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j ou rn a l h om epa g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t
Research
paper
Associations
between
a
locus
downstream
DRD1
gene
and
cerebrospinal
fluid
dopamine
metabolite
concentrations
in
psychosis
Dimitrios
Andreou
a,∗,
Erik
Söderman
a,
Tomas
Axelsson
b,
Göran
C.
Sedvall
a,
Lars
Terenius
a,
Ingrid
Agartz
a,c,d,
Erik
G.
Jönsson
aaDepartmentofClinicalNeuroscience,PsychiatrySection,HUBINProject,KarolinskaInstitutetandHospital,Stockholm,Sweden bDepartmentofMedicalSciences,MolecularMedicine,UppsalaUniversity,Uppsala,Sweden
cNORMENT,InstituteofClinicalMedicine,UniversityofOslo,Oslo,Norway dDepartmentofPsychiatricResearch,DiakonhjemmetHospital,Oslo,Norway
h
i
g
h
l
i
g
h
t
s
•DopaminereceptorD1genevariationissignificantlyassociatedwithcerebrospinalfluidhomovanillicacid.
•Thesignificantassociationsarerestrictedtopsychoticpatients.
•Theresultssupportthedopaminehypothesisofschizophrenia.
a
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e
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Articlehistory:
Received21December2015
Receivedinrevisedform13February2016 Accepted2March2016
Availableonline5March2016 Keywords:
Schizophrenia Psychiatricgenetics Homovanillicacid(HVA) DopaminereceptorD1gene(DRD1)
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Dopamineactivity,mediatedbythecatecholaminergicneurotransmitterdopamine,isprominentinthe humanbrainandhasbeenimplicatedinschizophrenia.Dopaminetargetsfivedifferentreceptorsandis thendegradedtoitsmajormetabolitehomovanillicacid(HVA).Wehypothesizedthatgenesencoding dopaminereceptorsmaybeassociatedwithcerebrospinalfluid(CSF)HVAconcentrationsinpatients withpsychoticdisorder.
Wesearchedforassociationbetween67singlenucleotidepolymorphisms(SNPs)inthefivedopamine receptorgenesi.e.,DRD1,DRD2,DRD3,DRD4andDRD5,andtheCSFHVAconcentrationsin74patients withpsychoticdisorder.NominallyassociatedSNPswerealsotestedin111healthycontrols.
Weidentifiedalocus,locateddownstreamDRD1gene,wherefourSNPs,rs11747728,rs11742274, rs265974and rs11747886,showedassociation withCSFHVAconcentrationsinpsychotic patients. Theassociationsbetweenrs11747728,whichisaregulatoryregionvariant,andrs11742274withHVA remainedsignificantaftercorrectionformultipletesting.Theseassociationswererestrictedtopsychotic patientsandwereabsentinhealthycontrols.TheresultssuggestthattheDRD1geneisimplicatedinthe pathophysiologyofpsychosisandsupportthedopaminehypothesisofschizophrenia.
©2016TheAuthors.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCCBY license(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Dopaminergicinnervationisprominentinthecentralnervous systemandiscriticallyimplicatedinmanycentralandperipheral functions[5].Thecatecholaminergicneurotransmitterdopamineis
Abbreviations:HVA,homovanillicacid;DRD2,dopaminereceptorD2gen;CSF, cerebrospinalfluid;CNS,centralnervoussystem;DRD1,dopaminereceptorD1 gene;DRD3,dopaminereceptorD3gene;DRD4,dopaminereceptorD4gene;DRD5, dopaminereceptorD5gene;SNPs,singlenucleotidepolymorphisms.
∗ Correspondingauthorat:DepartmentofClinicalNeuroscience,HUBINProject, KarolinskaInstitutetandHospital,R5:00,SE-17176Stockholm,Sweden.
E-mailaddress:dimitrios.andreou@sll.se(D.Andreou).
releasedfromthepresynapticterminalsofdopaminergicneurons andexertsitsactionbytargetingfivedifferentG-protein-coupled receptorslocatedbothpre-andpostsynaptically[5].Dopamineis thendegradedtoitsmajormetabolitehomovanillicacid(HVA)by catechol-O-methyltransferaseandmonoamineoxidase.
Dopaminergic dysfunction is considered to be implicated in various mental disorders, mainly schizophrenia [5,9,22]. Schizophreniaaffectsapproximately1%oftheworld’spopulation withaheritabilityupto80%[37].Manydopaminergicgene vari-antshavebeenassociatedwiththedisorder,howevertheresults havebeenambiguousanddifficulttoreplicateuntilrecently,when a genome-wide significant association was found betweenthe dopaminereceptor D2 (DRD2)gene and schizophrenia [1].The
http://dx.doi.org/10.1016/j.neulet.2016.03.005
associationbetweendopaminergicgenevariantsandmeasurable biologicalmarkersmaybemore robustandconsistentthanthe associationsbetweengenevariantsandthedisorderitself. More-over,thisapproachcanshedfurtherlighttotheunderstandingof genotype-phenotypeassociations.
