Table Of ContentCitation:TranslPsychiatry(2012)2,e67, doi:10.1038/tp.2011.66
&2012MacmillanPublishersLimited Allrightsreserved2158-3188/12
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NMDA receptor genotypes associated with the
vulnerability to develop dyskinesia
SAIvanova1,AJMLoonen2,3,PPechlivanoglou2,MBFreidin4,5,AFYAlHadithy2,6,EVRudikov1,IAZhukova7,NVGovorin8,
VASorokina9,OYFedorenko1,VMAlifirova7,AVSemke1,JRBJBrouwers2andBWilffert2
Dyskinesias are involuntary muscle movements that occur spontaneously in Huntington’s disease (HD) and after long-term
treatmentsforParkinson’sdisease(levodopa-induceddyskinesia;LID)orforschizophrenia(tardivedyskinesia,TD).Previous
studies suggested that dyskinesias in these three conditions originate from different neuronal pathways that converge on
overstimulationofthemotorcortex.WehypothesizedthatthesamevariantsoftheN-methyl-D-aspartatereceptorgenethatwere
previously associated withthe age of dyskinesia onsetin HD were also associated with the vulnerability for TD and not LID.
Genotyping patients with LID and TD revealed, however, that these two variants were dose-dependently associated with
susceptibility to LID, but not TD. This suggested that LID, TD and HD might arise from the same neuronal pathways, but TD
resultsfromadifferentmechanism.
TranslationalPsychiatry(2012)2,e67;doi:10.1038/tp.2011.66;publishedonline10January2012
Introduction shown that presence of a specific variant of the NMDA
receptorsubunitgeneisstronglyrelatedtotheageofonsetof
Dyskinesiasareinvoluntarymusclemovementsthatoccuras dyskinesias in HD.11–13 These variants could increase the
a symptom of Huntington’s disease (HD), but also as a vulnerabilitytodevelopdyskinesiabyincreasingthesensitiv-
complicationoflong-termtreatmentwithantipsychoticdrugs ity of indirect pathway MSN to excitotoxicity. As TD is also
(tardive dyskinesia, TD) or levodopa in Parkinson’s disease believed to be related to toxicity at indirect pathway MSN,
(on-offphenomena,levodopa-induceddyskinesia,LID).1–4In these variants might also increase the likelihood to develop
HD, these involuntary movements have been linked to TD. LID is believed to be related to increased sensitivity
degeneration of a specific subtype of striatal medium-sized of cortico-striatal synapses with direct pathway MSN, and
spiny neurons (MSNs) that constitute part of the indirect expected to be independent of excitotoxicity at indirect
pathway of the extrapyramidal circuit (Figure 1).5–7 Stimula- pathwayMSN.Wethereforehypothesizedthatthesevariants
tion of these indirect pathway MSN by cortico-striatal mightbeassociatedwiththelikelihoodtodevelopTDandnot
glutamatergic neurons results in inhibition of cortical motor LID. Our results, to our surprise, are opposite to this
areas at the end of this circuit (hypokinesia).8 In contrast to hypothesisandmakeustoquestiontheroleofdirectpathway
dyskinesiasinHD,LIDisbelievedtobeinducedbydopamine MSNinLID.
D1receptorbearingdirectpathwayMSN.4Pulsatilestimula-
tionoftheseD1receptorsmaycauselong-termpotentiationat
N-methyl-D-aspartate (NMDA) receptor of corresponding Subjectsandmethods
cortico-striatal synapses, which results in overactivity of the
directpathwayMSNs.Thisleadstoactivationofcorticalareas Patients. In order to test our hypotheses about the role
at the end of this circuit (hyperkinesia).8 The mechanism of the abovementioned NMDA receptor variants in TD and
underlyingTDisnotwellunderstood.Thismovementdisorder LID, we examined a total of 574 Caucasian patients
occursafterlong-termtreatmentwithD2receptorantagonists (Supplementary Table 1). Of them, 431 were from three
andisbelieved tobecausedbydamageof theD2 receptor psychiatrichospitalsinTomsk,KemerovoandChitaoblasts
bearingindirectpathwayMSNs.Ithasbeenhypothesizedthat inSiberia.Thesepatientsdemonstratedschizophrenia(N¼401,
this damage is caused by the formation of free radicals 95.1%)orschizotypicaldisordersaccordingtoICD-10criteria.
