Molecular Psychiatry (2014) 19, 880–889 & 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp

ORIGINAL ARTICLE Functional effects of transporter gene genotypes on in vivo functioning: a meta-analysis

SV Faraone1, TJ Spencer2, BK Madras3,4, Y Zhang-James1 and J Biederman2

Much psychiatric genetic research has focused on a 40-base pair variable number of tandem repeats (VNTR) polymorphism located in the 30-untranslated region (30UTR) of the dopamine active transporter (DAT) gene (SLC6A3). This variant produces two common alleles with 9- and 10-repeats (9R and 10R). Studies associating this variant with in vivo DAT activity in humans have had mixed results. We searched for studies using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) to evaluate this association. Random effects meta-analyses assessed the association of the 30UTR variant with DAT activity. We also evaluated heterogeneity among studies and evidence for publication bias. We found twelve studies comprising 511 subjects, 125 from PET studies and 386 from SPECT studies. The PET studies provided highly significant evidence that the 9R allele was associated with increased DAT activity in human adults. The SPECT studies were highly heterogeneous. As a group, they suggested no association between the 30UTR polymorphism and DAT activity. When the analysis was limited to the most commonly used ligand, [123I]b-CIT, stratification by affection status dramatically reduced heterogeneity and revealed a significant association of the 9R allele with increased DAT activity for healthy subjects. In humans, the 9R allele of the 30UTR polymorphism of SLC6A3 regulates dopamine activity in the striatal brain regions independent of the presence of neuropsychiatric illness. Differences in study methodology account for the heterogeneous results across individual studies.

Molecular Psychiatry (2014) 19, 880–889; doi:10.1038/mp.2013.126; published online 24 September 2013 Keywords: ADHD; dopamine transporter; genetics; meta-analysis; PET; SPECT

INTRODUCTION model of ADHD.3 Based on a meta-analysis of nine in vivo 15 The dopamine active transporter (DAT) is a key regulator of the SPECT and PET studies, Fusar-Poli et al. concluded that DAT dopamine system and the gene that encodes it (SLC6A3) has been activity was 14% higher in ADHD patients compared with controls the focus of much research in biological psychiatry, having been and that, among ADHD patients, DAT activity was higher among implicated in several disorders including attention deficit hyper- patients with a history of (although this latter activity disorder (ADHD),1–3 pediatric bipolar disorder,4 Tourette conclusion has been questioned due to incorrect coding of 16 syndrome5 and alcoholism,6 but not schizophrenia.7 Much medication status. ) research in this area has focused on the DAT gene (SLC6A3), Functional in vitro studies have shown mixed results as to especially a 40-base pair variable number of tandem repeats whether it is the 9R or 10R allele that increases DAT gene 17–21 (VNTR) polymorphism located in the 30-untranslated region expression. These results varied in the reporter gene designs (30UTR) of the gene, which has a regulatory role during and cell types used. A few studies measured in vivo striatal DAT gene transcription. This variant produces two common alleles with expression using postmortem brains, and the results were also 22–25 9- and 10-repeats (9R and 10R). In humans, meta-analyses suggest inconsistent. MRI and magnetic resonance spectroscopy studies 26–28 the 10R allele of this polymorphism is associated with ADHD in have also produced heterogeneous results. Areviewof youth8 whereas the 9R allele is associated with ADHD in adults.9 neuropsychological studies found little evidence supporting the 29 Meta-analysis also associates the 9R allele with alcoholism,6 a idea that the SLC6A3 30UTR is associated with deficits in cognition, 27 common comorbidity of ADHD in adults. with the possible exception of functions mediated by the striatum. The DAT was initially implicated in ADHD by the PET and SPECT neuroimaging studies have examined the drugs, which are efficacious for the disorder and block the DAT, association of the 30UTR polymorphism with in vivo striatal DAT thereby increasing the concentration of dopamine in the synaptic binding in humans. Such studies are particularly compelling cleft. These effects are most pronounced in the nucleus accum- because they directly measure the protein produced by the gene bens and dorsal striatum due to the high density of DATs in these rather than measuring mRNA level, or downstream effects of brain regions.10,11 Positron emission tomography (PET) studies in activation or cognition. DAT binding may be an intermediate humans show that both methylphenidate12 and amphetamine13 phenotype that mediates the effects of DAT gene variants on increase extracellular dopamine levels in the striatum. Single- dopamine-regulated brain functions and, ultimately, a wide array photon emission tomography (SPECT) and PET studies also show of behavior; including information processing, inhibition, emotion, that treatment blocks the DAT.14 Consistent with movement, salience and reward. Advances in molecular imaging this, methylphenidate normalizes elevated DAT densities in a rat and the development of highly specific DAT binding ligands allow

1Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA; 2Pediatric Psychopharmacology Unit, Psychiatry Service, Massachusetts General Hospital, Department of Psychiatry, Harvard Medical School, Boston, MA, USA; 3Division of Neuroscience, New England Primate Research Center, Southborough, MA, USA and 4Harvard Medical School, Boston, MA, USA. Correspondence: Dr SV Faraone, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA. E-mail: [email protected] Received 20 February 2013; revised 15 August 2013; accepted 16 August 2013; published online 24 September 2013 Meta-analysis of dopamine transporter gene associations SV Faraone et al 881

Records identified through Additional records identified database searching through other sources (n = 105) (n = 1) Identification

Records after duplicates removed (n =106)

Screening Records screened Records excluded (n =106) (n =88)

Full-text articles excluded, Full-text articles assessed with reasons for eligibility (n = 6) (n =18) Wrong type of study, i.e., no human dopamine Eligibility transporter genotype or imaging data Studies included in qualitative synthesis (n = 12)

Studies included in

Included quantitative synthesis (meta-analysis) (n = 12)

Figure 1. PRISMA flow diagram. Note: PRISMA ¼ Preferred reporting items for systematic reviews and meta-analyses (http://www.prisma- statement.org/). for the imaging of the DAT in humans facilitating the direct in vivo SPECT) and, for SPECT studies, type of ligand. As we show in this examination of the product of the DAT gene and its polymorph- manuscript, attending to these key issues provides a better isms in humans. Thus, studies of the SLC6A3 30UTR polymorphism understanding of the association between the SLC6A3 30UTR and DAT binding provide evidence as to whether this polymorph- polymorphism with in vivo striatal DAT binding in humans. ism regulates DAT functioning in living humans. Results from such in vivo studies could advance our under- standing of the genetic control of dopamine as an important neuromodulator of brain function. However, they have produced METHODS mixed results. Only two PET studies addressed this issue. These A PubMed literature search identified studies that met the following criteria: (1) use of SPECT or PET to assess DAT availability in the brains of studies of striatal structures reported that the 9R allele was 0 30,31 human subjects; (2) genotyping of the DAT gene (SLC6A3)3UTR VNTR. (3) associated with increased DAT activity. In contrast, ten SPECT reporting of means and standard deviations of DAT availability stratified by studies produced conflicting results. A meta-analysis of eight of genotype; (4) reporting of the numbers of subjects in each genotype these SPECT studies concluded that these studies did not provide group. We used the following search algorithm in PubMed: ‘dopamine support for the putative association between the SLC6A3 30UTR transporter’ [TIAB] OR DAT1[TIAB] OR SLC6A3[TIAB] AND (imaging [TIAB] OR polymorphism and DAT activity in the brain.32 single-photon [TIAB] OR SPECT [TIAB] OR PET [TIAB] OR ‘positron emission In vivo imaging of DAT is particularly relevant for ADHD given tomography’[TIAB]) AND (genetic [TIAB] OR genotype [TIAB] OR genotypes that DAT is the target of stimulant and, subsequently, [TIAB] OR allele [TIAB] OR alleles [TIAB] OR polymorphism [TIAB]). If the a target protein for studies of pathophysiology. Clarifying the reference sections of any of these articles suggested additional articles, nature of the association of SLC6A3 variants would provide a key these were also examined. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) diagram in Figure 1 describes the step towards identifying part of ADHD’s pathophysiology. number of articles identified and their disposition. If the required data Depending upon the strength of the relationship, it could point were not available in relevant articles, we contacted authors for that data toward a means of parsing ADHD’s heterogeneity, which could or extracted it from a prior summary of a subset of relevant studies.32 have implications for treatment development. We computed separate meta-analyses for the PET and SPECT studies. The goal of the present study was to clarify the PET and SPECT Our meta-analysis used the random effects model of DerSimonian and imaging studies by updating Costa et al.’s32 meta-analysis in Laird,35 which computes a pooled standardized mean difference weighted 2 several ways. Costa et al.’s32 meta-analysis is limited in several by sample size. We use the I index to assess the heterogeneity of effect 36 ways. It ignored two available SPECT studies,33,34 which could sizes. Its value lies between 0 and 100 and estimates the percentage of potentially add to our understanding of the magnitude of effects variation among effect sizes that can be attributed to heterogeneity. A significant I2 suggests that the effect sizes analyzed are not estimating and sources of heterogeneity and it was conducted prior to 30,31 the same population effect size. We used meta-analytic regression to publication of the two recent PET studies described above. assess the degree to which effect sizes varied with methodological Costa et al. did not (or could not) assess the degree to which features.37,38 The meta-analyses and meta-analytic regressions were heterogeneity of results could be accounted for by sample weighted by the reciprocal of the variance of the effect size. We used characteristics (affected vs. healthy), imaging method (PET vs. Egger’s39 method to assess for publication biases.

