caused problems (a) at home or with friends, or (b) at school. Second, parents and teachers completed the Impairment Rating Scale [IRS; Fabiano et al., 2006], in which the child s need for treatment in a variety of areas is rated using seven-point scales ranging from No problem; definitely does not need treatment (¼0) to Extreme problem; definitely needs treatment (¼6). Parents rated the child s need for treatment related specifically to problems with peers, siblings, and parents; academic progress at school, selfesteem, and impact on the family. Teachers rated the child s need for treatment related specifically to problems with classmates and teachers, academic progress, classroom disruption, and self-esteem. Parents and teachers also rated the child s need for treatment. IRS scores of 3 on at least one scale differentiated clinic and non-clinic children for both parents and teachers and 1 year test retest stability with different teachers for the six IRS scales is r ¼ [Fabiano et al., 2006]. Data Analysis Genotypes were coded to reflect the influence of the 10- repeat allele to test both additive and non-additive models of transmission in the same analyses. Given the case-control design of this study, we tested if DAT1 alleles were associated with demographic variables at baseline (age, sex, and raceethnicity) using Chi-square tests and if alleles were related to family income. The additive term for the 10-repeat allele was not associated with any demographic variable, but the nonadditive term was associated with family income (b ¼ 0.006, w 2 ¼ 8.70, P < 0.01). Therefore, the log of family income was controlled in all models. Race-ethnicity was also controlled to adjust for race-ethnicity differences in income. Interactions between time (wave) and genotype were modeled separately for each DBD symptom to determine if the slopes for ADHD, ODD, and CD symptoms differed by genotype. Primary analyses. We tested the hypothesis that the 10- repeat allele of DAT1 is associated with increased risk for DBD using longitudinal analyses. Four separate models were conducted, treating one of the four symptom dimensions of DBD as the response variable in each model: inattention, hyperactivity-impulsivity (HI), ODD, and CD. Counts of each of these kinds of symptoms obtained in seven assessments over 8 years were treated as repeated measures using generalized estimating equations (GEE) in SAS GENMOD. Statistical tests were based on the z-statistic (all tests df ¼ 1) and robust standard errors. The working distributions for counts of each kind of symptom were specified as Poisson distributions and correlations among the repeated measures over time were treated as unstructured. Because GEE longitudinal analyses estimate means in each assessment from available data, they accommodate missing data well, especially when retention is high and few participants drop out of the study completely, as in the present study. Two time-varying covariates were included in each longitudinal model to adjust for methodologic features of the study. First, we treated the blindness of the interviewer in each wave to information about the youth from previous assessments as a time-varying covariate. Interviewers of the parents sometimes had assessed the child or parent in the previous year or had entered data about a previous assessment. Thus, interviewers had access to varying amounts of diagnostic, social, academic, and impairment data about the child and family. Second, the number of informants in each wave (parent, teacher, or both) was treated as a time-varying covariate because fewer symptoms were reported when only one informant was assessed. Genotypes were coded to test additive and non-additive influences of the 10-repeat allele. Terms for additive and non-additive effects for the 10-repeat allele were entered simultaneously in each of the four longitudinal models for the different kinds of symptoms. The additive term was coded as 1, 0, and 1, testing linear differences among having 0, 1, or 2 copies of the 10-repeat allele. The non-additive term was coded as 1, 2, and 1, capturing any mode of non-additive transmission (i.e., recessive, dominant), or heterozygote disadvantage. The linear