Weconsiderthecerebrospinalfluid(CSF)HVAtobearelevant measurablemarkerasitreflectsthedopamineturnoverrateinthe centralnervoussystem(CNS),itispartiallygeneticallydetermined andithasbeenassociatedwithschizophrenia.Concentrationsof HVAinventricular,cisternal,andlumbarCSFshowa craniocau-dalgradient [25,28].Studiesin postmortemhumanbrainshave shown thatCSF HVAreflectsbrain HVAconcentrations [36,42]. StudiesinhumantwinsandotherprimateshaveshownthatHVA concentrationsarepartiallyundergeneticinfluence[30,32].HVA concentrationsaresignificantlylowerindrug-freepatientswith schizophreniarelativetocontrols[6,43].Moreover,quetiapineand olanzapineadministrationwasfoundtobeassociatedwitha sig-nificantincreaseinCSFHVA[26,33].
Dopaminereceptors,i.e.,D1,D2,D3,D4andD5,areclassified asD1-classdopaminereceptors,includingD1andD5[39],and D2-classdopaminereceptors,includingD2,D3andD4[5,40].D1and D2receptorshavethehighestlevelofexpressioninmultiplebrain regions,whereasD4receptorshavethelowestlevelofexpression inthebrain[5].Dopaminereceptorsactivation,mainlyD1andD2, affectsamongotherslocomotion,rewardmechanisms,psychotic symptomatology,sleepandattention[5,24].
ThedopaminereceptorsD1,D2,D3,D4and D5areencoded bythedopaminereceptor D1gene (DRD1),theDRD2 gene,the dopaminereceptor D3gene (DRD3),thedopaminereceptor D4 gene(DRD4)andthedopaminereceptorD5gene(DRD5), respec-tively.DRD1andDRD5havenointrons,whereasDRD2,DRD3and DRD4containsix,fiveand threeintronsintheircodingregions, respectively,providingsplicevariants[16].DRD1islocatedon chro-mosome5q35.1,DRD2islocatedonchromosome11q23.1,DRD3is locatedonchromosome3q13.3,DRD4islocatedonchromosome 11p15.5andDRD5islocatedonchromosome4p16.1.
Inthepresentstudy,wehavesearchedforassociationbetween DRD1,DRD2, DRD3, DRD4and DRD5 singlenucleotide polymor-phisms(SNPs)anddopamineturnoverrateintheCNS,asreflected bytheCSFconcentrationsofthemajordopaminemetaboliteHVA, inpatientswithpsychosis.
2. Materialandmethods
2.1. Subjects
Thesubjectsof thepresent studyhave beeninvestigated as previously described[4].Patients withpsychoticdisorderwere recruitedamong inpatientsatfourpsychiatricuniversityclinics inStockholmCountybetween1973and1987andwereaskedto participateinpharmacologicaland/orbiologicalresearchprojects
[8].Theparticipantswereobservedforatleast48hwithoutany antipsychoticmedicationandCSFsamples(12.5ml)weredrawn bylumbarpuncture.
Threeto34 years afterthefirstinvestigation,patientswere askedto participate in geneticresearch studiesand blood was drawnforgenotyping.Patientswerethenaskedtoparticipateina diagnosticstructuredinterview[35]andpermittheresearchersto retrievetheirmedicalrecords.Availablerecordswerescrutinized byresearcherstoobtainalife-timediagnosisaccordingto DSM-III-RandDSM-IV.In2010,hospitaldischargediagnoseswerealso obtainedfromtheSwedishpsychiatricinpatientregister,aregister coveringallinpatienthospitalizationsinSwedensince1973.For eachhospitalizationthediagnosiswasrecordedaccordingtothe InternationalClassificationofDiseases,8th,9thor10threvisions.
Themajorityoftheparticipantshadexperiencedseveral hospital-izations,butonlyonediagnosiswasgivenperparticipant,following a diagnostic hierarchy[14,41].The final diagnoses, used in the present study,werebased ontheSwedishpsychiatricinpatient register,asitwasnotpossibletoretrieveallmedicalrecordsand severalofthepatientswerenotwillingtoparticipateinadiagnostic interview.
Analyses in healthy controls were conducted for SNPs that werenominallyassociatedwithHVAconcentrationsinpsychotic patients, inordertoevaluatewhethertheeffects ofthe associ-atedSNPswererestrictedtopatientswithpsychosis.CSFsamples weredrawnbylumbarpuncturefromunrelatedhealthyCaucasians between1973and1987.Eightto20yearsafterthefirst investi-gation,thesubjectswereinterviewedtore-assesstheabsenceof psychiatricmorbidity[18]andwholebloodwasdrawnfor geno-typing.
ThestudywasconductedinaccordancewiththeDeclarationof HelsinkiandapprovedbytheEthicsCommitteeoftheKarolinska UniversityHospital.Writteninformedconsentwasobtainedfrom alltheparticipatingsubjects.
2.2. CSFmonoaminemetaboliteconcentrations
CSFsamples(12.5ml)weredrawnbylumbarpuncturewiththe patientsandcontrolsinasittingorrecumbentpositionbetween8 and9a.m,afteratleast8hofbed-restandabsenceoffoodintake orsmoking.HVAconcentrationsweremeasuredbymass fragmen-tographywithdeuterium-labeledinternalstandards[38].
