Molecular Imaging Genetics ofMethylphenidate Response in ADHD

and Substance Use ComorbidityCLAUDIA M. SZOBOT,1,2,3* TATIANA ROMAN,4 MARA H. HUTZ,4 JULIA P. GENRO,4

MING CHI SHIH,5 MARCELO Q. HOEXTER,5 NEIVO JUNIOR,6 FLAVIO PECHANSKY,2

RODRIGO A. BRESSAN,5 AND LUIS A.P. ROHDE1,7

1ADHD Outpatient Clinic, Hospital de Clınicas de Porto Alegre (HCPA), Universidade Federal do Rio Grande doSul (UFRGS). Porto Alegre (POA), Rio Grande do Sul (RS), Brasil

2Center for Drug and Alcohol Research, HCPA, UFRGS, POA, Brasil3Universidade Luterana do Brasil, Pos-graduacao em Saude Coletiva, RS, Brasil

4Departamento de Genetica, UFRGS, POA, RS, Brasil5Laboratorio Interdisciplinar de Neurociencias Clınicas (LINC), Universidade Federal de Sao Paulo (UNIFESP),

Sao Paulo, Brasil6Irmandade Santa Casa de Misericordia, POA, RS, Brasil

7Instituto Nacional de Psiquiatria do Desenvolvimento (INPD), Brasil

KEY WORDS attention-deficit/hyperactivity disorder; substance use disorders;methylphenidate; SPECT; DRD4; DAT1

ABSTRACT Purpose: Attention-deficit/hyperactivity disorder (ADHD) and sub-stance use disorders (SUDs) are highly comorbid and may share a genetic vulnerabil-ity. Methylphenidate (MPH), a dopamine transporter (DAT) blocker, is an effectivedrug for most ADHD patients. Although dopamine D4 receptor (DRD4) and dopaminetransporter (DAT1) genes have a role in both disorders, little is known about howthese genes influence brain response to MPH in individuals with ADHD/SUDs. Thegoal of this study was to evaluate whether ADHD risk alleles at DRD4 and DAT1genes could predict the change in striatal DAT occupancy after treatment with MPHin adolescents with ADHD/SUDs. Methods: Seventeen adolescents with ADHD/SUDsunderwent a SPECT scan with [Tc99m]TRODAT-1 at baseline and after three weeks onMPH. Caudate and putamen DAT binding potential was calculated. Comparisons onDAT changes were made according to the subjects’ genotype. Results: The combina-tion of both DRD4 7-repeat allele (7R) and homozygosity for the DAT1 10-repeat allele(10/10) was significantly associated with a reduced DAT change after MPH treatmentin right and left caudate and putamen, even adjusting the results for potential con-founders (P � 0.02; R2 from 0.50 to 0.56). Conclusions: In patients with ADHD/SUDs, combined DRD4 7R and DAT1 10/10 could index MPH reduced DAT occupancy.This might be important for clinical trials, in terms of better understanding individualvariability in treatment response. Synapse 65:154–159, 2011. VVC 2010 Wiley-Liss, Inc.

INTRODUCTION

Brain imaging studies in attention-deficit/hyperac-tivity disorder (ADHD) strongly support the involve-ment of fronto-striatal-cerebellar circuits (Krain andCastellanos, 2006), where dopamine (DA) plays animportant function. Imaging studies with positronemission tomography (PET) and single photon emis-sion computed tomography (SPECT) have shown ahigher striatal dopamine transporter (DAT) density insubjects with ADHD (Dougherty et al., 1999; Larischet al., 2006). Methylphenidate (MPH) is considered afirst-line medication for ADHD (Pliszka et al., 2006)and studies have shown that this drug induces signif-

icant decrease in [11C]raclopride binding at the D2

receptor, presumably due to increased synaptic DAlevels, which is believed to mediate the therapeuticeffects of MPH (Schlaepfer et al., 1997; Volkow et al.,1995). Dresel (2000) found that specific binding of

*Correspondence to: Claudia M. Szobot. Programa de Deficit de Atencao/Hiperatividade, Hospital de Clınicas de Porto Alegre, Rua Ramiro Barcelos,2350, sala 2201, CEP: 90035-003. Porto Alegre, RS, Brasil. E-mail: cmszobot@terra.com.br

Received 15 March 2010; Accepted 14 May 2010

DOI 10.1002/syn.20829

Published online 30 June 2010 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2010 WILEY-LISS, INC.