inducedbylong-termtreatment.9However,treatmentwiththe All these patients were on long-term treatment with anti-
antioxidant,vitaminE,didnothaveconvincingeffectsonTD.9 psychotic drugs. For comparison, antipsychotic medication
Alternatively, Konitiostis and others reported that NMDA doses were converted into chlorpromazine equivalents.14
antagonists showed therapeutic effects in an animal model Patients were assessed for the presence or absence of
of TD.10 Recently, two independent research groups have dyskinesia,accordingtotheabnormalinvoluntarymovement
1MentalHealthResearchInstitute,Tomsk,Russia;2DepartmentofPharmacy,UniversityofGroningen,Groningen,TheNetherlands;3MentalHealthInstituteWestelijk
Noord-Brabant,Halsteren,TheNetherlands;4ResearchInstituteforMedicalGenetics,Tomsk,Russia;5NationalHeartandLungInstitute,ImperialCollegeLondon,
London,UK;6ErasmusUniversityMedicalCenter,Rotterdam,TheNetherlands;7SiberianStateMedicalUniversity,Tomsk,Russia;8ChitaStateMedicalAcademy,
Chita,Russiaand9KemerovoRegionalClinicalPsychiatricHospital,Kemerovo,Russia
Correspondence:ProfessorAJMLoonen,DepartmentofPharmacy,UniversityofGroningen,AntoniusDeusinglaan1,Groningen9713AV,TheNetherlands.
E-mail:[email protected]
Keywords:extra-pyramidal;GRIN2A;levodopa-induceddyskinesia;mediumspinyneuron;NMDA;tardivedyskinesia
Received1September2011;revised7November2011;accepted6December2011
Dyskinesia-associatedglutamatereceptorgenotypes
SAIvanovaetal
2
The selection method was previously described by Xu
and Taylor (freely available at http://www.niehs.nih.gov/
snpinfo).17 We selected only tag SNPs that captured at
least 10 SNPs, including finally 15 SNPs that covered the
GRIN2A gene and 9 that covered the GRIN2B gene. For
comparison,wealsoselectedasetofvariantsof6unrelated
genes that have been reported to be of interest for
developingTD.Ourfinalpanelconsistedof48tagSNPs.
Statistical analysis. In order to be more certain about
thevalidityoftheresults,weusedtwostatisticalapproaches
to analyze the data. Both biostatisticians (MF and PP)
independently developed different strategies to account for
missing data and interactions between different SNPs. The
Hardy–Weinbergequilibriumtestwasappliedtogroupswith
and without dyskinesia. Thereafter, the first biostatistician
used the more classical logistic regression and log-linear
regression approach. The second biostatistician used the
permutation tests and logic regression method.18 Because
the genotyping for GRIN2A and GRIN2B was definitely
hypothesis-driven, no correction for multiple testing was
necessaryontheprimaryoutcomemeasure.Allcalculations
were performed in the R statistical environment with the
SNPassoc19andLogicReg18packageandbasicRfunctions.
Results
Inpatientswithneurologicaldisorders(n¼143),permutation
tests indicated that potential associations existed between
LID and the candidate SNPs or combinations of SNPs. In
Figure 1 Schematic model of the cortico-striatal-thalamic-cortical (formerly models focused on patients with diagnosed Parkinson’s
extrapyramidal) circuits, including the indirect and direct pathways; ENK, disease (n¼101), 9% of permutations yielded a deviance-
enkephalin;GPe,Globuspallidus,externalsegment;GPi,Globuspallidus,internal based score smaller than that obtained with the best-fitted
segment; SNc, Substantia nigra, pars compacta; SNr, substantia nigra, pars model. In models focused on patients with Parkinson’s
reticulata; SP/DYN, substance P/dynorphin; STh, subthalamic nucleus; D1, D2, disease that displayed only SNPs of the GRIN2A gene,
medium-sizedspinyneuronswithD1orD2receptors.
4.8%ofpermutations yieldedasmaller score;thisindicated
possibleassociationsbetweentheGRIN2ASNPsandLID.
AsubsequentMonteCarlologicregressionanalysisrevea-
scale (AIMS).15 The AIMS scores were transformed into a led that two GRIN2A variants, rs7192557 and rs8057394,
binaryform(presenceorabsenceofdyskinesia)withSchooler were most frequently associated with LID observed in all
andKane’scriteria.16Inaddition,abloodsamplewastakenfor body locations (‘all locations’). It is important to notice that
DNA isolation and genotyping. We also examined 143 rs7192557 is a tagging SNP for rs1969060, which was
patients with spontaneous extrapyramidal disorders (101 previously associated with the age of dyskinesia onset
(70.6%) of them had Parkinson’s disease, 21 (14.7%) had in,11,12 and that SNP rs8057394 was the other variant
dystonia, 21 had tremor) from the neurology department of associatedwithHD.13
the Siberian State Medical University of Tomsk. These In an alternative analytical approach, logistic regression
patients had not been treated with dopamine antagonists wasusedtotestassociationsbetweenSNPsanddyskinesia.