2.3. DNAanalysis
GenomicDNAwasextractedfromwholeblood.Totally,67SNPs were genotyped in DRD1 (n=17), DRD2 (n=23), DRD3 (n=18), DRD4(n=7)andDRD5(n=2).TheseSNPswereeithercandidate SNPs reported to be associated with mental disorders, mainly schizophrenia,enzymefunctionormonoaminemetabolite concen-trations,ortag-SNPsselectedusingHapMaptocoverthegenesof interestwithanr2thresholdof0.8.Thegenotypingwasperformed
usingtheIlluminaBeadStation500GXandthe768-plexIllumina GoldenGateassay(IlluminaInc.,SanDiego,CA,USA)[15].
2.4. Statisticalanalysis
The associations between SNPs and HVA CSF monoamine metaboliteconcentrationsweretestedwithmultiplelinear regres-sion(STATA12.1),whereconcentrationwasmodeledasalinear function ofthe allelecountand three tofivecovariates. In the analysesofpsychoticpatients,back-length, gender,ageat lum-bar puncture, diagnosis (i.e., schizophrenia spectrum psychosis or otherpsychosis)and useof antipsychoticswereincludedas covariates.Back-lengthwasdefinedasthedistancebetweenthe externaloccipitalprotuberanceandthepointofneedleinsertion. Antipsychotictreatmentwasconsideredaspresentifthepatient hadtakenantipsychoticsduringathree-weekperiodpriortothe lumbarpuncture.Intheanalysesofhealthycontrols,back-length, genderandageatlumbarpuncturewereincludedascovariates. Hardy–Weinberg(HW) equilibrium was tested usingexact sig-nificance asimplementedinSTATA12.1.Normality ofresiduals wascheckedgraphicallywithSTATA12.1.Adjustmentsfor multi-pletestingwereperformedusingBonferronicorrectiontakinginto accountthenumberoftestsconducted(␣=0.05/67=7.5×10−4).
3. Results
Psychoticpatients(n=74,45menand29women)participated in thepresent study.Themeanage ofdiseaseonset±standard
Table1
Minorallelefrequencies(MAF),exactsignificanceoftestingforHardy–Weinbergequilibrium(HWE)andp-values(P)frommultiplelinearregressionsofsinglenucleotide polymorphisms(SNPs)nominallyassociatedwithhomovanillicacid(HVA)concentrationsinthecerebrospinalfluidofpsychoticpatientsandthecorrespondingassociation statisticsamonghealthycontrols.
Psychoticpatients(n=74;45men,29women) Healthycontrols(n=111;63men,48women)
HVAmean(S.D) 178.6(79.3)nmol/l 167.5(68.4)nmol/l
Gene SNP MAF(%) HWE P MAF(%) HWE P
DRD1 rs11747728 14 0.339 0.0004a 12 0.640 0.129 DRD1 rs11742274 18 1.000 0.0005a 20 0.235 0.340 DRD1 rs265974 36 0.311 0.001 39 0.431 0.221 DRD4 rs3758653 15 1.000 0.002 16 0.468 0.910 DRD2 rs2234689 23 0.096 0.016 18 0.046 0.437 DRD1 rs11747886 18 1.000 0.047 15 0.705 0.961
aSignificantassociationsaftercorrectionformultipletesting.
deviationwas27.6±7.8years,whereastheirmeanage±standard deviationwas30.4±7.2yearsatlumbarpuncture.Twenty-sixof thepatientsweretreatedwithantipsychoticsatthetimeoflumbar puncture,whereasthirty-sixpatientswerefreefromantipsychotic medicationsincethreeweeksorlonger.Sixty-fourpatientswere diagnosedwithschizophreniaspectrumdisorder(schizophrenia n=60,schizoaffectivedisordern=4)andtenwithotherpsychosis (psychosisnototherwisespecifiedn=7,delusionaldisordern=1, bipolardisordern=1,alcoholinducedpsychoticdisordern=1).
InordertoevaluatehowtheSwedishin-patientresister-based diagnosesconformedtootherdiagnostictools,separateanalyses wereconducted.Evaluationsoriginatingfromthemedicalrecords in52ofthepatientsresultedinadiagnosisofapsychoticdisorderin 98%oftheseindividuals.Of44patientsparticipatingina diagnos-ticinterview91%displayedapsychoticdisorder[35].Theseresults areinaccordancewithpreviousreportsshowingthatthe register-baseddiagnosesofschizophreniaspectrumpsychosishaveahigh validity,with85%to94% ofthepatientsdisplayingthese diag-noseswhendiagnosticevaluationsusinginformationfrommedical recordsandastructuredclinicalinterviewweremade[41].