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 880 – 889 Meta-analysis of dopamine transporter gene associations SV Faraone et al 882 results across all observations. The test for publication bias was not Table 1. Description of studies providing data significant (t ¼ 1.3, P ¼ 0.2). Consistent with the finding of no Study Method Ligand Ethnicity heterogeneity, meta-analysis regression found that the SMDs were not associated with age (F(1, 9) ¼ 1.9, P ¼ 0.22), sex (F(1, 9) ¼ 1.4, Cheon34 SPECT [123I]IPT Korean P ¼ 0.26) brain region (F(6, 4) ¼ 2.80, P ¼ 0.17), affection status Contin87 SPECT [123I]FP-CIT C (F(1, 9) ¼ 0.02, P ¼ 0.88) or ligand (F(1, 9) ¼ 1.38, P ¼ 0.27). The non- Heinz88 SPECT [123I] b-CIT C significant PET findings should be viewed cautiously given that 41 Jacobsen SPECT [123I] b-CIT AA, C there were only two PET studies. Krause75 SPECT 89MTC-TRODAT–1 C 90 Figure 3 shows the results for SPECT studies. Across all studies, Lafuente SPECT [123I]FP-CIT C the SMD of 0.00 was not statistically significant (z ¼ 0.00, P ¼ 0.99). Lynch43 SPECT 89MTC-TRODAT–1 C, AA, other Martinez42 SPECT [123I]b-CIT AA, C The SMD was positive and significant for the healthy group (0.46, Shumay30 PET [11Cocaine]PET AA, C, Other z ¼ 2.1, P ¼ 0.035) and negative but not significant for the affected Spencer31 PET [11Altropane] AA, C group (–0.40, z ¼ 1.7, P ¼ 0.093). The test for publication bias was 2 Van Dyck40 SPECT [123I]b-CIT C not significant (t ¼ –2.0, P ¼ 0.06). The I statistic was high and Van de Giessen91 SPECT [123I] b-CIT C significant for the entire SPECT group (I2 ¼ 73.8%, Po0.0009). Although meta-analysis regression confirmed that one source of Abbreviatons: AA, African–American; C, Caucasian; PET, positron emission tomography; SPECT, speeded photon emission tomography. heterogeneity was affection status (F(1, 13) ¼ 5.04, P ¼ 0.04), heterogeneity remained high when analyzing the affected (I2 ¼ 71.2%, P ¼ 0.004) and healthy groups (I2 ¼ 63.1%, P ¼ 0.006) separately. Neither sex (F(1, 13) ¼ 0.1, P ¼ 0.8) nor age were RESULTS significant predictors of the SMDs (F(1, 13) ¼ 0.5, P ¼ 0.48). Table 1 gives the characteristics of each study’s sample. Ten We explored two additional sources of heterogeneity. As studies used SPECT and these used four different ligands. Two Figure 3 shows, the findings of Cheon et al.34 are markedly studies used PET, one using 11Altropane as the ligand and the discrepant from the other SPECT studies. Their study was the only other 11Cocaine. Six studies were of Caucasian samples, one one to study children (seven 10/10R homozygotes, two 9/10 used a Korean sample and the others were of mixed ethnicity. heterozygotes and two 10/11 heterozygotes). They were the only As reported by Costa et al.,32 data from van Dyck et al.40 investigators to use [123I]IPT as a ligand and basal ganglia as the overlapped with data reported by Jacobsen et al.41 and Martinez region of interest. Consistent with this, meta-analysis regression et al.42 For our meta-analyses, we used the non-overlapping found significant effects of brain region F(4, 10) ¼ 3.86, P ¼ 0.038) van Dyck40 data that had been presented by Costa et al.32 The and ligand F(3, 11) ¼ 5.9, P ¼ 0.01). After excluding Cheon et al.34 twelve studies comprised 511 subjects, 125 from PET studies and from the analysis, the overall I2 statistic remained high and 386 from SPECT studies. significant for the entire SPECT group (I2 ¼ 64.7%, Po0.001) but Table 2 describes the data used in the meta-analysis. Some was reduced to non-significance in the affected group (I2 ¼ 10.9%, studies provided data for both healthy and affected groups. P ¼ 0.35). The SMDs for the overall group and the affected group Several disorders were studied: ADHD, schizophrenia, alcoholism did not achieve significance (P’s40.25). and Parkinson’s disease. Most studies imaged the striatum or As suggested by the meta-analysis regression, another potential subareas of the striatum. The only exception was Cheon et al.,34 source of heterogeneity among SPECT studies was the choice of who studied the left and right basal ganglia. If a study provided ligand. However, only one ligand, [123I]b-CIT, was used with data on a structure (for example, putamen) and subdivisions of the sufficient frequency to be analyzed separately. In this analysis, the structure (for example, left and right putamen), we only analyzed overall I2 statistic remained high and significant for the entire the substructures. Each study compared a 10 repeat (10R) SPECT group (I2 ¼ 70.7%, Po0.001) but was low and non- genotype group with a 9 repeat (9R) genotype group. significant when separately considering the affected (I2 ¼ 15.9%, As Table 2 shows, nearly all studies defined the 10R group as P ¼ 0.35) and healthy groups (I2 ¼ 0.0%, P ¼ 0.47). The SMD was those carrying two 10R alleles (10R/10R). The only exception was significant and positive for the healthy group (0.67, z ¼ 5.2, Lynch et al.,43 who also included 10R heterozygotes carrying a rare Po0.009). In contrast, it was nearly significant and negative for the allele other than the 9R allele. The 9R genotype groups primarily affected group (–0.52, z ¼ 1.9, P ¼ 0.057). comprised 9R homozygotes and 9R/10R heterozygotes. However, some rare genotypes that did not include a 10R allele were also included in some samples. DISCUSSION In the analyses that follow, differences between genotype The prior meta-analysis of the association between the SLC6A3 groups are expressed as the standardized mean difference (SMD) 30UTR polymorphism with in vivo striatal DAT binding in humans effect size. Positive SMDs favor the 9R allele as being associated concluded that there was no evidence to support the hypothe- with increased DAT activity; negative SMDs favor the 10R allele. sized association. In contrast, by analyzing a larger sample and Figure 2 shows the results for PET studies. In Figure 2, the dot incorporating relevant covariates, our meta-analyses have yielded gives the relative risk and the horizontal line gives the 95% several firm conclusions based on 12 studies comprising 511 confidence interval. The diamonds give the weighted SMD across subjects. Although limited by the existence of only two studies, studies and the width of the diamond gives its 95% confidence the PET studies provided highly significant evidence indicating interval. The first two diamonds give pooled results for the that the 9R allele is associated with increased DAT binding in the affected and healthy subgroups. The last diamond gives results for striatal brain regions. Although the effect size was similar for the all studies with the left and right ends of the diamond marking the healthy and affected samples, the latter effect size did not achieve 95% confidence interval. statistical significance, probably due to the smaller number of Across all PET study observations, the SMD of 0.31 was observations. Notably, all observations from PET studies, albeit statistically significant (z ¼ 3.61, Po0.0009). The SMD was sig- individually not significant, were consistent with the 9R allele nificant for the healthy group (0.31, z ¼ 3.1, P ¼ 0.002). Although predicting greater DAT binding in humans. This consistency of the magnitude of the SMD was the same for the affected (all results was reflected in finding zero heterogeneity across these ADHD) group, it was only marginally significant, perhaps due to the observations. smaller sample size (0.33, z ¼ 1.9, P ¼ 0.056). The low and non- In contrast to the consistency of findings across PET study significant I2 statistic of 0.0% indicates very low heterogeneity of observations, the data from SPECT studies was highly and

Molecular Psychiatry (2014), 880 – 889 & 2014 Macmillan Publishers Limited &

04McilnPbihr Limited Publishers Macmillan 2014 Table 2. Data used in the meta-analysis