term for time (assessment waves) was controlled in all analyses. Exploratory analyses. To complement the primary analyses focused on the 10-repeat allele, two additional sets of exploratory analyses were conducted using the same longitudinal methods. First, because two previous papers [Young et al., 2002; Todd et al., 2005] have suggested that the 9-repeat VNTR of DAT1 is a risk allele, we repeated the four primary longitudinal analyses (one for each symptom dimension), but this time coding the additive and non-additive terms based on the 9-repeat allele to test the alternative hypothesis that the 9-repeat VNTR is the risk allele. Second, to provide additional preliminary information that might ultimately lead to an understanding of why previous studies have inconsistently indicated that either the 9- or the 10-repeat alleles of DAT1 may be associated with increased risk for DBD, we

4 DAT1 and Disruptive Behavior Disorders 313 conducted a set of longitudinal analyses for each of the four symptom dimensions in an exploratory spirit. In each of the latter four models, three groups were compared based on the presence of 9- and 10-repeat alleles: 9/9, 9/10, and 9/9. The few youth with other numbers of repeats were excluded from this second set of exploratory analyses. RESULTS Hardy Weinberg Equilibrium Before conducting the analyses of interest, we performed Chi-square tests of Hardy Weinberg equilibrium evaluating statistical significance via simulation. The genotype frequencies met the assumption of Hardy Weinberg equilibrium (10- repeat allele genotypes: w 2 ¼ 3.23, one-tailed P ¼ 0.19; 9-repeat allele genotypes: w 2 ¼ 1.14, one-tailed P ¼ 0.39). Primary Analyses: 10-Repeat Allele of DAT1 Based on previous findings, we tested if the 10-repeat allele of DAT1 is associated with the two dimensions of ADHD symptoms and with ODD and CD symptoms. Controlling for time (assessment waves) and the methodologic and demographic covariates, the non-additive term for DAT1 was significantly related to HI symptoms (b ¼ 0.09, z ¼ 2.24, P < 0.05) but the additive term was not (b ¼ 0.08, z ¼ 0.84, P ¼ 0.40). This indicates that the number of 10-repeat alleles is not associated linearly with more HI symptoms. For inattention symptoms, neither the non-additive nor the additive term for DAT1 was significant (b ¼ 0.05, z ¼ 1.19, P ¼ 0.24, b ¼ 0.12, z ¼ 1.29, P ¼ 0.20). For ODD symptoms, neither the non-additive (b ¼ 0.09, z ¼ 1.69, P ¼ 0.09) nor the additive term for DAT1 was significant at the P < 0.05 level (b ¼ 0.17, z ¼ 1.37, P ¼ 0.17). Similarly, neither the nonadditive nor the additive term for DAT1 was significantly related to CD symptoms (b ¼ 0.09, z ¼ 1.20, P ¼ 0.23 and b ¼ 0.29, z ¼ 1.63, P ¼ 0.10, respectively). Interactions between time and the 10-repeat allele were tested to determine if the associations between genotype and DBD symptoms changed with the age of the child. Separate genotype time interaction terms were created for the additive and non-additive genotypes of the 10-repeat allele (additive wave, non-additive wave). When the interactions were tested, the models simultaneously included both interaction terms, both additive and non-additive main effects, and the covariates described above into GEE models for each of the four DBD dimensions. The additive wave interaction terms were not significant for HI, inattention, and CD symptoms (b ¼ 0.02, z ¼ 1.25, P ¼ 0.21, b ¼ 0.02, z ¼ 1.68, P ¼ 0.09, and b ¼ 0.03, z ¼ 0.87, P ¼ 0.38, respectively) but there was a significant interaction with time (wave) for ODD symptoms (b ¼ 0.06, z ¼ 3.06, P ¼ 0.00). This reflects the finding that individuals with more copies of the 10-repeat allele had a significantly more negative slope for ODD symptoms over time. None of the non-additive wave interactions were significant (b ¼ 0.01, z ¼ 0.79, P ¼ 0.43, b ¼ 0.01, z ¼ 1.39, P ¼ 0.17, b ¼ 0.01, z ¼ 1.32, P ¼ 0.19, and b ¼ 0.004, z ¼ 0.24, P ¼ 0.81 for HI, inattention, ODD, and CD symptoms, respectively). Exploratory Analyses: Comparisons Among 10-Repeat Allele Groups To contribute to the empirical basis for testable hypotheses regarding the relation between DAT1 alleles and DBD symptoms, differences in the mean number of each of the