SYNAPSE 65:154–159 (2011)

[Tc99m]TRODAT-1 to DAT decreased significantly inadults with ADHD after MPH treatment.

Pharmacogenetic studies of ADHD represent agrowing area of interest. Investigations have tried topredict both clinical and brain responses to MPH.There are studies demonstrating, for instance, thatafter MPH treatment, the homozygosis of the 10-repeat allele at DAT gene (DAT1), DAT1-10/10 wasassociated with a poorer clinical response to MPH insome studies, but there are conflicting results (Romanet al., 2004). A previous SPECT study from our groupfound a higher regional cerebral blood flow in medialfrontal and left basal ganglia areas in children withDAT1-10/10 after MPH use (Rohde et al., 2003). Anassociation between DAT1-10/10, DAT availabilityand poor response to MPH was later described byanother group (Cheon et al., 2005). The presence ofthe 7-repeat (7R) allele at the dopamine D4 receptorgene (DRD4), DRD4-7, was also related to MPHresponse. Hamarman (2004) documented that chil-dren and adolescents with ADHD who possessed atleast one 7R allele required higher MPH doses toachieve more robust clinical improvement. Most ofthese studies either do not account for the effect ofcomorbidities, or do not include individuals with cer-tain additional disorders, such as substance use disor-ders (SUDs). This might result in some limitations,since comorbidity in ADHD is more a rule than anexception (Ollendick et al., 2008).

The co-occurrence of ADHD and SUDs is notewor-thy, as the negative impact of ADHD on SUDs out-come (Szobot and Bukstein, 2008). ADHD is associ-ated, for instance, with both earlier and more fre-quent alcohol relapses (Ercan et al., 2003) and lowerlikelihood of cannabis treatment completion in adoles-cents (White et al., 2004). Both DAT1 and DRD4genes have been associated with SUDs. Guindaliniet al. (2006) demonstrated an association between co-caine dependence and a VNTR allele in SLC6A3.Shao et al. (2006) suggested that DRD4 VNTR poly-morphism contributes to cue-elicited craving in heroindependence. However, little is known about howgenetics affect brain response to MPH in subjectswith ADHD/SUDs, despite the dopaminergic actionsof drugs of abuse (Volkow et al., 2004), and despitefindings from epigenetic studies showing that drugexposure might influence gene expression (Renthaland Nestler, 2008).

We recently conducted a study using [Tc99m]TRODAT-1 and SPECT before and after three weekson MPH-SODAS (Spheroidal Oral Drug AbsorptionSystem), documenting that MPH significantly reducedDAT availability in adolescents with ADHD/SUDs(Szobot et al., 2008). This was an important findingsince most placebo-controlled studies on adults withADHD and SUDs show no clinical response to MPHon ADHD symptoms (Carpentier et al., 2005; Levin

et al., 2006, 2007), on the contrary of pharmacologicaltrials on ADHD without drug addicts (Adler et al.,2009; Medori et al., 2008). That is, we are able todemonstrate, by a brain imaging technique, thatMPH binding to striatal DAT was preserved in a con-text of drug abuse, reinforcing the debate about possi-ble reasons for different responses to MPH betweendrug addicts and nondrug addicts with ADHD.Assuming that individual genetic variation may beassociated with the variability in response to pharma-cological treatment, in this study our goal was to ver-ify whether dopaminergic genes (DAT1 and DRD4)could help to predict MPH DAT occupancy in thissame group of subjects.