9R genotype N with 9R Mean of 9R SD of 9R 10R genotype N with 10R Mean of 10R SD of 10R group Study Sample Mean age and range Brain region group genotypes group group group genotypes group

Shumay30 Healthy 34.5 Caudate 9R/9R 9R/10R 9R/11R 47 0.79 0.14 10R/10R 44 0.76 0.15 AA: 21.5–45.5 CE: 20.5–49.5 Others: 22.2–40.3 Shumay30 Healthy 34.5 Putamen 9R/9R 9R/10R 9R/11R 47 0.96 0.13 10R/10R 44 0.9 0.13 AA: 21.5–45.5 CE: 20.5–49.5 Others: 22.2–40.3 Shumay30 Healthy 34.5 Ventral 9R/9R 9R/10R 9R/11R 47 0.81 .2 10R/10R 44 0.8 0.19 AA: 21.5–45.5 striatum CE: 20.5–49.5 Others: 22.2–40.3 Spencer31 Healthy 27.8 Left caudate 9R/9R 9R/10R 9R/11R 18 3.589 0.847 10R/10R 16 3.336 0.6202 18–49

Spencer31 ADHD 32.8 Left caudate 9R/9R 9R/10R 9R/11R 15 3.401 0.7424 10R/10R 19 2.988 0.3448 18–52.3 Spencer31 Healthy 27.8 Left putamen 9R/9R 9R/10R 9R/11R 15 3.349 0.643 10R/10R 19 3.174 0.3701 18–49 Spencer31 ADHD 32.8 Left putamen 9R/9R 9R/10R 9R/11R 18 3.386 0.6246 10R/10R 16 3.312 0.4996 18–52.3 Spencer31 Healthy 27.8 Right caudate 9R/9R 9R/10R 9R/11R 15 3.266 0.5557 10R/10R 19 3.051 0.343 18–49 Spencer31 ADHD 32.8 Right caudate 9R/9R 9R/10R 9R/11R 18 3.664 0.62 10R/10R 16 3.247 0.538 18–52.3 Spencer31 Healthy 27.8 Right putamen 9R/9R 9R/10R 9R/11R 15 3.296 0.5265 10R/10R 19 3.19 0.335 18–49 Spencer31 ADHD 32.8 Right putamen 9R/9R 9R/10R 9R/11R 18 3.355 0.7085 10R/10R 16 3.245 0.6071 18–52.3 SPECT [123I]b-CIT studies 42 ±

Martinez Schizophrenia 39.2 9 Striatum 9R/9R 9R/10R 7 7.9 2.1 10R/10R 14 7.8 1.5 associations Faraone gene SV transporter dopamine of Meta-analysis Martinez42 Healthy 40.0±9 Striatum 9R/9R 9R/10R 9R/11R 6 8.2 1.5 10R/10R 15 8.2 1.3 Van Dyck40 Healthy 49.9 Striatum 9R/9R 9R/10R 30 7.5 1.9 10R/10R 35 6.6 1.6 18–88 Van de Giessen91 Healthy/prior 22.0 Putamen 9R/9R 9R/10R 32 11.4 2.72 10R/10R 45 9.6 2.06 Ecstasy use 18–35 Van de Giessen91 Healthy 22.0 Caudate 9R/9R 9R/10R 32 13.5 3.25 10R/10R 45 11.4 2.42 18–35 al et Heinz88 Alcoholics/controls 36.7±7 Caudate 9R/10R 10 2399 750 10R/10R 15 2895 724 Heinz88 Alcoholics/controls 36.7±7 Putamen 9R/10R 10 2226 635 10R/10R 15 2840 743 Jacobsen41 Healthy 37±9.3 Striatum 9R/9R 9R/10R 9 8.2 1 10R/10R 18 7.1 1

SPECT [123I]FP-CIT studies Contin87 Parkinson’s 60.4 9–10 (N ¼ 14): 63±11 9–9 (N ¼ 6): Putamen 9R/9R 9R/10R 20 0.92 0.54 10R/10R 16 0.91 0.63 58±9 10–10 (N ¼ 16)59±7 oeua scity(04,80–889 – 880 (2014), Psychiatry Molecular Lafuente90 Schizophrenia 24.0 Striatum 9R/9R 9R/10R 8 4.6 0.5 10R/10R 6 4.4 0.5 9–9: 26±0 9–10: 22.6±3 10–10: 24.8±6.5 SPECT89 MTC-TRODAT–1 studies Krause75 ADHD 37.6 Striatum 9R/9R 9R/10R 12 1.31 .27 10R/10R 17 1.28 .34 19–54 Lynch43 Healthy 46.5 Striatum 9R/10R 9R/*R 49 1.175 .26 10R/10R 10R/*R 32 1.25 .22 18.3–83.3 Lynch43 Parkinson’s 60.8 Striatum 9R/10R 9R/*R 58 .65 .215 10R/10R 10R/*R 42 .65 .2 38.3–84.2 SPECT [123I]IPT studies Cheon34 ADHD 9.8 Left basal 10R/11R 9R/10R 4 2.22 1.39 10R/10R 7 6.91 2.5 6–12 ganglia Cheon34 ADHD 9.8 Right basal 10R/11R 9R/10R 4 2 1.2 10R/10R 7 7.1 1.95 6–12 ganglia