four DBD dimensions were tested in exploratory analyses among the three groups defined on the basis of the number of 10-repeat alleles (i.e., 0, 1, or 2, 10-repeats). Controlling for time and the methodologic and demographic covariates, the three groups were found to differ significantly in the number of HI symptoms (w 2 ¼ 9.19, df ¼ 2, P ¼ 0.01), inattention symptoms (w 2 ¼ 6.43, df ¼ 2, P < 0.05), ODD symptoms (w 2 ¼ 8.57, df ¼ 2, P ¼ 0.01), and CD symptoms (w 2 ¼ 6.56, df ¼ 2, P < 0.05) over 8 years. We then conducted specific follow-up comparisons of the individual groups. For all DBD dimensions, youth with one copy of the 10-repeat allele had significantly higher levels of hyperactivity, inattention, ODD, and CD symptoms (b ¼ 0.35, z ¼ 3.14, P < 0.01; b ¼ 0.25, z ¼ 2.48, P ¼ 0.01; b ¼ 0.42, z ¼ 2.97, P < 0.01; and b ¼ 0.56, z ¼ 2.63, P < 0.01, respectively) than the 10-repeat homozygotes. The 9-repeat homozygotes did not differ significantly from the other groups on any DBD dimension. Exploratory Analyses: 9-Repeat Allele of DAT1 Because two previous studies reported an association between having at least 1 allele of the 9-repeat allele and externalizing behavior [Young et al., 2002; Todd et al., 2005], all analyses for the 10-repeat allele were repeated coding the additive and non-additive terms based on the number of 9- repeat alleles, and by creating three groups based on the number of 9-repeat alleles. Controlling for time and methodologic and demographic covariates, the non-additive term for the 9-repeat allele was significant (b ¼ 0.09, z ¼ 2.17, P < 0.05), but the additive term was not (b ¼ 0.06, z ¼ 0.58, P ¼ 0.56) for HI symptoms. The non-additive and additive terms based on the 9-repeat allele were not significant for inattention symptoms (b ¼ 0.06, z ¼ 1.54, P ¼ 0.12, b ¼ 0.08, z ¼ 0.82, P ¼ 0.41, respectively). For ODD symptoms, the non-additive term for the 9-repeat allele was significant (b ¼ 0.11, z ¼ 1.98, P < 0.05), but the additive term was not (b ¼ 0.10, z ¼ 0.72, P ¼ 0.47). Neither the additive nor the non-additive term for the 9-repeat allele was significant for CD symptoms (b ¼ 0.27, z ¼ 1.35, P ¼ 0.18 and b ¼ 0.09, z ¼ 1.10, P ¼ 0.27, respectively). Using the same data analytic approach described above, we created interaction terms between additive and non-additive 9-repeat allele genotypes and time. Once again, the only additive wave interaction was significant for ODD symptoms (b ¼ 0.05, z ¼ 2.13, P < 0.05) but not for HI, inattention, and CD symptoms (b ¼ 0.02, z ¼ 1.27, P ¼ 0.21; b ¼ 0.02, z ¼ 1.49, P ¼ 0.14; and b ¼ 0.03, z ¼ 0.56, P ¼ 0.57, respectively). The interaction indicates that having more copies of the 9-repeat allele was associated with a less negative slope for ODD symptoms over time than individuals with fewer copies of the 9-repeat allele. For the non-additive interaction with wave, there was no significant association with the DBD dimensions (b ¼ , z ¼ 0.03, P ¼ 0.97; b ¼ 0.01, z ¼ 1.64, P ¼ 0.10; b ¼ 0.01, z ¼ 0.86, P ¼ 0.39; and b ¼ 0.01, z ¼ 0.28, P ¼ 0.78 for HI, inattention, ODD, and CD symptoms, respectively). Exploratory Analyses: Comparisons Among 9-Repeat Allele Groups When the three groups defined by the number of 9-repeat alleles were compared, they differed significantly in the number of HI (w 2 ¼ 8.87, df ¼ 2, P < 0.05), inattention (w 2 ¼ 7.01, df ¼ 2, P < 0.05), ODD (w 2 ¼ 8.45, df ¼ 2, P < 0.05), and CD symptoms (w 2 ¼ 6.44, df ¼ 2, P < 0.05) over 8 years, controlling for time and the methodologic and demographic covariates. When the three 9-repeat allele groups were contrasted on DBD dimensions, the one 9-repeat allele group showed significantly higher levels of all DBDs relative to the group with zero 9-repeat alleles (b ¼ 0.34, z ¼ 3.13, P < 0.01; b ¼ 0.28, z ¼ 2.78, P < 0.01; b ¼ 0.43, z ¼ 3.13, P < 0.01; and b ¼ 0.60, z ¼ 2.96, P < 0.01 for hyperactivity, inattention, ODD, and CD symptoms, respectively). The 9-repeat homozgytoes