(antipsychotic or antiemetic drugs) for at least 3 years. Of The results indicated that the same two SNPs mentioned
both the groups, other inclusion criteria were no addictions, abovehadthehighestsignificance.Whenthepatientsample
no current clozapine treatment, no organic disorders and a was limited to patients with diagnosed Parkinson’s disease
highqualityDNAsample. (Supplementary Table 2), a higher odds ratio was observed
than that for the entire group of patients with neurological
DNA analysis. DNA extraction and the Veracode Assay disorders (n¼143). For example, the odds ratio for an
wereconductedaccordingtostandardprotocols.Apartfrom association between rs7192557 and the ‘all locations’ LID
the to-be studied variants of the genes that encoded two was3.21(95%CI:1.37–7.51,P¼0.0062)forpatientsdiagnosed
subunits of the glutamatergic NMDA receptor, NR2A and with Parkinson’s disease, and 2.71 (95% CI: 1.35–5.46,
NR2B (GRIN2A and GRIN2B, respectively), we selected a P¼0.0046)forthewholegroupofpatientswithneurological
subset of informative SNPs, or tag SNPs, that would disorders. For rs8057394, these figures were 3.59 (95%
accurately represent the majority of SNPs for these genes. CI: 1.48–8.71; P¼0.0033) and 2.86 (95% CI: 1.42–5.76,
TranslationalPsychiatry
Dyskinesia-associatedglutamatereceptorgenotypes
SAIvanovaetal
3
P¼0.0028), respectively. These two SNPs were also signi- striatalMSNsthatformtheindirectpathway.5–7Accordingly,
ficantly(Po0.05)associatedwiththeseverityofLID. thisimpliesthatLIDmayarisefromdamagetoMSNsinthe
Thus, two different statistical methods, the permutation indirect pathway. Our hypothesis is supported by the
analysis with logic regression and the logistic regression pathological changes observed in HD, where damage only
providedsimilarresults. occurstoMSNsintheindirectpathway.
In the patient population with psychiatric disorders, no Incontrast,ourresultsshowedthatTDwasnotassociated
relationshipcouldbedemonstratedbetweendyskinesiaand with the same NMDA receptor variants. However, antipsy-
theGRIN2Avariants,rs7192557andrs8057394.Moreover, chotic drugs are also known to affect MSNs that carry
inthispatientpopulation,thetwodifferentstatisticalanalysis dopamine D2 receptors in the indirect pathway.8 Therefore,
methods produced inconsistent results. Logic regression the MSNs in the indirect pathwaymay be involvedin TD as
revealedthattheGRIN2Bvariantrs2192970andtheGRIN2A well,butthroughadifferentmechanism.
variant rs1345423 were the most influential of the SNPs On the basis of our results for LID, we speculate that the
tested; however, permutation tests showed almost no MSNsintheindirectpathwaymaybeparticularlysusceptible
association between the SNPs and the outcome. A logistic to excitotoxicity during long-term treatment with levodopa
regressionanalysisalsoidentifiedthesetwoSNPs,butonly owing to the same mechanism that results in progressive
rs1345423 consistently showed highly significant associa- degeneration of nigrostriatal dopaminergic neurons in Par-
tionswiththepresenceofTDandwiththeseverityofTD. kinson’s disease.23 A well-known theory states that Parkin-
son’s disease-induced degeneration is related to the
genetically-determined susceptibility of catecholamine-
Discussion
expressing neurons to oxidative stress.23 Therefore, we
Our results showed that the susceptibility to LID was propose that long-term exposure to the catecholamines
associated with two NMDA receptor (GRIN2A) variants, derivedfromlevodopamaycausethedeathofMSNsdueto
identified by SNPs rs7192557 and rs8057394, which were a combination of increased susceptibility to oxidative stress
previouslyfoundtobeassociatedwiththeageofdyskinesia andthepresenceofavariantNMDAreceptorthataccelerates
onsetinHD.11–13WealsofoundthatthesusceptibilitytoTD excitotoxicity.