Abbreviations: 9R, 9 repeat allele; 10R, 10 repeat allele; *R, other allele; xR/yR ¼ x repeat allele/y repeat allele genotype. ‘Mean of 9R group’ refers to the mean DAT activity for the 9R group and the ‘s.d.’ column gives the standard deviations. 883 Meta-analysis of dopamine transporter gene associations SV Faraone et al 884 significantly heterogeneous. Meta-analysis regression suggested 9R allele in the 30UTR as a regulator of SLC6A3 expression. The that heterogeneity among SPECT studies could be due to the brain association of the 9R allele with higher DAT binding site density region, affection status or choice of ligand. Due to the distribution could result from a number of possible pathways: interaction of these features across samples, it was not possible to assess their of the 30UTR with regulatory proteins or microRNAs, shunting of joint effects. However, when the analysis was limited to [123I]b-CIT, mRNA to distinct compartments in the neuron, regulation of the most commonly used ligand, which was used in 5 of the 10 mRNA stability, turnover, increases in translational efficiency,52,53 SPECT studies, stratifying by affection status dramatically reduced or even remote interaction with regulatory elements of the heterogeneity of results. This sub-analysis was consistent with other genes that may affect SLC6A3 expression, stability and the PET studies in finding a significant association of the 9R allele trafficking.54 A parsimonious interpretation of the functional with increased DAT activity for the healthy group. consequences of elevated DAT is more efficient in clearing Unlike the PET studies, which showed a trend association of the extracellular dopamine, yielding lower extracellular levels and 9R allele and DAT activity for the affected group, for SPECT studies, reduced dopamine signaling. It may be feasible to test this the effect for the affected group favored the 10R allele and was hypothesis by monitoring extracellular dopamine indirectly, using nearly significant. This difference is likely due to the difference in displacement of D2 receptor occupancy with [11C]raclopride as a the composition of the affected sub-samples. For the PET studies, surrogate for direct measures of dopamine, in 9R and 10R carriers. these were all ADHD patients. For SPECT studies, a variety of The role of the DAT in pathophysiology and therapeutic disorders were studied: ADHD, alcoholism, schizophrenia and response has catalyzed research into the association of DAT Parkinson’s disease. density with pathology, and the interrogation of whether SLC6A3 Our results suggest that heterogeneity of findings across studies alleles are relevant to SLC6A3 expression, regulation and can be explained by methodological differences. DAT binding membrane transporter density. Based on a meta-analysis of 9 varies considerably in different brain regions, and within the SPECT and PET studies, Fusar-Poli et al.15 concluded that DAT striatum, density gradients are detected from superior to inferior, density was 14% higher in ADHD patients compared with controls. medial to lateral and anterior to posterior regions, particularly in Mill et al.25 measured dopamine transporter mRNA levels in the the caudate nucleus.44 Several studies report differences between cerebellum, temporal lobe and lymphocytes and reported that DAT binding in caudate and putamen.31 In a study of ADHD, dopamine transporter mRNA expression increased with the Jucaite et al.45 reported decreased DAT binding in the midbrain number of 10R alleles. Brookes et al.22 also found that the 10R but increased DAT binding in the caudate. Due to poor spatial allele increased levels of SLC6A3 mRNA in human postmortem discrimination in SPECT, most of the SPECT studies report a midbrain tissue. However, Zhou et al.24 and Pinsonneault et al.23 combined striatal value that may obscure apparent effects, if failed to find a differential effect of 9R and 10R alleles on SLC6A3 discrete regional expression of SLC6A3 is modified by the VNTR expression in postmortem brains. (Table 2, Figure 3). In vitro studies of the functional effects of the 30UTR have One must also consider variability among DAT radioligands produced conflicting results. A study of HEK–293 cells reported when interpreting the heterogeneity of findings across studies. that cells containing the 10R allele had a SLC6A3 expression that Although the core structures of these potent DAT radioligands are was 50% greater than cells with the 9R allele.18 Similarly, using a derived from CFT (or WIN 35,428), only Altropane contains a luciferase reporter system in COS–7 cells Fuke et al.17 found 4-fluoro substituent on the phenyl ring, rendering it relatively greater SLC6A3 mRNA expression for constructs containing the selective, 28-fold, for the DAT over the and 10R allele compared with other alleles. In contrast to these displaying favorable kinetic properties.46,47 Among all the studies findings suggesting that the 10R allele is associated with greater included in our meta-analysis, only Spencer et al.31 used altropane. transcription, using the human neuroblastoma cell line, Inoue- The more commonly used ligands, CIT, IPT, FP-CIT and TRODAT, Murayama, et al.21 reported that the 9R allele led to more SLC6A3 contain 4-chloro or 4-iodo substituents, which markedly reduce mRNA expression than the 10R allele. Increased DAT gene DAT:serotonin transporter specificity (TRODAT 3:1, CIT: 1:1, FP-CIT expression associated with the 9R allele was also reported by 3:1, IPT: 5:1) and require varying lengths of time for the Miller and Madras55 using HEK–293 cells transfected with 30UTR radioligand to wash out from serotonin transporter sites and variants using two different promoters. Greenwood and Kelsoe20 other non-specific sites.48–50 In addition, differences in lipophilic found that the 9R allele led to non-significantly increased properties may affect the ability of the ligands to detect transcriptional regulation in dopaminergic substantia nigra intracellular vs. membrane-bound DAT. (SN4741) cell lines. Mill et al.19 also reported non-significantly Combining disorders may also obscure findings, as different greater mRNA expression for 9R compared with 10R constructs pathophysiologies may be associated with greater or lesser DAT evaluated in SH-SY5Y and HEK–293 cell lines. In agreement with binding. These could be due to either other genetic and these findings, if the 9-repeat allele was subcloned upstream of environmental risks or medications that alter membrane SLC6A3 the viral promoter coupled to a green fluorescent protein reporter, expression. Other variables that are hard to control for include age the construct enhanced transcription in an immortalized dopami- and smoking status, which are known to affect DAT dramati- nergic cell line derived from mouse substantia nigra.56 cally.40,51 DAT densities also decline as a function of age,30 and it These differences among studies could be due to using remains unclear whether the expression levels of 9R and 10R DAT different experimental constructs to introduce the mutation into alleles are equally affected by age. In our analyses, age was not cell lines, the amount of flanking sequence included in the predictive of SMDs, which suggests that age effects do not construct, choice of reporter gene, along with variable presence of moderate the effects of SLC6A3 alleles. This finding is, however, other SLC6A3 transcription regulators across different cell tempered by the fact that we only had access to mean ages from lines.19,57 Clarifying this issue will require detailed analyses of each study, which ranged from 9.8 to 60.8. Shumay et al.’s30 PET polymorphisms of length or of single nucleotides in the DAT gene study found that the association of 30UTR genotypes and DAT that conceivably contribute to the dynamic processes regulating binding was significant across all regions (caudate, putamen, DAT density in the brain. The grouping together of multiple alleles ventral striatum) for younger subjects but not in older subjects by the number/length of repeat sequences of the 30UTR could despite the use of comparable sample sizes for both groups. Their mask the relevance of other sequence variations, which contribute study also found that the age-related decline in DAT levels was to DAT gene regulation, SLC6A3 expression levels and phenotype, greatest for carriers of the 9R/9R 30UTR genotype. either in conjunction with, or independently of the 30UTR. The strength of the PET-derived data together with consistency The inconsistencies from in vitro and postmortem brain with SPECT imaging data that used CIT as a probe, insinuates the expression studies support in vivo imaging as a direct method

Molecular Psychiatry (2014), 880 – 889 & 2014 Macmillan Publishers Limited Meta-analysis of dopamine transporter gene associations SV Faraone et al 885 Values greater than zero suggest the Brain Author/Date Sample 9R allele is associated with increased Region DAT binding Diseased Spencer 2013 Lft Caudate ADHD Spencer 2013 Lft Putamen ADHD Spencer 2013 Rt Caudate ADHD Spencer 2013 Rt Putamen ADHD Subtotal (I-squared = 0.0%, p = 0.627) . Healthy Shumay 2011 Caudate Healthy Shumay 2011 Putamen Healthy Shumay 2011 Ventral Striatum Healthy Spencer 2013 Lft Caudate Healthy Spencer 2013 Lft Putamen Healthy Spencer 2013 Rt Caudate Healthy Spencer 2013 Rt Putamen Healthy Subtotal (I-squared = 0.0%, p = 0.674) . Overall (I-squared = 0.0%, p = 0.834)

-1 0 1 2 Standardized Mean Difference Figure 2. Meta-analysis of the dopamine transporter gene 30UTR VNTR polymorphism with in vivo dopamine transporter activity assessed by PET. Note: For each comparison, the dot gives the relative risk and the horizontal line gives the 95% confidence interval; the diamonds give the weighted SMD across studies and the width of the diamond gives its 95% confidence interval. The first two diamonds give pooled results for the affected and healthy subgroups. The last diamond gives results for all studies with the left and right ends of the diamond marking the 95% confidence interval. I-squared is a measure of heterogeneity among studies.

Values greater than zero suggest the Brain Author/Date Sample 9R allele is associated with increased Region DAT binding Diseased Cheon 2005 Lft Basal Ganglia ADHD Cheon 2005 Rt Basal Ganglia ADHD Contin 2004 Putamen Parkinson's Heinz 2000 Caudate Alcoholics/Controls Heinz 2000 Putamen Alcoholics/Controls Krause 2006 Striatum ADHD Lafuente 2007 Striatum Schizophrenia Lynch 2002 Striatum Parkinson's Martinez 2001 Striatum Schizophrenia Subtotal (I-squared = 63.1%, p = 0.006) . Healthy Jacobsen 2000 Striatum Healthy Lynch 2002 Striatum Healthy Martinez 2001 Striatum Healthy Van Dyck 2005 Striatum Healthy Van de Giessen 2009 Caudate Healthy Van de Giessen 2009 Putamen Healthy Subtotal (I-squared = 71.2%, p = 0.004) . Overall (I-squared = 73.8%, p = 0.000)

-5 -4 -3 -2 -1 0 1 2 Standardized Mean Difference Figure 3. Meta-analysis of the dopamine transporter Gene 30UTR VNTR polymorphism with in vivo dopamine transporter activity assessed by SPECT. Note: For each comparison, the dot gives the relative risk and the horizontal line gives the 95% confidence interval; The diamonds give the weighted standardized mean difference across studies and the width of the diamond gives its 95% confidence interval. The first two diamonds give pooled results for the affected and healthy subgroups. The last diamond gives results for all studies with the left and right ends of the diamond marking the 95% confidence interval. I-squared is a measure of heterogeneity among studies. to monitor the relevance of 9R and 10R genotypes on SLC6A3 was not, however, an association between SLC6A3 genotypes and regulation and function. In a functional magnetic resonance striatal hypoactivation. In a magnetic resonance spectroscopy imaging study, ADHD patients homozygous for the 10R allele study, Sherk et al28 reported that the 10R allele was associated showed significant hypoactivation in the left dorsal anterior with higher ratios of NAA/Cho and NAA/Cr in the left putamen. cingulate cortex compared with 9R carriers.26 In another functional They concluded that the 30UTR VNTR polymorphism modulates magnetic resonance imaging study, carriers of a haplotype dopaminergic activity, and neuronal function in putamen. including the 10R allele showed differentially modulated neural Our meta-analyses suggest that, in humans, the 9R allele of the activation to reward-predicting cues in the caudate nucleus.58 In a 30UTR polymorphism leads to increased DAT activity in the striatal sample of adults, Hoogman et al.59 found that the 9–6 haplotype brain regions. These results imply that some of the DAT (and of SLC6A3 was associated with ADHD and that ADHD adults therefore dopamine) regulation could be due to the presence (or showed striatal hypoactivation during reward anticipation. There absence) of the 9R allele. The relationships among the 30UTR