MATERIALS AND METHODSStudy design and participants

This was a three-week study on the effects of dopa-minergic genotype on MPH-SODAS action on DAT oc-cupancy, assessed by SPECT with [Tc99m]TRODAT-1,in 17 outpatient male adolescents with both ADHD/SUD. All subjects underwent two brain scans: one atbaseline and another after three weeks on escalateddoses of MPH-SODAS. The study was conducted inthe city of Porto Alegre, Brazil, and approved by theInstitutional Review Board (IRB) of Hospital de Clıni-cas de Porto Alegre (approved as an IRB by the Officefor Human Research Protections, United States ofAmerica—IRB 00000921). Written informed consentwas obtained from all participants and their parents.

Subjects were recruited from a previous studyassessing ADHD in a large community sample of ado-lescents with SUD (Szobot et al., 2008) (n 5 14) and byadvertisements in local newspapers and radio broad-casts (n 5 3). Inclusion criteria were: (a) age rangebetween 15 and 21 years old; (b) male gender; (c) cur-rent DSM-IV diagnosis of abuse/dependence of mari-juana or cocaine; (d) current DSM-IV diagnosis ofADHD; (e) stimulant-naive subjects. Exclusion criteriaincluded: (a) lack of a responsible adult to informabout possible childhood psychopathology or to takeresponsibility for the medication; (b) need for inpatienttreatment for drug abuse or psychiatric comorbidities;(c) presence of a primary psychiatric condition thatrequired immediate outpatient treatment (such asmoderate/severe depression); (d) inhalant use.

Study medication procedures

Medication was taken once a day (in the morning),by oral administration. Study compliance was assessedby self-report, mother’s report and pill counting. Sub-jects received 0.3 mg/kg/day of MPH-SODAS at week1, 0.7 mg/kg/day at Week 2 and 1.2 mg/kg/day at Week3. On the day of the second scan, all subjects receivedthe medication by a staff member at the moment of[Tc99m]TRODAT-1 injection, thus ensuring that all

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subjects had the same time interval between MPH-SODAS intake and brain imaging acquisition (4 h).

Each MPH-SODAS capsule contains a 50:50 mix-ture of immediate release (IR) and enteric-coateddelayed release (EC-DR) beads, resulting in a bimodalrelease pattern which is equivalent to twice daily im-mediate-release MPH. The IR beads yield an initialpeak at about 2 h and the EC-DR beads yield a sec-ond peak on average at 6.5 h (Liu et al., 2005).

Diagnostic procedures

The diagnoses of ADHD and comorbid mental disor-ders were confirmed by semistructured interviews(Portuguese version of the Schedule for Affective Dis-orders and Schizophrenia for School - Age Children,Epidemiological Version - K-SADS-E) (Mercadanteet al., 1995) with the parents, and clinical interviewswith the adolescent and the parents conducted by achild psychiatrist (CMS). Detailed description of thediagnostic process in our ADHD clinic can be foundelsewhere (Rohde et al., 2005). The diagnoses of SUDrelied on the drug section of the Mini InternationalNeuropsychiatry Interview (MINI), Brazilian version,which generates diagnoses of abuse or dependenceaccording to DSM-IV criteria (Amorim, 2000). Partici-pants had drug use confirmed by urinary tests (can-nabis and/or cocaine), at baseline and after threeweeks. ADHD severity was based on the Swanson,Nolan and Pelham Scale – version IV (SNAP-IV),which is a 26-item scale, based on DSM-IV ADHDand oppositional defiant disorder (ODD) symptoms,for ADHD severity (Swanson et al., 2001). Items arescored on a 4-point scale (from 0, never, to 3, very of-ten). The instrument has its Portuguese version al-ready assessed (Mattos et al., 2006). In the presentstudy, the SNAP-IV evaluation was based on themother’s report. Drug use severity was consideredaccording to the number of days with drug use,according to subjects’ report. A more detailed descrip-tion from our sample and diagnostic procedures canbe found elsewhere (Szobot et al., 2008).