Inthe74psychoticpatients,67 SNPsinfive genesencoding dopaminereceptorswereselectedandgenotyped.Theresultsare illustratedinSupplementaryTableS1(seeSupplementaryTable S1intheonlineversionDOI:10.1016/j.neulet.2016.03.005).The minorallelefrequenciesfortheselectedmarkersrangedfrom2% to50%.Inpsychoticpatients,themean(standarddeviation) con-centrationofHVAwas178.6(79.3)nmol/L.Sixoftheinvestigated polymorphismswerefoundtobenominallyassociatedwithCSF HVAconcentrations and noneof theseSNPs showeddeparture fromHardyWeinbergequilibrium(p-value<0.05)(Table1).The residualsofthenominalassociationswereapproximatelynormally distributed.
Takingintoaccountthetotal numberoftestsconducted,we appliedaBonferronicorrection(˛=0.05/67=7.5×10−4)andtwo of the nominal associations i.e., rs11747728 (p=4×10−4) and rs11742274(p=5×10−4)remainedsignificantaftercorrectionfor multipletesting(Table1).
Thesix SNPs that wereassociated withHVA concentrations inpsychoticpatientsweretestedin111healthyCaucasians(63 menand48women).Theirmeanages±standarddeviationwere 28.4±7.5yearsatlumbarpuncture.Inhealthycontrols,themean (standarddeviation)concentrationofHVAwas167.5(68.4)nmol/L. Theminorallelefrequencyforthesixselectedmarkersrangedfrom 12%to39%.DeparturefromHardyWeinbergequilibrium(p<0.05) wasfoundinoneoftheSNPsanalyzed.Theresidualswere approxi-matelynormallydistributed.Noassociationswerefoundinhealthy individualsbetweentheselectedSNPsandHVA(Table1).
PreliminaryanalysisexcludingtheSNPsshowedthatHVA con-centrationswerenotassociatedwithantipsychotictreatmentin psychoticpatients.Ourindependentvariables,i.e.,theSNPs,are notexpected tobe associatedwith thepresence orabsence of
antipsychotictreatmentandmoreoverwehaveincludedtheuse ofantipsychoticsasacovariateinouranalyses.Thus,theuseof antipsychoticsin acluster ofpatientsshouldnot confound our analyses.
4. Discussion
To our knowledge, there is only one previously published studysearchingforassociationsbetweengenevariantsand CSF monoaminemetaboliteconcentrations,includingHVA,inpatients withpsychosis.Inthatstudy,nominalassociationsbetweengenes encodingenzymesimplicatedinthemonoaminemetabolismand CSFmonoaminemetaboliteconcentrationswerefound[4].No pre-viousstudieshavesearchedfor associationsbetweendopamine receptorgenevariantsandCSFmonoaminemetabolite concentra-tionsinpsychoticpatients.
4.1. DRD1
Almostallindependentstudiesand onemeta-analysisfailed toshowevidenceofassociationbetweenDRD1genevariantsand schizophrenia(http://www.szgene.org).DRD1hasbeenassociated withbipolardisorder[11,34],attention-deficit/hyperactivity dis-order[7]andautism[17].
DRD1rs4532hasbeenassociatedwithtreatmentresponseto antipsychoticdrugs[29]andtardivedyskinesiainpatientswith schizophrenia[21].Inthepresentstudy,rs4532wasnotassociated withCSFHVAconcentrations.
WeidentifiedfourSNPs,i.e.,rs11747728,rs11742274,rs265974 andrs11747886,locatedinaregion3.5–8kbpdownstreamDRD1, thatwereassociated withCSF HVAconcentrationsin psychotic patients.Theassociationsbetweenrs11747728(pvalue=0.0004) andrs11742274(pvalue=0.0005)withCSFHVAremained signif-icantaftercorrectionformultipletesting.TheSNPwiththelowest pvalue,rs11747728,isaregulatoryregionvariant,located4kbp downstreamDRD1.Theseassociationswererestrictedtopsychotic patientsandwereabsentinhealthycontrols.TheseSNPshavenot beenascribedanyassociationwithschizophreniaorothermental disorders.
D1receptorsareexpressedatahighlevelofdensityin vari-ousregionsinCNS[5]andthelackofassociationbetweenDRD1 andschizophrenialeadstootherapproaches,suchasthe investi-gationofintermediatephenotypes,inordertoimplicateDRD1in thediseaseprocesses.Arecentstudydisplayedthattheexpression ofDRD1mRNAwasdecreasedindorsolateralprefrontalcortexof patientswithschizophreniacomparedtocontrols[19].TheDRD1 densityindifferentbrainregionsinpatientswithschizophrenia andhealthy controlshasalsobeenstudiedwithpositron emis-siontomographyscanwithsomestudiesfindinganincreasedDRD1 availabilityinprefrontalcortexinpatientsrelativetocontrols[2], mainlyinthecaseofdrug-naïvepatients[3].Thisincreasemay
rep-resentacompensatoryupregulationsecondarytoamesocortical dopaminefunctiondeficiency.Theidentificationofalocus down-streamDRD1affectingthemajordegradationproductofdopamine supportsthehypothesisoftheimplicationofDRD1inpsychosis andgenerallythedopaminehypothesisofschizophrenia.
4.2. DRD2
DRD2gene variationshave beenextensivelyinvestigated for associations with schizophrenia, with numerous positive stud-ies,threepositivemeta-analyses(www.szgene.org)andapositive genome-wide associationstudy[1].DRD2 hasalsobeen associ-atedwithmooddisorders,substanceusedisorders[44],Tourette’s syndromeandpost-traumaticstressdisorder[27].