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 880 – 889 Meta-analysis of dopamine transporter gene associations SV Faraone et al 886 polymorphism, DAT binding and pathophysiology remain com- previous studies. Moreover, DAT is expressed in a region-specific plex. This complexity is well illustrated by the ADHD literature for manner in the brain81 and demonstrates an age-dependent which meta-analyses show: (a) significantly increased DAT density profile,82 with multiple alternative transcription initiation and in ADHD, which was greatest for those having had prior stimulant splice isoforms existing. The SLC6A3 gene region is enriched for treatment;15 (b) an increased prevalence of the 10R allele in ADHD transcription factor and miRNA-binding sites and DNA youth;8 and (c) an increased prevalence of the 9R allele in ADHD methylation sites. Many of these regulatory sites are co-localized adults.60 Because the positive studies in our meta-analysis all used with known variants such as sequence repeats, single-nucleotide adult samples, our results are consistent with the adult association polymorphisms (SNPs) and copy number variations.80 studies. We cannot, however, explain why the 10R allele has been For example, the number of the 30UTR VNTR repeats can change associated with ADHD in youth, which is either a false-positive the length of the transcribed mRNA, which may alter the secondary finding or reflects the complex regulation of SLC6A3, which we structure and degradation rate of the mRNA. This may also alter the discuss below. Given current sample sizes, reconciling apparent efficiency of the miRNA-binding sites due to changes of sequence genetic differences between childhood and persistent ADHD has and of secondary structure. This cascade of signaling could not been feasible. It is, however, notable that the only in vivo DAT continue with changes in transcription and degradation rates. imaging study to find a significant association of the 10R allele Furthermore, miRNA expression itself is often tissue specific and with DAT binding was also the only study of children. This pattern developmentally regulated.83 Some miRNAs simultaneously is consistent with the pattern of genetic association seen in adult interact with both the 30 and 50UTR regions.84 Shumay et al80 and child studies. showed the presence of such sites in the 30UTR region of SLC6A3, None of the studies analyzed addressed other mechanisms that indicating that variations in the 50 regulatory region of the gene might influence DAT binding. Additional mechanisms were may influence the function of the 30UTR VNTR via long-range implicated in a study by our group, which reported that, while interactions. Consistent with this, Brookes et al.1 reported repli- the 30UTR polymorphism increased DAT binding regardless of cated associations between ADHD and SNPs in the 50 regulatory ADHD status, ADHD made an additional, independent contribution region of SLC6A3. Drgon et al.85 reported that haplotypes of two to DAT binding.31 This suggests that there are additional ADHD- SNPs in the 50 regulatory region were associated with in vivo associated genetic or non-genetic mechanisms that influence DAT DAT activity measured by [11Cocaine]PET and also with striatal binding. For example, DAT is constitutively recycled through the DAT activity in postmortem brain samples. endosome.61 Although majority of DAT is sequestered intracellularly Signals from the ENCODE chromatin interaction analysis with in the recycling endosome, only membrane-associated DAT is paired-end tag sequencing (ChIA-PET) also indicate various functionally available for the reuptake of dopamine. An endosome long-range physical interactions of the 30UTR VNTR. By changing sodium–hydrogen exchanger protein, encoded by SLC9A9,has the length and sequence of the mRNA 30UTR, the VNTR may alter been implicated in ADHD62–66 and disruptive genetic variants of these long-range interactions in synergy with other 50-haplotypes SLC9A9 have been found in ADHD and autism cases.67,68 Ameta- that together regulate SLC6A3 transcription and splicing. These analysis of ADHD GWAS data sets implicated the CHMP7 gene long-range regulatory interactions, mediated through complex (charged multivesicular body protein 7), another protein involved in transcription factor interactions, are often sensitive to cell-type endosomal sorting/recyling pathway.69 These findings suggest specificity and developmental stage.86 These interactions offer that endosomal pathway genes may be compromised in ADHD another explanation for some of the discrepancies seen between and that DAT membrane density could be altered due to mutations adults and children in genetic association studies and among the in these genes.70,71 in vitro studies. The variability of our DAT binding findings in affected vs. Our conclusions are limited by methodological issues. Because healthy subjects, in child vs. adult samples and in various different only two PET studies were available, the power for these studies, brain regions could be influenced by both genetic and non- which comprised 125 subjects, was lower than the power for the genetic factors, as well as their interactions, about which we do SPECT studies, which comprised 386 subjects. Although this low not currently have a full understanding. Non-genetic mechanisms power does not vitiate the statistically significant results, the lack that alter the striatal DAT density include , cigarette of significance for the affected group should be viewed cautiously. smoking and alcohol consumption.51,72–74 The effect of stimulant A range of disorders in which dopamine dysregulation has been medications on DAT density is of considerable interest given implicated (Parkinson’s schizophrenia, ADHD, alcoholism) were Fusar-Poli’s15 report that increased DAT density in ADHD could be represented in our analyses. The variability in data from these accounted for by stimulant treatment. Their conclusion, however, cohorts may conceivably reflect DAT regulatory processes, which has been questioned due to incorrect coding of medication compensate for dysfunctional dopamine signaling. Cheon et al.’s status.16 Our study cannot shed much light on this issue because study,34 the most prominent outlier, was the only study conducted only three of the studies examined ADHD patients and these all in children and used IPT as a SPECT probe. IPT shows an unusually used treatment-naı¨ve samples.31,34,75 high sensitivity to age-dependent DAT decline.49 Information on There are several potential genetic mechanisms underlying DAT the age of each 9R or 10R subject would improve interpretation of variability. Some studies suggest that the effects of the SLC6A3 these data. Like all meta-analyses, our analyses of covariates were 30UTR VNTR are limited to specific haplotypes formed with an limited by the information provided in the papers we reviewed. intron 8 VNTR.2,9,29,76–78 In their study of DAT density using Notably, we did not have sufficient information about any single [11Cocaine]PET, Shumay et al.,30 showed that the intron 8 VNTR disorder to draw firm conclusions about their potential was associated with DAT levels in caudate and putamen. In moderating effects on SLC6A3 DAT associations. As is apparent contrast, Guindalini et al.79 using SPECT with TRODAT–1 did not from the Tables, our analyses were also limited by the variability of detect this association. Haplotype analyses by Shumay et al.,30 brain regions across studies. None of the studies in the meta- suggested that higher DAT levels were associated with the 5R analysis adjusted their analyses for genomic background using intron 8 allele. Thus, it may be the 10R–5R haplotype formed by ancestrally informative SNPs. We could not correct the meta- the 30UTR/intron8 haplotype is worthy of further study as the analysis for ethnicity (a coarse measure of genomic background), variant increases DAT density. as there were too many ‘mixed’ ethnic samples. If the SLC6A3 SLC6A3 has not been highly conserved during evolution, polymorphism we studied is associated with genomic background especially as regards the 30UTR VNTR region.80 This would be and if genomic background is associated with DAT availability, expected to cause variability in gene expression and functionality. these results could be spurious. Caution about ethnic effects is Such effects could contribute to the inconsistencies among suggested by the work of Shumay et al.,30 which found