DAT imaging acquisitions

SPECT scans were performed with [Tc99m]-TRO-DAT-1, a radiotracer with high selectivity and speci-ficity for the DAT. TRODAT-1 kits were producedby the Institute of Nuclear Energy Research (INER-Taiwan R.O.C.) and labeled according to Choi (1999).Images were acquired 4 h after the injection of 740MBq (674) of [Tc99m]-TRODAT-1 using a dual-headgamma camera ‘‘ECAM’’ (Siemens Medical System,USA), fitted with low-energy, general purpose, fanbeam-hole collimators. For each scan a total of 128projections (30 s per frame) were collected in a step-and-shoot mode on circular orbit with the mean ra-dius of rotation of 15 cm.

The image data were reconstructed by standard fil-tered backprojection using a Butterworth filter (cut-off frequency 0.35 Nq) and attenuation was correctedusing Chang’s first-order method (attenuation coeffi-cient m [1/4] 0.12 cm_1).

Image data analysis

All the SPECT images are converted from dicom toanalyze file through MRIcro Program (www.mri-cro.com) and transfer to nifti file by Stastistic Para-metric Mapping 5 program (SPM5) mounted in MAT-LAB 7.0 platform. Reorientation was corrected for allSPECT imaging and spatially normalized to a stand-ardized image space based on a select subject image,after the four-time repeat process, creating the spe-cific template image for the study. Spatial normaliza-tion is performed by linear and nonlinear transforma-tion algorithm with parameters consistent and theimaging data was then applied to match each imageto the template provided from all imaging data study.The imaging data was also smoothed with an iso-tropic Gaussian filter to improve the signal-to-noiseratio and to reduce errors attributed to individualvariation in striatum anatomy. Resulting transforma-tions were postprocessed to generate maps of thedeformation at each voxel during the normalizationprocess.

Volumetric regions of interest (vROIs) in striatum(caudate and putamen) and occipital lobe are man-ually delineated by radiologist and nuclear medicinephysician using neuroanatomy atlas (www.sph.sc.edu/comd/rorden/anatomy/home.html) onto the templateusing SPM5 Volume toolbox. DAT occupancy (O) wascalculated with binding potential (BP) in the striatum(STR) separately as caudate and putamen, and in theoccipital cortex (OCC) as background, by the followingformula: [(STR–OCC)]/OCC. Estimates of DAT O weremade relative to baseline using the following formula:

O ¼ BPMPH�BPDNBPDN

100%, where BPDN is the BP values

while patients where MPH-naive and BPMPH is theBP value after three weeks of MPH.

Genetic analysis

High molecular weight genomic DNA was extractedfrom the whole blood by a salting out procedure(Lahiri and Nurnberger, 1991). Genotyping of the 48base pair (bp) variable number of tandem repeats(VNTR) polymorphism at the DRD4 gene and the40bp VNTR at the DAT1 gene were performed as al-ready described (Roman et al., 2001).

Statistical analysis

Allele frequencies for both loci were estimated bycounting. For analyses, patients were grouped accord-ing to the presence of at least one risk allele at DRD4

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locus (DRD4-7) and of risk genotype at DAT1 locus(DAT1-10/10), resulting in three tested genotypegroups: (a) DRD4-7 (n 5 9, 53%); (b) DAT1-10/10 (n 58, 47%); (c) the combination of both DRD4-7 andDAT1-10/10 (n 5 5, 29%).

Linear regression analysis was used to test whetherthe changes in DAT O from baseline to Week 3 onstriatum could be predicted by genotype, adjustingthe results for confounders (baseline ADHD, MPHdose, and drug use severity). These comparisons wereperformed considering patients with one genotypecondition vs. all other patients. A significant level ofP � 0.05 was established for all analyses.