ThemostrobustassociationfoundwasbetweenDRD2rs6277 andschizophreniawithapositivemeta-analysiswithanoddsratio of1.29(www.szgene.org).Rs6277hasbeenincludedasacandidate SNPinthepresentstudyandhasnotbeenfoundtobeassociated withCSFHVAconcentrationsinpsychoticpatients.Another inter-estingresultistheidentificationofaSNP,i.e.,rs2514218,located 47kbpdownstreamDRD2foundtobegenome-widesignificantly associatedwithschizophrenia[1].Rs2514218wasnotincludedin thepresentstudy.
Inthepresentstudy,DRD2 rs2234689wasnominally associ-ated withCSF HVAconcentrations in psychoticpatients. DRD2 rs2234689isadownstreamgenevariantandhasnotbeenascribed anyfunctionalityorassociationwithmentaldisorders.
4.3. DRD3
DRD3genevariantshavebeenassociatedwithschizophreniain manyindependentstudies.However,allmeta-analysesconducted failedtoconfirmsignificantassociations(www.szgene.org).DRD3 SNPshavealsobeenassociatedwithothermentaldisorderssuch asautism[10]andunipolardepression[12].
Inthepresentstudy,noDRD3SNPwasassociatedwithCSFHVA concentrationsinpatientswithpsychoticdisorder.
4.4. DRD4
DRD4genevariantshavebeenassociatedwithschizophreniain someindependentstudiesandtwometa-analyses(www.szgene. org),aswellaswithothermentaldisorders,mainlyADHD[31].
In the present study, DRD4 rs3758653 wasnominally asso-ciatedwithCSFHVAconcentrations inpatientswithpsychosis. Rs3758653is an upstreamgene variantpreviously reportedto beassociatedwithDNAmethylation acrosstheDRD4 promoter regioninbothlymphoblastoidcelllinesandpost-mortembrain tis-sue[13].Rs3758653hasbeenalsoreportedtobeassociatedwith Alzheimer’sdisease[23].
4.5. DRD5
DRD5genevariantshavebeenassociated withschizophrenia (www.szgene.org)andADHD[20]inindependentstudies.Inthe presentstudy,noDRD5SNPwasassociatedwithCSFHVA concen-trationsinpsychoticpatients.
4.6. Limitations
Thepresentstudysuffersfromsomelimitations.First,beinga geneticstudythenumbersofparticipantsissmall.Moreover,the assumptionthataBonferronicorrectionforthetotalnumberof testsconductedisasufficientcorrectionforageneticstudymay beconsideredasalimitation,asthereisnosufficientprior evi-dencetolimittheanalysistothespecificgenes.However,aswe
havenottestedthewholegenomeandhaveusedastronga pri-orihypothesis,i.e.,thattheselecteddopamine-relatedgenesmay beregardedlikelytoinfluencedopaminemetabolite concentra-tionsinpatientswithpsychosis,wehavenotappliedthestandard thresholdforgenome-widesignificancebutaBonferroni correc-tiontakingintoaccountthetotalnumberoftestsconducted.The resultsmustbeseenastentative,andthereisneedforindependent replication.Also,severalofthepatientshadbeenmedicatedwith antipsychoticsdrugs.However,fromastatisticalpointofview,the varioususeofantipsychoticsamongthepatientsshouldnot con-foundtheresultsasthepresenceofantipsychoticshasnotbeen foundtobeassociated withHVAconcentrationsin thepresent sampleandisnotexpectedtobeassociatedwiththeinvestigated SNPs.
5. Conclusions
Inpsychoticpatients,wefoundtwosignificantandtwo nomi-nalassociationsbetweenSNPslocatedatalocusdownstreamDRD1 andtheCNSdopamineturnoverrate,asreflectedbytheCSF con-centration of HVA.These associations were present in patients withpsychoticdisorderandabsentinhealthycontrols.Thepresent studysuggeststhattheDRD1geneisimplicatedinthe pathophys-iologyofschizophreniaandsupportsthedopaminehypothesisof schizophrenia.
Authors’contributions
DAcontributedtotheconceptionanddesignofthestudy, par-ticipatedinsubjectassessment,subjectcharacterizationandthe statisticalanalysis,managedtheliteraturesearchandweb-based databasesearchesanddraftedthearticle.ESperformedthe sta-tisticalanalysis.TAwasinchargeofthegenotypingprocedures. GCSmadeacontributiontotheconceptionanddesignofthestudy andtotheacquisitionofdata.LTandIAcontributedtothe concep-tionanddesignofthestudy.EGJcontributedtotheconceptionand designofthestudy,theacquisitionandtheinterpretationofdata. Allauthorsrevisedthearticlecriticallyforimportantintellectual contentandapprovedthefinalmanuscript.