Molecular Psychiatry (2014), 880 – 889 & 2014 Macmillan Publishers Limited Meta-analysis of dopamine transporter gene associations SV Faraone et al 887 significantly different SLC6A3 genotype distributions between Alza, AstraZeneca, Bristol Myers Squibb, Celltech, Cephalon, Eli Lilly and Co., Esai, Caucasians and African–Americans. In their study, the association Forest, Glaxo, Gliatech, Janssen, McNeil, Merck, NARSAD, NIDA, New River, NICHD, between SLC6A3 genotypes and DAT density was significant for NIMH, Novartis, Noven, Neurosearch, Organon, Otsuka, Pfizer, Pharmacia, The Caucasians but not African–Americans. Prechter Foundation, Shire, The Stanley Foundation, UCB Pharma, Inc. and Wyeth. Moreover, heterogeneity of genomic background or of envir- Yanli Zhang-James, MD, PhD, does not have any conflict of interest. onmental exposures relevant to DAT binding might have accounted for the heterogeneity of findings if they had been REFERENCES measured. We also could not adjust our analyses for smoking, which was not consistently reported. Taken together, these 1 Brookes KJ, Xu X, Anney R, Franke B, Zhou K, Chen W et al. Association of ADHD problems limited our ability to find significant covariate effects, with genetic variants in the 5’-region of the dopamine transporter gene: Evidence for allelic heterogeneity. Am J Med Genet B Neuropsychiatr Genet 2008; 147B: but they would not have created spurious results. 1519–1523. Our results along with the limitations of our work highlight 2 Asherson P, Brookes K, Franke B, Chen W, Gill M, Ebstein RP et al. Confirmation directions for future functional studies of genetic variants using that a specific haplotype of the dopamine transporter gene is associated with in vivo studies of DAT density. PET imaging has clearly been most combined-type ADHD. Am J Psychiatry 2007; 164: 674–677. effective in producing replicable results. Thus, attempting to 3 Roessner V, Sagvolden T, Dasbanerjee T, Middleton FA, Faraone SV, Walaas SI et al. ‘replicate’ these findings with other imaging methods would not Methylphenidate normalizes elevated dopamine transporter densities in an be appropriate. Future studies should genotype ancestrally animal model of the attention-deficit/hyperactivity disorder combined type, but informative SNPs to assure that differences in genomic back- not to the same extent in one of the attention-deficit/hyperactivity disorder ground do not affect results. Careful documentation of medica- inattentive type. Neuroscience 2011; 167: 1183–1191. 4 Mick E, Kim JW, Biederman J, Wozniak J, Wilens T, Spencer T et al. Family based tions, smoking history and alcohol use is also essential. And, given association study of pediatric bipolar disorder and the dopamine transporter the complex regulation of SLC6A3, genotyping tag SNPs in all gene (SLC6A3). Am J Med Genet B Neuropsychiatr Genet 2008; 147B: 1182–1185. regulatory regions would be a real advance. 5 Tarnok Z, Ronai Z, Gervai J, Kereszturi E, Gadoros J, Sasvari-Szekely M et al. Despite these limitations, our meta-analyses suggest that in Dopaminergic candidate genes in Tourette syndrome: association between tic human adults, the 9R allele of the 30UTR polymorphism of the DAT severity and 3’UTR polymorphism of the dopamine transporter gene. Am J Med gene regulates dopamine activity in the striatal brain regions Genet B Neuropsychiatr Genet 2007; 144B: 900–905. independent of the presence of ADHD or other disorders. Future 6 Du Y, Nie Y, Li Y, Wan YJ. The association between the SLC6A3 VNTR 9-repeat in vivo neuroimaging studies of the DAT should attend to the allele and alcoholism-a meta-analysis. Alcohol Clin Exp Res 2011; 35: 1625–1634. methodological features we highlighted as contributing to the 7 Gamma F, Faraone SV, Glatt SJ, Yeh YC, Tsuang MT. Meta-analysis shows schi- zophrenia is not associated with the 40-base-pair repeat polymorphism of the heterogeneity of findings across studies. dopamine transporter gene. Schizophr Res 2005; 73: 55–58. 8 Faraone SV, Mick E. Molecular genetics of attention deficit hyperactivity disorder. Psychiatr Clin North Am 2010; 33: 159–180. CONFLICT OF INTEREST 9 Franke B, Vasquez AA, Johansson S, Hoogman M, Romanos J, Boreatti-Hummer A In the past year, Dr Faraone received consulting income and/or research support et al. Multicenter analysis of the SLC6A3/DAT1 VNTR haplotype in persistent from Akili Interactive Labs, VAYA Pharma and SynapDx and research support from the ADHD suggests differential involvement of the gene in childhood and persistent National Institutes of Health (NIH). His institution is seeking a patent for the use of ADHD. Neuropsychopharmacology 2010; 35: 656–664. sodium–hydrogen exchange inhibitors in the treatment of ADHD. In previous years, 10 Kuczenski R, Segal DS. Exposure of adolescent rats to oral methylphenidate: he received consulting fees or was on Advisory Boards or participated in continuing preferential effects on extracellular and absence of sensitization medical education programs sponsored by: Shire, Alcobra, Otsuka, McNeil, Janssen, and cross-sensitization to . J Neurosci 2002; 22: 7264–7271. Novartis, Pfizer and Eli Lilly. Dr Faraone receives royalties from books published by 11 Segal DS, Kuczenski R. Repeated binge exposures to and Guilford Press: Straight Talk about Your Child’s Mental Health and Oxford University methamphetamine: behavioral and neurochemical characterization. J Pharmacol Press: Schizophrenia: The Facts. In the last two years, Dr Thomas Spencer has been an Exp Ther 1997; 282: 561–573. Advisor or on an Advisory Board of the following sources: Alcobra, Ironshore, the 12 Volkow ND, Wang GJ, Fowler JS, Ding YS. Imaging the effects of methylphenidate Department of Defense and the National Institute of Mental Health. In the last two on brain dopamine: new model on its therapeutic actions for attention-deficit/ years, Dr Thomas Spencer has received research support from of the following hyperactivity disorder. Biol Psychiatry 2005; 57: 1410–1415. sources: Shire Laboratories Inc, Cephalon, Eli Lilly & Company, Janssen, McNeil 13 Riccardi P, Baldwin R, Salomon R, Anderson S, Ansari MS, Li R et al. Estimation Pharmaceutical, Novartis Pharmaceuticals and the Department of Defense. In of baseline dopamine D(2) receptor occupancy in striatum and extrastriatal previous years, Dr Thomas Spencer has received research support from, has been regions in humans with positron emission tomography with [(18)F] Fallypride. Biol a speaker for or on a speaker bureau or has been an Advisor or on an Advisory Board Psychiatry 2008; 63: 241–244. of the following sources: Shire Laboratories, Inc, Eli Lilly & Company, Glaxo-Smith 14 Spencer T, Biederman J, Ciccone P, Madras B, Dougherty D, Bonab A et al. A PET Kline, Janssen Pharmaceutical, McNeil Pharmaceutical, Novartis Pharmaceuticals, study examining pharmacokinetics, detection and likeability, and dopamine Cephalon, Pfizer and the National Institute of Mental Health. Dr Spencer receives transporter receptor occupancy of short and long-acting orally administered research support from Royalties and Licensing fees on copyrighted ADHD scales formulations of methylphenidate in adults. Am J Psychiatry 2006; 163: 387–395. through MGH Corporate Sponsored Research and Licensing. Dr Spencer has a US 15 Fusar-Poli P, Rubia K, Rossi G, Sartori G, Balottin U. Striatal dopamine transporter Patent Application pending (Provisional Number 61/233686), through MGH corporate alterations in ADHD: pathophysiology or adaptation to psychostimulants? A meta- licensing, on a method to prevent stimulant abuse. Bertha K Madras, PhD, has the analysis. Am J Psychiatry 2012; 169: 264–272. following financial interests: She is patent holder of 19 patents, including 11C- or 16 Spencer TJ, Madras BK, Fischman AJ, Krause J, La Fougere C. Striatal dopamine 131I-altropane, other DAT imaging agents and DAT inhibitors, the majority of which transporter binding in adults with ADHD. Am J Psychiatry 2012; 169: 665 author are licensed to Alseres. Alseres licensed Altropane from Harvard University; Navidea reply 6. Biopharmaceuticals, a radiopharmaceutical developer, is evaluating an option to 17 Fuke S, Suo S, Takahashi N, Koike H, Sasagawa N, Ishiura S. The VNTR poly- license Altropane from Alseres. In the past year, she has received consulting fees from morphism of the human dopamine transporter (DAT1) gene affects gene Prexa Pharmaceuticals, NIDA, research support from NIDA, has been an advisor to expression. Pharmacogenomics J. 2001; 1: 152–156. NIDA Council, CDC, and a non-reimbursed advisor to the Hilton Foundation and 18 VanNess SH, Owens MJ, Kilts CD. The variable number of tandem repeats element Convecta. In 2012, she received speaker fees from the following sources: McGill in DAT1 regulates in vitro dopamine transporter density. BMC Genet 2005; 6:55. University, Dartmouth University, BOLD Coalition, Student Assistant Services and 19 Mill J, Asherson P, Craig I, D’Souza UM. Transient expression analysis of allelic royalties as editor or author of four books, from Cold Spring Harbor Press, variants of a VNTR in the dopamine transporter gene (DAT1). BMC Genet 2005; 6:3. Neuroscience-Net, American Psychological Association. Joseph Biederman, MD is 20 Greenwood TA, Kelsoe JR. Promoter and intronic variants affect the transcri- currently receiving research support from the following sources: Elminda, Janssen, ptional regulation of the human dopamine transporter gene. Genomics 2003; 82: McNeil, and Shire. In 2010, Dr Joseph Biederman did not receive any outside income. 511–520. In 2009, Dr Joseph Biederman received a speaker’s fee from the following sources: 21 Inoue-Murayama M, Adachi S, Mishima N, Mitani H, Takenaka O, Terao K et al. Fundacion Areces, Medice Pharmaceuticals and the Spanish Child Psychiatry Variation of variable number of tandem repeat sequences in the 3’-untranslated Association. In previous years, Dr Joseph Biederman received research support, region of primate dopamine transporter genes that affects reporter gene consultation fees or speaker’s fees for/from the following additional sources: Abbott, expression. Neurosci Lett 2002; 334: 206–210.