RESULTS

All 17 participants had a cannabis SUDs diagnosis,of whom five (29%) were also cocaine users. There wereno differences in baseline striatum [Tc99m]TRODAT-1binding and in ADHD severity and drug use measures(number of days with drug use per week), as well inADHD and drug use improvement for each testedgenotype group (P � 0.09).

We first assessed whether presence of the DRD4-7(independently of the genotype at DAT1) could predictstriatal DAT occupancy after MPH, adjusting theresults for baseline SNAP, baseline drug use andMPH dosage at the last week of the protocol. Thesame analyses were done for the DAT1-10/10 (inde-pendently of presence of the 7R allele at DRD4). Nosignificant findings emerged (all P values � 0.08 for

both DAT1-10/10 and DRD4-7). Thus, we furthertested whether the combination of both DRD4-7 andDAT1-10/10 could predict striatal DAT occupancy af-ter MPH, adjusting the results for the same confound-ers. The combination of these risk alleles was signifi-cantly associated with a smaller DAT occupancy incaudate and putamen, bilaterally (Table I). Drug usehad also an independent effect in all brain areasexcept for LP. There was no significant effect forMPH dose and for ADHD severity at baseline. In thismultivariable model, R2 ranged from 0.50 (LP andRC) to 0.56 (LC and RP). If genotype is not includedin the analysis, R2 falls from 0.56 to 0.14 in the LC,for instance. According to Beta standardized values,genotype was the most powerful variable in all stria-tal areas, except for RP, where genotype and drug usehad the same impact.

DISCUSSION

This study provides connection between pharmaco-genetics and molecular brain imaging on stimulanttreatment of individuals with ADHD/SUDs. Ourresults suggest a smaller striatal DAT blockade afterthree weeks of MPH in individuals with both DRD4-7and DAT1-10/10, even adjusting the results for con-founders. In a SPECT study, a different pattern ofbrain activation was detected in ADHD children withDAT1-10/10 after four days on MPH (Rohde et al.,2003), but this study did not include drug addicts,therefore limiting comparisons. DAT, mostly located

TABLE I. Regression analysis of the effect of the presence of both 7R allele at DRD4 and homozygosisfor the 10-repeat allele at DAT1 on DAT occupancy after 3 weeks on MPH-SODAS in 17 adolescents

with ADHD/SUDs, adjusted for confounders

B B Standardized P value

95% CI

Lower Upper

RPDAT1 10-10 1 DRD4 7R 0.28 0.65 0.02 0.06 0.50SNAP <0.01 0.19 0.35 20.003 <0.01Drug use 0.08 0.65 0.01 0.02 0.13MPH dosage at week 3 20.003 20.14 0.54 20.01 <0.01

R2 5 0.56RC

DAT1 10-10 1 DRD4 7R 0.38 0.70 0.015 0.09 0.68SNAP <0.01 0.06 0.67 20.007 <0.01Drug use 0.09 0.60 0.02 0.02 0.17MPH dosage at week 3 <0.01 20.04 0.86 20.02 0.01

R2 5 0.50LP

DAT1 10-10 1 DRD4 7R 0.28 0.69 0.02 0.06 0.49SNAP <0.01 0.17 0.43 20.004 <0.01Drug use 0.05 0.42 0.09 20.009 0.1MPH dosage at week 3 20.003 20.14 0.55 20.01 0.01

R2 5 0.50LC

DAT1 10-10 1 DRD4 7R 0.37 0.79 0.006 0.13 0.61SNAP <0.01 0.02 0.91 20.006 <0.01Drug use 0.08 0.60 0.02 0.02 0.14MPH dosage at week 3 <0.01 0.02 0.93 20.01 0.01

R2 5 0.56

B, regression coefficient; B standardized, the strength of the effect of each independent variable on the dependentvariable; R2, R squared (coefficient of determination); DAT, dopamine transporter; MPH-SODAS, methylphenidatespheroidal oral drug absorption system; SUDs, illicit substance use disorder; ADHD, attention deficit/hyperactivitydisorder; SNAP, Swanson, Nolan and Pelham Scale – version IV; RP, right putamen; RC, right caudate; LP, left puta-men; LC, left caudate. SNAP and drug use refer to baseline measures.