Acknowledgements
We thank the patients and controls for their participation. ThepresentstudywasfinancedbytheSwedishResearch Coun-cil(K2007-62X-15077-04-1,K2008-62P-20597-01-3, K2010-62X-15078-07-2,K2012-61X-15078-09-3),theregionalagreementon medicaltrainingandclinicalresearchbetweenStockholmCounty CouncilandtheKarolinskaInstitutet,theKnutandAlice Wallen-bergFoundation,andtheHUBINproject.WethankAlexandraTylec, AgnetaGunnar,MonicaHellberg,andKjerstinLindfortechnical assistance.GenotypingwasperformedbytheSNP&SEQTechnology PlatforminUppsala,Sweden(www.genotyping.se)whichis sup-portedbyUppsalaUniversity,UppsalaUniversityHospital,Science forLifeLaboratory—UppsalaandtheSwedishResearchCouncil.
References
[1]SchizophreniaWorkingGroupofthePsychiatricGenomicsConsortium, Biologicalinsightsfrom108schizophrenia-associatedgeneticloci,Nature 511(2014)421–427.
[2]A.Abi-Dargham,O.Mawlawi,I.Lombardo,R.Gil,D.Martinez,Y.Huang,D.R. Hwang,J.Keilp,L.Kochan,R.VanHeertum,J.M.Gorman,M.Laruelle, PrefrontaldopamineD1receptorsandworkingmemoryinschizophrenia,J. Neurosci.22(2002)3708–3719.
[3]A.Abi-Dargham,X.Xu,J.L.Thompson,R.Gil,L.S.Kegeles,N.Urban,R. Narendran,D.R.Hwang,M.Laruelle,M.Slifstein,Increasedprefrontalcortical D(1)receptorsindrugnaivepatientswithschizophrenia:aPETstudywith [(1)(1)C]NNC112,J.Psychopharmacol.26(2012)794–805.
[4]D.Andreou,E.Soderman,T.Axelsson,G.C.Sedvall,L.Terenius,I.Agartz,E.G. Jonsson,Polymorphismsingenesimplicatedindopamine,serotoninand noradrenalinmetabolismsuggestassociationwithcerebrospinalfluid monoaminemetaboliteconcentrationsinpsychosis,Behav.BrainFunct.10 (2014)26.
[5]J.M.Beaulieu,R.R.Gainetdinov,Thephysiologysignaling,andpharmacology ofdopaminereceptors,Pharmacol.Rev.63(2011)182–217.
[6]L.Bjerkenstedt,G.Edman,L.Hagenfeldt,G.Sedvall,F.-A.Wiesel,Plasma aminoacidsinrelationtocerebrospinalfluidmonoaminemetabolitesin schizophrenicpatientsandhealthycontrols,Br.J.Psychiatry147(1985) 276–282.
[7]A.J.Bobb,A.M.Addington,E.Sidransky,M.C.Gornick,J.P.Lerch,D.K. Greenstein,L.S.Clasen,W.S.Sharp,G.Inoff-Germain,F.Wavrant-DeVrieze, M.Arcos-Burgos,R.E.Straub,J.A.Hardy,F.X.Castellanos,J.L.Rapoport, SupportforassociationbetweenADHDandtwocandidategenes:NET1and DRD1,Am.J.Med.Genet.BNeuropsychiatr.Genet.134B(2005)67–72. [8]A.Carlborg,J.Jokinen,A.-L.Nordström,E.G.Jönsson,P.Nordström,CSF 5-HIAA,attemptedsuicideandsuicideriskinschizophreniaspectrum psychosis,Schizophr.Res.112(2009)80–85.
[9]A.Carlsson,N.Waters,S.Holm-Waters,J.Tedroff,M.Nilsson,M.L.Carlsson, Interactionsbetweenmonoamines,glutamate,andGABAinschizophrenia: newevidence,Annu.Rev.Pharmacol.Toxicol.41(2001)237–260.
[10]M.deKrom,W.G.Staal,R.A.Ophoff,J.Hendriks,J.Buitelaar,B.Franke,M.V.de Jonge,P.Bolton,D.Collier,S.Curran,H.vanEngeland,J.M.vanRee,Acommon variantinDRD3receptorisassociatedwithautismspectrumdisorder,Biol. Psychiatry65(2009)625–630.
[11]M.DelZompo,V.DeLuca,G.Severino,X.Ni,S.Mulas,D.Congiu,M.P.Piccardi, J.L.Kennedy,HaplotypeassociationstudybetweenDRD1geneandbipolar typeIaffectivedisorderintwosamplesfromCanadaandSardinia,Am.J.Med. Genet.BNeuropsychiatr.Genet.144B(2007)237–241.
[12]D.G.Dikeos,G.N.Papadimitriou,D.Avramopoulos,G.Karadima,E.G. Daskalopoulou,D.Souery,J.Mendlewicz,D.Vassilopoulos,C.N.Stefanis, AssociationbetweenthedopamineD3receptorgenelocus(DRD3)and unipolaraffectivedisorder,Psychiatr.Genet.9(1999)189–195.
[13]S.J.Docherty,O.S.Davis,C.M.Haworth,R.Plomin,U.D’Souza,J.Mill,Agenetic associationstudyofDNAmethylationlevelsintheDRD4generegionfinds associationswithnearbySNPs,Behav.BrainFunct.8(2012)31.