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 880 – 889 Meta-analysis of dopamine transporter gene associations SV Faraone et al 888 22 Brookes KJ, Neale BM, Sugden K, Khan N, Asherson P, D’Souza UM. Relationship 46 Madras BK, Gracz LM, Fahey MA, Elmaleh D, Meltzer PC, Liang AY et al. Altropane, between VNTR polymorphisms of the human dopamine transporter gene and a SPECT or PET imaging probe for dopamine neurons: III. Human dopamine expressionin post-mortem midbrain tissue. Am J Med Genet B Neuropsychiatr transporter in postmortem normal and Parkinson’s diseased brain. Synapse 1998; Genet 2007; 144B: 1070–1078. 29: 116–127. 23 Pinsonneault JK, Han DD, Burdick KE, Kataki M, Bertolino A, Malhotra AK et al. 47 Fischman AJ, Bonab AA, Babich JW, Palmer EP, Alpert NM, Elmaleh DR et al. Rapid Dopamine transporter gene variant affecting expression in human brain is detection of Parkinson’s disease by SPECT with altropane: a selective ligand for associated with bipolar disorder. Neuropsychopharmacology 2011; 36: 1644–1655. dopamine transporters. Synapse 1998; 29: 128–141. 24 Zhou Y, Michelhaugh SK, Schmidt CJ, Liu JS, Bannon MJ, Lin Z. Ventral midbrain 48 Abi-Dargham A, Gandelman MS, DeErausquin GA, Zea-Ponce Y, Zoghbi SS, correlation between genetic variation and expression of the dopamine trans- Baldwin RM et al. SPECT imaging of dopamine transporters in human brain with porter gene in -abusing versus non-abusing subjects. Addict Biol advance iodine–123-fluoroalkyl analogs of beta-CIT. J Nucl Med 1996; 37: 1129–1133. online publication, 26 October 2011; doi:10.1111/j.1369-1600.2011.00391.x 49 Mozley PD, Kim HJ, Gur RC, Tatsch K, Muenz LR, McElgin WT et al. Iodine–123-IPT (e-pub ahead of print). SPECT imaging of CNS dopamine transporters: nonlinear effects of normal aging 25 Mill J, Asherson P, Browes C, D’Souza U, Craig I. Expression of the dopamine on striatal uptake values. J Nucl Med 1996; 37: 1965–1970. transporter gene is regulated by the 3’UTR VNTR: evidence from brain and 50 Kung MP, Stevenson DA, Plossl K, Meegalla SK, Beckwith A, Essman WD et al. lymphocytes using quantitative RT-PCR. Am J Med Genet 2002; 114: 975–979. [99mTc]TRODAT–1: a novel technetium–99 m complex as a dopamine transporter 26 Brown AB, Biederman J, Valera EM, Doyle AE, Bush G, Spencer T et al. Effect imaging agent. Eur J Nucl Med 1997; 24: 372–380. of dopamine transporter gene (SLC6A3) variation on dorsal anterior cingulate 51 Krause KH, Dresel SH, Krause J, Kung HF, Tatsch K, Ackenheil M. Stimulant- function in attention-deficit/hyperactivity disorder. Am J Med Genet B like action of on striatal dopamine transporter in the brain of adults Neuropsychiatr Genet 2010; 153B: 365–375. with attention deficit hyperactivity disorder. Int J Neuropsychopharmacol 2002; 5: 27 Paloyelis Y, Asherson P, Mehta MA, Faraone SV, Kuntsi J. DAT1 and COMT effects 111–113. on delay discounting and trait impulsivity in male adolescents with attention 52 D’Souza UM, Craig IW. Functional polymorphisms in dopamine and serotonin deficit/hyperactivity disorder and healthy controls. Neuropsychopharmacology pathway genes. Hum Mutat 2006; 27: 1–13. 2010; 35: 2414–2426. 53 Hahn MK, Blakely RD. The functional impact of SLC6 transporter genetic variation. 28 Scherk H, Backens M, Schneider-Axmann T, Kraft S, Kemmer C, Usher J et al. Annu Rev Pharmacol Toxicol 2007; 47: 401–441. Dopamine transporter genotype influences N-acetyl-aspartate in the left 54 Sanyal A, Lajoie BR, Jain G, Dekker J. The long-range interaction landscape of gene putamen. World J Biol Psychiatry 2009; 10(4 Pt 2): 524–530. promoters. Nature 2012; 489: 109–113. 29 Rommelse NN, Altink ME, Arias-Vasquez A, Buschgens CJ, Fliers E, Faraone SV et al. 55 Miller GM, Madras BK. Polymorphisms in the 30-untranslated region of human A review and analysis of the relationship between neuropsychological measures and and monkey dopamine transporter genes affect reporter gene expression. Mol DAT1 in ADHD. Am J Med Genet B Neuropsychiatr Genet 2008; 147B: 1536–1546. Psychiatry 2002; 7: 44–55. 30 Shumay E, Chen J, Fowler JS, Volkow ND. Genotype and ancestry modulate brain’s 56 Michelhaugh SK, Fiskerstrand C, Lovejoy E, Bannon MJ, Quinn JP. The dopamine DAT availability in healthy humans. PLoS One 2011; 6: e22754. transporter gene (SLC6A3) variable number of tandem repeats domain enhances 31 Spencer TJ, Biederman J, Faraone SV, Madras BK, Bonab AA, Dougherty DD et al. transcription in dopamine neurons. J Neurochem 2001; 79: 1033–1038. Functional genomics of attention-deficit/hyperactivity disorder (ADHD) Risk 57 D’Souza UM, Russ C, Tahir E, Mill J, McGuffin P, Asherson PJ et al. Functional alleles on dopamine transporter binding in ADHD and healthy control subjects. effects of a tandem duplication polymorphism in the 5’flanking region of the Biol Psychiatry 2012. DRD4 gene. Biol Psychiatry 2004; 56: 691–697. 32 Costa A, Riedel M, Muller U, Moller HJ, Ettinger U. Relationship between SLC6A3 58 Paloyelis Y, Mehta MA, Faraone SV, Asherson P, Kuntsi J. Striatal sensitivity during genotype and striatal dopamine transporter availability: a meta-analysis of human reward processing in attention-deficit/hyperactivity disorder. J Am Acad Child single photon emission computed tomography studies. Synapse 2011; 65: 998–1005. Adolesc Psychiatry 2012; 51: 722–32 e9. 33 Lynch WJ, Roth ME, Carroll ME. Biological basis of sex differences in drug abuse: 59 Hoogman M, Onnink M, Cools R, Aarts E, Kan C, Arias Vasquez A et al. The preclinical and clinical studies. Psychopharmacology 2002; 164: 121–137. dopamine transporter haplotype and reward-related striatal responses in adult 34 Cheon KA, Ryu YH, Kim JW, Cho DY. The homozygosity for 10-repeat allele at ADHD. Eur Neuropsychopharmacol 2013; 23: 469–478. dopamine transporter gene and dopamine transporter density in Korean children 60 Franke B, Hoogman M, Arias Vasquez A, Heister JG, Savelkoul PJ, Naber M et al. with attention deficit hyperactivity disorder: relating to treatment response to Association of the dopamine transporter (SLC6A3/DAT1) gene 9–6 haplotype with methylphenidate. Eur Neuropsychopharmacol 2005; 15: 95–101. adult ADHD. Am J Med Genet B Neuropsychiatr Genet 2008; 147B: 1576–1579. 35 DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986; 7: 61 Loder MK, Melikian HE. The dopamine transporter constitutively internalizes and 177–188. recycles in a protein kinase C-regulated manner in stably transfected PC12 cell 36 Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in lines. J Biol Chem 2003; 278: 22168–22174. meta-analyses. BMJ 2003; 327: 557–560. 62 Lasky-Su J, Neale BM, Franke B, Anney RJ, Zhou K, Maller JB et al. Genome-wide 37 Hedges LV, Olkin I. Statistical Methods for Meta-Analysis. Academic Press: Orlando, association scan of quantitative traits for attention deficit hyperactivity disorder 1985, p 369 ill.; 24 cm. p. identifies novel associations and confirms candidate gene associations. Am J Med 38 Hunter JE, Schmidt FL. Methods of Neta-Analysis: Correcting Error and Bias in Genet B Neuropsychiatr Genet 2008; 147B: 1345–1354. Research Findings. Sage Publications: Newbury Park, CA, 1990, p 592. 63 Lasky-Su J, Anney RJ, Neale BM, Franke B, Zhou K, Maller JB et al. Genome-wide 39 Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected association scan of the time to onset of attention deficit hyperactivity disorder. by a simple, graphical test. Br Med J 1997; 315: 629–634. Am J Med Genet B Neuropsychiatr Genet 2008; 147B: 1355–1358. 40 van Dyck CH, Malison RT, Jacobsen LK, Seibyl JP, Staley JK, Laruelle M et al. 64 Markunas CA, Quinn KS, Collins AL, Garrett ME, Lachiewicz AM, Sommer JL et al. Increased dopamine transporter availability associated with the 9-repeat allele of Genetic variants in SLC9A9 are associated with measures of attention-deficit/ the SLC6A3 gene. JNuclMed2005; 46: 745–751. hyperactivity disorder symptoms in families. Psychiatr Genet 2010; 20: 73–81. 41 Jacobsen LK, Staley JK, Zoghbi SS, Seibyl JP, Kosten TR, Innis RB et al. Prediction 65 Franke B, Neale BM, Faraone SV. Genome-wide association studies in ADHD. of dopamine transporter binding availability by genotype: a preliminary report. Hum Genet 2009; 126: 13–50. Am J Psychiatry 2000; 157: 1700–1703. 66 Mick E, Todorov A, Smalley S, Hu X, Loo S, Todd RD et al. Family-based genome- 42 Martinez D, Gelernter J, Abi-Dargham A, van Dyck CH, Kegeles L, Innis RB et al. The wide association scan of attention-deficit/hyperactivity disorder. J Am Acad Child variable number of tandem repeats polymorphism of the dopamine transporter Adolesc Psychiatry 2010; 49: 898–905 e3. gene is not associated with significant change in dopamine transporter pheno- 67 de Silva MG, Elliott K, Dahl HH, Fitzpatrick E, Wilcox S, Delatycki M et al. Disruption type in humans. Neuropsychopharmacology 2001; 24: 553–560. of a novel member of a sodium/hydrogen exchanger family and DOCK3 is 43 Lynch DR, Mozley PD, Sokol S, Maas NM, Balcer LJ, Siderowf AD. Lack of effect of associated with an attention deficit hyperactivity disorder-like phenotype. JMed polymorphisms in dopamine metabolism related genes on imaging of TRODAT–1 Genet 2003; 40: 733–740. in striatum of asymptomatic volunteers and patients with Parkinson’s disease. 68 Morrow EM, Yoo SY, Flavell SW, Kim TK, Lin Y, Hill RS et al. Identifying autism loci Mov Disord 2003; 18: 804–812. and genes by tracing recent shared ancestry. Science 2008; 321: 218–223. 44 Kaufman MJ, Madras BK. Distribution of cocaine recognition sites in monkey 69 Neale BM, Medland SE, Ripke S, Asherson P, Franke B, Lesch KP et al. Meta-analysis brain: II. Ex vivo autoradiography with [3H]CFT and [125I]RTI–55. Synapse 1992; of genome-wide association studies of attention-deficit/hyperactivity disorder. 12: 99–111. J Am Acad Child Adolesc Psychiatry 2010; 49: 884–897. 45 Jucaite A, Fernell E, Halldin C, Forssberg H, Farde L. Reduced midbrain dopamine 70 Zhang-James Y, Dasbanerjee T, Sagvolden T, Middleton FA, Faraone SV. SLC9A9 transporter binding in male adolescents with attention-deficit/hyperactivity dis- mutations, gene expression, and protein-protein interactions in rat models of order: association between striatal dopamine markers and motor hyperactivity. attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet Biol Psychiatry 2005; 57: 229–238. 2011; 156: 835–843.