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on basal ganglia, is under the influence of severaladaptative factors. Psychoactive drugs can modify thestriatal DA balance and cause more prolongedchanges in DAT regulation (Daws et al., 2002).Although presence of DRD4-7 allele was associatedwith poorer clinical response to stimulants in individ-uals with ADHD (Hamarman et al., 2004), little isknown about the associated brain imaging patterns.DRD4-7 has been associated with lower DA receptionefficiency (Oak et al., 2002). It was documented thatsubjects without the DRD4-7 allele who smoked nico-tine during PET scan had a greater smoking-inducedDA release in ventral caudate and nucleus accumbens(Brody et al., 2006). Thus, although DRD4 locatesmostly in the frontal cortex, this receptor seems toplay an important role in striatum DA balance, whichcan also be measured by brain imaging studies.

The effect of the combination of risk conditions atDAT1 and DRD4 was more powerful than each geno-type considered separately. One possible explanationis a Type II error. However, the DRD4-7 allele had aneffect on MPH response just along with the serotonintransporter (SERT) gene in another investigation(Seeger et al., 2001). Since ADHD etiology is underthe influence of several genes of small effect (Faraoneand Khan, 2006), it should not be surprising thatmore than just one gene is necessary in order to rec-ognize a brain response pattern. We could speculatethat there was an additional effect of the combinationof both DAT1-10/10 and DRD4-7 in our study, withDAT1-10/10 determining a less efficient MPH occu-pancy and DRD4-7 promoting a smaller DA signal infrontal cortex. Together, it would result in a smallerchange in striatal DA balance, as detected by ourbrain imaging technique.

Our protocol has limitations, such as inclusion ofonly males. The sample size is small, but not smallerthan other studies on brain imaging, genetics andresponse to medication (Cheon et al., 2005; Potkinet al., 2003; Rohde et al., 2003). This study, and noneof the above mentioned, had a control group. As aconsequence, our findings cannot be generalized toadolescents with ADHD without SUDs. It is not possi-ble to know whether this same genotype could indexa poorer brain response to MPH in individuals withADHD without SUDs; however, it does not invalidatethe finding for the group of individuals with thecomorbidity ADHD/SUDs. Treatment compliancerelied on subjects’ and their mothers’ report sincethere were no MPH plasma levels. However, in thelast day of the protocol, when the second scan wasperformed, medication was taken at the hospital, inthe presence of one member of our staff.

This is the first study documenting how genotypescan predict brain response to MPH in adolescentswith ADHD/SUDs. Based on R2 values, the presenceof both DRD4-7 and DAT1-10/10 conferred a robust

contribution for the understanding of the variance ofstriatal dopaminergic balance after MPH use,strongly suggesting dopaminergic genotypes as pre-dictors in further studies evaluating MPH effects inindividuals with ADHD/SUDs. The combination ofthe risk allele at DRD4 and risk genotype at DAT1might indicate a subgroup of individuals with areduced MPH striatal DAT O. The clinical meaning ofour finding should be better evaluated with largersamples and clinical outcomes. Even so, our studymight be important for future clinical trials onpharmacological treatment of ADHD/SUDs, mostlybecause clinical trials with adults with ADHD/SUDshave not shown very encouraging results, as alreadydescribed. Assuming that individual genetic variationmay be associated with individual variability inresponse to pharmacological treatment, our studycould help to identify individuals who are less likelyto respond to MPH, which has strong clinical applica-tion in view of the fact that drug addicts are at higherrisk for treatment dropout (Walker, 2009).

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