5 314 Lee et al. did not differ significantly from the other two groups on any DBD dimension. Effect Sizes for Mean Differences Among the Genotypes For our primary analyses, mean levels of the four dimensions of DBD symptoms over the seven waves of data collection were calculated. Specifically, we contrasted two non-additive models of influence based on the 10-repeat allele. In the first model, we compared individuals with one copy of the 10-repeat allele (n ¼ 61) versus those with zero or two copies of the 10-repeat allele (n ¼ 122). To quantify the magnitude of the differences, Cohen s d was calculated using means and standard deviations of the number of each of the four dimensions of DBD symptoms averaged over the seven assessments. The effect sizes were 0.30 for inattention and ODD symptoms, 0.32 for CD symptoms, and 0.41 for hyperactivity symptoms (the one 10- repeat allele group showing more DBD symptoms than the group comprising zero or two copies of the 10-repeat allele). In the second model, we contrasted individuals with zero copies of the 10-repeat allele (n ¼ 17) with individuals with one or two copies of the 10-repeat allele (n ¼ 166). Effect sizes were modest, ranging from 0.02 for ODD to 0.12 for CD symptoms. For our exploratory analyses, we calculated effect sizes comparing individuals with one 9-repeat allele (n ¼ 60) versus those with zero or two copies of the 9-repeat allele (n ¼ 123). Effect sizes were 0.31 for inattention and CD symptoms, 0.32 for ODD, and 0.40 for HI symptoms with the one 9-repeat allele group showing the highest levels of DBDs. Our second comparison contrasted one group with zero copies of the 9- repeat allele (n ¼ 110) versus individuals with one or two copies of the 9-repeat allele (n ¼ 73). Higher DBD symptoms were evident in the group with one or more copies of the 9-repeat allele with effect sizes of 0.28 for inattention and ODD, 0.36 for hyperactivity, and 0.32 for CD symptoms. Given the non-additive influence of the 10-repeat allele on HI symptoms and the non-additive influence of the 9-repeat allele on HI and ODD symptoms, these findings raise an additional hypothesis about the DAT1 VNTR. Specifically, heterozygotes (one copy of the 10-repeat allele and one copy of the 9-repeat allele) may actually show the highest levels of DBD symptoms. We investigated effect sizes based on the three most common genotypes (9/9, 9/10, and 10/10). Cohen s d was 0.53 for HI symptoms, 0.43 for inattention symptoms, 0.44 for ODD symptoms, and 0.50 for CD symptoms for the 9/10 genotype relative to the 10/10 genotype (Figs. 1 4). Fig. 2. Mean number of inattention symptoms reported by the parent and teacher (P þ T) during annual assessments for three primary DAT1 genotypes (9/9, 9/10, and 10/10). DISCUSSION Controlling for demographic variables and time-varying methodologic covariates, we confirmed the hypothesis based on previous studies that the 10-repeat DAT1 VNTR is associated with the number of HI symptoms across childhood, but not with the number of inattention, ODD, or CD symptoms. Only the non-additive term for the 10-repeat allele was significant, however, suggesting that the genetic effect is non-additive. When children with 0, 1, or 2, 10-repeat alleles were compared in an exploratory follow-up analysis, the group with one 10- repeat allele exhibited markedly higher levels of all DBD dimensions over time than the group with two copies of the 10- repeat allele, including HI symptoms. In addition, based on recent published reports of the association of the 9-repeat allele with the DBDs, we supplemented our primary analyses based on the 10-repeat allele with identical analyses based on the 9-repeat allele. There was a significant non-additive association between the number of 9-repeat alleles and levels of HI and ODD symptoms over time. Specifically, individuals with one copy of the 9-repeat allele had higher levels of all DBD symptoms than individuals with zero copies of the 9-repeat allele. Finally, although power requirements are high to detect interactions [McClelland and Judd, 1993], there were two significant interactions between additive Fig. 1. Mean number of hyperactivity-impulsivity symptoms reported by the parent and teacher (P þ T) during annual assessments for three primary DAT1 genotypes (9/9, 9/10, and 10/10). Fig. 3. Mean number of ODD symptoms reported by the parent and teacher (P þ T) during annual assessments for three primary DAT1 genotypes (9/9, 9/10, and 10/10).