Molecular Psychiatry (2014), 880 – 889 & 2014 Macmillan Publishers Limited Meta-analysis of dopamine transporter gene associations SV Faraone et al 889 71 Zhang-James Y, Middleton FA, Sagvolden T, Faraone SV. Differential expression of 81 Ciliax BJ, Drash GW, Staley JK, Haber S, Mobley CJ, Miller GW et al. SLC9A9 and interacting molecules in the hippocampus of rat models for attention Immunocytochemical localization of the dopamine transporter in human brain. deficit/hyperactivity disorder. Dev Neurosci 2012; 34: 218–227. J Comp Neurol 1999; 409: 38–56. 72 Methner DN, Mayfield RD. Ethanol alters endosomal recycling of human dopa- 82 Cruz-Muros I, Afonso-Oramas D, Abreu P, Perez-Delgado MM, Rodriguez M, mine transporters. J Biol Chem 2010; 285: 10310–10317. Gonzalez-Hernandez T. Aging effects on the dopamine transporter expression 73 Pandolfo P, Machado NJ, Kofalvi A, Takahashi RN, Cunha RA. Caffeine regulates and compensatory mechanisms. Neurobiol Aging 2009; 30: 973–986. frontocorticostriatal dopamine transporter density and improves attention and 83 Tani S, Kuraku S, Sakamoto H, Inoue K, Kusakabe R. Developmental expression cognitive deficits in an animal model of attention deficit hyperactivity disorder. and evolution of muscle-specific microRNAs conserved in vertebrates. Evol Dev Eur Neuropsychopharmacol 2013; 23: 317–328. 2013; 15: 293–304. 74 Roessner V, Sagvolden T, Dasbanerjee T, Middleton FA, Faraone SV, Walaas SI et al. 84 Lee I, Ajay SS, Yook JI, Kim HS, Hong SH, Kim NH et al. New class of microRNA Methylphenidate normalizes elevated dopamine transporter densities in an targets containing simultaneous 5’-UTR and 3’-UTR interaction sites. Genome Res animal model of the attention-deficit/hyperactivity disorder combined type, but 2009; 19: 1175–1183. not to the same extent in one of the attention-deficit/hyperactivity disorder 85 Drgon T, Lin Z, Wang GJ, Fowler J, Pablo J, Mash DC et al. Common human 5’ inattentive type. Neuroscience 2010; 167: 1183–1191. dopamine transporter (SLC6A3) haplotypes yield varying expression levels in vivo. 75 Krause J, Dresel SH, Krause KH, La Fougere C, Zill P, Ackenheil M. Striatal dopa- Cell Mol Neurobiol 2006; 26: 875–889. mine transporter availability and DAT–1 gene in adults with ADHD: no higher DAT 86 Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H et al. availability in patients with homozygosity for the 10-repeat allele. World J Biol Systematic localization of common disease-associated variation in regulatory Psychiatry 2006; 7: 152–157. DNA. Science 2012; 337: 1190–1195. 76 Sanchez-Mora C, Ribases M, Casas M, Bayes M, Bosch R, Fernandez-Castillo N et al. 87 Contin M, Martinelli P, Mochi M, Albani F, Riva R, Scaglione C et al. Exploring DRD4 and its interaction with SLC6A3 as possible risk factors for Dopamine transporter gene polymorphism, spect imaging, and levodopa adult ADHD: a meta-analysis in four European populations. Am J Med Genet B response in patients with Parkinson disease. Clin Neuropharmacol 2004; 27: Neuropsychiatr Genet 2011; 156B: 600–612. 111–115. 77 Hawi Z, Kent L, Hill M, Anney RJ, Brookes KJ, Barry E et al. ADHD and DAT1: further 88 Heinz A, Goldman D, Jones DW, Palmour R, Hommer D, Gorey JG et al. evidence of paternal over-transmission of risk alleles and haplotype. Am J Med Genotype influences in vivo dopamine transporter availability in human striatum. Genet B Neuropsychiatr Genet 2010; 153B: 97–102. Neuropsychopharmacology 2000; 22: 133–139. 78 Altink ME, Slaats-Willemse DI, Rommelse NN, Buschgens CJ, Fliers EA, Arias-Vas- 89 Gunnar MR, Brodersen L, Krueger K, Rigatuso J. Dampening of adrenocortical quez A et al. Effects of maternal and paternal smoking on attentional control in responses during infancy: Normative changes and individual differences. Child children with and without ADHD. Eur Child Adolesc Psychiatry 2009; 18: 465–475. Dev 1996; 67: 877–889. 79 Guindalini C, Martins RC, Andersen ML, Tufik S. Influence of genotype on 90 Lafuente A, Bernardo M, Mas S, Crescenti A, Aparici M, Gasso P et al. Dopamine dopamine transporter availability in human striatum and sleep architecture. transporter (DAT) genotype (VNTR) and phenotype in extrapyramidal symptoms Psychiatry Res 2010; 179: 238–240. induced by . Schizophr Res 2007; 90: 115–122. 80 Shumay E, Fowler JS, Volkow ND. Genomic features of the human dopamine 91 van de, Giessen E, de Win MM, Tanck MW, van den Brink W, Baas F et al. transporter gene and its potential epigenetic states: implications for phenotypic Striatal dopamine transporter availability associated with polymorphisms in the diversity. PLoS One 2010; 5: e11067. dopamine transporter gene SLC6A3. J Nucl Med 2009; 50: 45–52.

& 2014 Macmillan Publishers Limited Molecular Psychiatry (2014), 880 – 889