[14]B.Ekholm,A.Ekholm,R.Adolfsson,M.Vares,U.Ösby,G.C.Sedvall,E.G. Jönsson,EvaluationofdiagnosticproceduresinSwedishpatientswith schizophreniaandrelatedpsychoses,Nord.J.Psychiatry59(2005)457–464. [15]J.B.Fan,A.Oliphant,R.Shen,B.G.Kermani,F.Garcia,K.L.Gunderson,M.
Hansen,F.Steemers,S.L.Butler,P.Deloukas,L.Galver,S.Hunt,C.McBride,M. Bibikova,T.Rubano,J.Chen,E.Wickham,D.Doucet,W.Chang,D.Campbell,B. Zhang,S.Kruglyak,D.Bentley,J.Haas,P.Rigault,L.Zhou,J.Stuelpnagel,M.S. Chee,HighlyparallelSNPgenotyping,ColdSpringHarb.Symp.Quant.Biol.68 (2003)69–78.
[16]J.A.Gingrich,M.G.Caron,Recentadvancesinthemolecularbiologyof dopaminereceptors,Annu.Rev.Neurosci.16(1993)299–321. [17]J.A.Hettinger,X.Liu,C.E.Schwartz,R.C.Michaelis,J.J.Holden,ADRD1
haplotypeisassociatedwithriskforautismspectrumdisordersinmale-only affectedsib-pairfamilies,Am.J.Med.Genet.BNeuropsychiatr.Genet.147B (2008)628–636.
[18]E.G.Jönsson,J.Bah,J.Melke,R.AbouJamra,J.Schumacher,L.Westberg,R.Ivo, S.Cichon,P.Propping,M.M.Nothen,E.Eriksson,G.C.Sedvall,Monoamine relatedfunctionalgenevariantsandrelationshipstomonoaminemetabolite concentrationsinCSFofhealthyvolunteers,BMCPsychiatry4(2004)4. [19]S.S.Kaalund,E.N.Newburn,T.Ye,R.Tao,C.Li,A.Deep-Soboslay,M.M.
Herman,T.M.Hyde,D.R.Weinberger,B.K.Lipska,J.E.Kleinman,Contrasting changesinDRD1andDRD2splicevariantexpressioninschizophreniaand affectivedisorders,andassociationswithSNPsinpostmortembrain,Mol. Psychiatry19(2013)1256–1258.
[20]B.N.Kim,D.Kang,S.C.Cho,T.W.Park,M.H.Lim,Y.C.Chung,J.W.Kim,J.W. Hwang,H.J.Yoo,U.S.Chung,J.W.Son,J.C.Yang,S.K.Chung,J.Y.Lee,Y.W.Jung, ShorterdinucleotiderepeatlengthintheDRD5geneisassociatedwith attentiondeficithyperactivitydisorder,Psychiatr.Genet.19(2009)57. [21]I.C.Lai,G.H.Mo,M.L.Chen,Y.C.Wang,J.Y.Chen,D.L.Liao,Y.M.Bai,C.C.Lin,
T.T.Chen,Y.J.Liou,AnalysisofgeneticvariationsinthedopamineD1receptor (DRD1)geneandantipsychotics-inducedtardivedyskinesiainschizophrenia, Eur.J.Clin.Pharmacol.67(2011)383–388.
[22]M.Laruelle,Schizophrenia:fromdopaminergictoglutamatergic interventions,Curr.Opin.Pharmacol.14C(2014)97–102.
[23]W.Y.Lin,B.T.Wu,C.C.Lee,J.J.Sheu,S.H.Liu,W.F.Wang,C.H.Tsai,H.P.Liu,F.J. Tsai,Associationanalysisofdopaminergicgenevariants(Comt,Drd4And Dat1)withAlzheimersdisease,J.Biol.Regul.Homeost.Agents26(2012) 401–410.
[24]C.Missale,S.R.Nash,S.W.Robinson,M.Jaber,M.G.Caron,Dopamine receptors:fromstructuretofunction,Physiol.Rev.78(1998)189–225. [25]A.T.Moir,G.W.Ashcroft,T.B.Crawford,D.Eccleston,H.C.Guldberg,Cerebral
metabolitesincerebrospinalfluidasabiochemicalapproachtothebrain, Brain93(1970)357–368.
[26]G.Nikisch,P.Baumann,G.Wiedemann,B.Kiessling,H.Weisser,A.Hertel,T. Yoshitake,J.Kehr,A.A.Mathe,Quetiapineandnorquetiapineinplasmaand cerebrospinalfluidofschizophrenicpatientstreatedwithquetiapine: correlationstoclinicaloutcomeandHVA,5-HIAA,andMHPGinCSF,J.Clin. Psychopharmacol.30(2010)496–503.
[27]E.P.Noble,TheDRD2geneinpsychiatricandneurologicaldisordersandits phenotypes,Pharmacogenomics1(2000)309–333.
[28]C.Nordin,B.Siwers,L.Bertilsson,Siteoflumbarpunctureinfluenceslevelsof monoaminemetabolites,Arch.Gen.Psychiatry39(1982)1445.