6 DAT1 and Disruptive Behavior Disorders 315 Fig. 4. Mean number of CD symptoms reported by the parent and teacher (P þ T) during annual assessments for three primary DAT1 genotypes (9/9, 9/10, and 10/10). genotype terms and time for ODD symptoms. Individuals with more copies of the 10-repeat allele had steeper reductions in ODD symptoms over time whereas individuals with more copies of the 9-repeat allele had a more stable pattern of ODD symptoms over time. Overall, the present findings support previous studies that the 10-repeat allele of the DAT1 VNTR polymorphism is a susceptibility locus for the HI dimension of ADHD symptoms. On the other hand, the exploratory analyses are also consistent with more recent findings [Young et al., 2002; Todd et al., 2005] on the significant association between the 9-repeat allele and the DBDs. Including this study, the 9-repeat allele has been implicated across a wide range of phenotypes including the CBCL externalizing scale, a latent class phenotype of severe combined ADHD, and actual DSM-IV symptoms count of the ADHD, ODD, and CD. Taken together, these studies suggest the potential importance of the 9-repeat allele. Many previous studies ignored the 9-repeat allele by focusing on a specific twogroup contrast (e.g., 10-repeat homozygotes vs. all other genotypes). Because the 9-repeat allele is less common in the population, studies with larger sample sizes are essential. Although our primary analyses focused on the 10-repeat allele, our exploratory secondary analyses addressed the possible influence of the 9-repeat allele. Indeed, the finding that the 9/10 heterozygotes exhibited the highest levels of maladaptive behavior suggests a testable hypothesis regarding the possibility of heterosis between DAT1 genotypes and DBD symptoms. Heterosis is defined by higher or lower levels of a phenotype in heterozygotes than homozygotes. This is a well-known phenomenon that has been detected in humans and non-human animals for many genes and phenotypes. For example, a review of studies of relations between a dopamine receptor gene (DRD2) and behavioral phenotypes, including DBD symptoms, pathological gambling, and alcoholism, suggested the importance of heterosis for this marker [Comings and MacMurray, 2000]. In addition, a more recent study found higher levels of smoking in males heterozygous for the DRD2 gene, although females with the same genotype showed lower levels of smoking relative to either homozygous class [Lee, 2003]. If 9/10 heterozygotes are actually at greatly increased risk for DBDs, but heterosis is not considered, studies might inconsistently find an association with the 9- repeat allele, or the 10-repeat allele, or null findings altogether, as has been the case for studies of DAT1 to date. It is possible that previous studies of DAT1 and DBDs yielded inconsistent findings because the relation involves heterosis rather than additive effects of either the 10- or the 9- repeat allele. The present findings suggest a testable hypothesis, but by no means support a conclusion of heterosis for DAT1. It should be emphasized that the present findings are only partly consistent with heterosis. Although the 9/10 group exhibited higher levels of all types of DBD symptoms than the 10/10 group, the 9/9 group also would need to exhibit significantly fewer symptoms than the 9/10 group to demonstrate heterosis. Unfortunately, the small size of the 9/9 group prevents firm conclusions regarding this group. This means that we cannot rule out the alternative hypothesis of a dominant effect of the 9-repeat allele. If larger samples revealed significantly more DBD symptoms in the 9/9 than the 10/10 genotype, but the 9/9 and 9/10 groups did not differ significantly, the findings would suggest a dominant effect of the 9-repeat allele. This possibility cannot be ruled out in the present sample because the small 9/9 group did not differ significantly from the other two genotype groups. The underlying genetic architecture of DAT1 also complicates attempts to measure simple models of genetic influence. Variants in or near DAT1 that are in LD with the DAT1 3 0 UTR VNTR may be relevant. Recently, Feng et al. [2005] identified a single nucleotide polymorphism (SNP) (MspI) within DAT1 that was preferentially transmitted in an ADHD sample. Identifying