[29]V.K.Ota,L.N.Spindola,A.Gadelha,A.F.dosSantosFilho,M.L.Santoro,D.M. Christofolini,F.T.Bellucco,A.K.Ribeiro-dos-Santos,S.Santos,J.MariJde,M.I. Melaragno,R.A.Bressan,A.SmithMde,S.I.Belangero,DRD1rs4532 polymorphism:apotentialpharmacogenomicmarkerfortreatmentresponse toantipsychoticdrugs,Schizophr.Res.142(2012)206–208.
[30]G.Oxenstierna,G.Edman,L.Iselius,L.Oreland,S.B.Ross,G.Sedvall, Concentrationsofmonoaminemetabolitesinthecerebrospinalfluidoftwins andunrelatedindividuals—ageneticstudy,J.Psychiatr.Res.20(1986)19–29. [31]R.Ptacek,H.Kuzelova,G.B.Stefano,DopamineD4receptorgeneDRD4andits
associationwithpsychiatricdisorders,Med.Sci.Monit.17(2011) RA215–RA220.
[32]J.Rogers,L.J.Martin,A.G.Comuzzie,J.J.Mann,S.B.Manuck,M.Leland,J.R. Kaplan,Geneticsofmonoaminemetabolitesinbaboons:overlappingsetsof genesinfluencelevelsof5-hydroxyindolaceticacid,
3-hydroxy-4-methoxyphenylglycol,andhomovanillicacid,Biol.Psychiatry 55(2004)739–744.
[33]F.E.Scheepers,C.C.Gispen-deWied,H.G.Westenberg,R.S.Kahn,Theeffectof olanzapinetreatmentonmonoaminemetaboliteconcentrationsinthe cerebrospinalfluidofschizophrenicpatients,Neuropsychopharmacology25 (2001)468–475.
[34]J.Shi,J.A.Badner,E.Hattori,J.B.Potash,V.L.Willour,F.J.McMahon,E.S. Gershon,C.Liu,Neurotransmissionandbipolardisorder:asystematic family-basedassociationstudy,Am.J.Med.Genet.BNeuropsychiatr.Genet. 147B(2008)1270–1277.
[35]R.L.Spitzer,J.B.W.Williams,M.Gibbon,M.B.First,StructuredClinical InterviewforDSM-III-R−PatientVersion(SCID-P),BiometricsResearch DepartmentNewYork,StatePsychiatricInstituteNewYork,1988. [36]M.Stanley,L.Traskman-Bendz,K.Dorovini-Zis,Correlationsbetween
aminergicmetabolitessimultaneouslyobtainedfromhumanCSFandbrain, LifeSci.37(1985)1279–1286.
[37]P.F.Sullivan,M.J.Daly,M.O’Donovan,Geneticarchitecturesofpsychiatric disorders:theemergingpictureanditsimplications,Nat.Rev.Genet.13 (2012)537–551.
[38]C.-G.Swahn,B.Sandgärde,F.-A.Wiesel,G.Sedvall,Simultaneous
determinationofthethreemajormonoaminemetabolitesinbraintissueand bodyfluidsbyamassfragmentographicmethod,Psychopharmacology(Berl.) 48(1976)147–152.
[39]M.Tiberi,K.R.Jarvie,C.Silvia,P.Falardeau,J.A.Gingrich,N.Godinot,L. Bertrand,T.L.Yang-Feng,R.T.FremeauJr.,M.G.Caron,Cloning,molecular characterization,andchromosomalassignmentofageneencodingasecond D1dopaminereceptorsubtype:differentialexpressionpatterninratbrain comparedwiththeD1Areceptor,Proc.Natl.Acad.Sci.U.S.A.88(1991) 7491–7495.
[40]D.Vallone,R.Picetti,E.Borrelli,Structureandfunctionofdopaminereceptors, Neurosci.Biobehav.Rev.24(2000)125–132.
[41]M.Vares,A.Ekholm,G.C.Sedvall,H.Hall,E.G.Jönsson,Characterisationof patientswithschizophreniaandrelatedpsychosis:evaluationofdifferent diagnosticprocedures,Psychopathology39(2006)286–295.
[42]P.Wester,U.Bergstrom,A.Eriksson,C.Gezelius,J.Hardy,B.Winblad, Ventricularcerebrospinalfluidmonoaminetransmitterandmetabolite concentrationsreflecthumanbrainneurochemistryinautopsycases,J. Neurochem.54(1990)1148–1156.
[43]I.M.Wieselgren,L.H.Lindstrom,CSFlevelsofHVAand5-HIAAindrug-free schizophrenicpatientsandhealthycontrols:aprospectivestudyfocusedon theirpredictivevalueforoutcomeinschizophrenia,PsychiatryRes.81(1998) 101–110.
[44]Y.F.Zou,F.Wang,X.L.Feng,W.F.Li,Y.H.Tian,J.H.Tao,F.M.Pan,F.Huang, AssociationofDRD2genepolymorphismswithmooddisorders:a meta-analysis,J.Affect.Disord.136(2012)229–237.