haplotypes (alleles in close proximity that are inherited together) should provide greater statistical power for detecting the genes that influence ADHD. The findings of relationships between haplotypes including the DAT1 3 0 UTR VNTR suggest either a combination of variants within DAT1 VNTR or the presence of a yet undetermined functional variant in LD with the DAT1 3 0 UTR VNTR [Feng et al., 2005; Brookes et al., 2006] influence differences in the level of DBD symptoms. The strengths of the present study derive from having repeated measurements of disruptive behavior on a wellcharacterized sample. Several limitations to this study should be kept in mind, however. First, although case-control designs allow for tests of association by examining allelic differences between affected and control individuals, results can be spurious if allele frequencies are significantly different between the populations from which cases and controls were drawn. However, in the present study, there was neither evidence of significant population substructure based on allele distributions for the categories of race-ethnicity used in this study nor evidence that race-ethnicity was associated with DBD dimensions [both are required for population stratification; Hutchison et al., 2004]. Because family income was related to ADHD symptoms, however, we controlled for its influence in all statistical models. Within-family association methods, such as the transmission disequilibrium test (TDT) are not influenced by population stratification because the nontransmitted alleles within the family are used as controls as the transmitted alleles are used as cases. TDT has the added advantage of not requiring multiple affected family members or unaffected siblings which are costly and difficult to collect. Unfortunately, we were unable to collect DNA from both parents. For case-control designs, genomic control is suggested as a viable method of dealing with population substructure [Devlin et al., 2001]. Genomic control requires additional markers in the sample to be genotyped to test for independence for both null and candidate loci. Significance levels are then adjusted for candidate loci tests. Unfortunately, we were unable to implement additional genotyping in the present study. It is also important to note that the majority of probands in this sample were clinic-referred preschool-aged children. Samples derived from clinic referrals are associated with more severe psychopathology than population-based samples [Goodman et al., 1997]. Furthermore, all of the children with

7 316 Lee et al. externalizing disorders in this study met criteria for earlyonset CD, which is thought to be more heritable and correlated with a wider range of risk indicators such as neuropsychological deficits [Moffitt et al., 2001]. Third, the number of girls in the sample did not provide enough power to fully test hypotheses about the influence of sex on distributions of alleles. Fourth, the inconsistency of previous findings regarding genetic influences on psychopathology raises the possibility of the importance of environmental factors. If specific genes exert stronger influence in the presence of environmental risk, participants in the study must have been sufficiently exposed to the variable to detect the genetic effect. Thus, large population-based studies that cover a wide range of environmental risk variables or more targeted samples of individuals with known exposure to putative environmental risk factors (e.g., a sample of low birthweight children) may be required to have enough statistical power to detect genetic main effects and interactions with the environment [Moffitt et al., 2005]. Overall, these preliminary findings suggest that both the 10-repeat allele and 9-repeat allele of the DAT1 VNTR polymorphism is associated with HI symptoms. The 9-repeat allele was also associated with ODD symptoms. In each case, the association was non-additive, suggesting the testable hypothesis that heterosis may be the actual mode of transmission. Therefore, re-analysis of existing samples, particularly those with larger samples with sufficient statistical power, should test the hypothesis that the 9/10 heterozygotes are at the greatest risk for engaging disruptive behavior. 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