was associated with two different GRIN2B and GRIN2A However,beforetheabovehypothesiscanbetested,itis
variants identified by SNPs rs2192970 and rs1345423, necessary to replicate our finding and to investigate the
respectively. However, the latter associations were by far functionofthevariantGRIN2Areceptorgeneingreaterdetail.
lessconvincing.Itshouldbeemphasizedthatourhypothesis ItshouldbeestablishedwhetherthevariantNMDAreceptoris
only addressed a possible role for the previously identified moreactiveininducingexcitotoxicityand,inaddition,whether
SNPs. We selected a panel of 48 SNPs in eight candidate itissensitiveforNMDAantagonists(forexample,memantine
genes. These included 24 SNPs in the genes that encoded or hemantane). It may then be possible to prevent LID
GRIN2AandGRIN2B.Therefore,atleastsomeassociations by blocking activation of these variant NMDA receptors.
should be attributed to chance findings owing to multiple Thus, these NMDA antagonists may also be an effective
testing. However, when truly associated, the GRIN2A prophylactic for preventing LID in patients that are prone to
(rs1345423) and GRIN2B (rs2192970) variants are unlikely excitotoxicity during levodopa treatment. Interestingly, the
toberelatedtothepolymorphismspreviouslyfoundingenetic NMDAantagonistamantadineisknowntohaveacuteeffects
associationstudiesinschizophrenia20ortheGRIN2Bvariants on LID,24 but memantine lacks this effects.25 This can be
thatwerepreviouslystudiedinTD21andLID.22 explainedbypostulatingNMDAantagonismprimarilyatdirect
Itshouldbenoticedthatlimb-truncalandalllocationsLID andindirectpathwayMSNs,respectively.
showanassociationwithrs7192557andrs8057394,andthat
orofacialLIDshowsnoassociationwithanySNPatall.This
Conflictofinterest
maycorrespondwiththeobservationthatinHDandLIDoften
large muscle groups are affected and in TD more subtle Theauthorsdeclarenoconflictofinterest.
movementsarepresent,mostoftenintheorofacialarea.
Animportantweaknessofourstudyistherestrictionofthe Acknowledgements. WethanktheGenomeAnalysisFacility(DrPvan
patientpopulationtoCaucasiansfromSiberiainRussia.This derVlies)oftheGeneticsDepartmentoftheUniversityMedicalCenter,Groningen
couldalsobeconsideredasstrengthofthestudyasamore (UMCG)fortheirassistanceandDrPKretchmer,SanFranciscoEdit,MillValley,
CA(www.sfedit.net)foreditingthemanuscript.WewishtothankDrsLarissaArning
homogenous sample was used. Nevertheless, the need for
(Ruhr-University, Bochum, Germany), Anton Bespalov (Abbott, Ludwigshafen,
replicationofourfindingsinanindependentpatientsampleis
Germany),BernardLerer(HebrewUniversityMedicalCenter,Jerusalem,Israel),
obvious.Inaddition,theinfluenceofgenderandraceshould and Hans-Ju¨rgen Mo¨ller (Ludwig-Maximilians-University, Munich, Germany) for
bestudiedinmoredetailinalargerpatientgroup.Moreover, reading the manuscript and their fruitful comments. This research project was
the measures to assess TD in general show important supported by the Groningen Center of Drug Research Fund with no external
limitations.15However,thelargenumberofstudiedpsychia- financialsupportinRussiaortheNetherlands.
tricpatientsandouradaptationstotheassessmentscalegive
1. KaneJM.Tardivedyskinesiacirca2006.AmJPsychiatry2006;163:1316–1318.
ourclassificationsufficientreliability.
2. MargoleseHC,ChouinardG,KolivakisTT,BeauclairL,MillerR.Tardivedyskinesiainthe
OurresultsindicatethatHDandLID,butnotTD,mayresult eraoftypicalandatypicalantipsychotics.part1:Pathophysiologyandmechanismsof
from the same biochemical processes involved in the induction.CanJPsychiatry2005;50:541–547.
3. NovakMJ,TabriziSJ.Huntington’sdisease.BMJ2010;340:c3109.
pathology of the extrapyramidal circuit. In HD, this process
4. ThanviB,LoN,RobinsonT.Levodopa-induceddyskinesiainparkinson’sdisease:Clinical
was linked to NMDA receptor-induced excitotoxicity in the features,pathogenesis,preventionandtreatment.PostgradMedJ2007;83:384–388.
TranslationalPsychiatry
Dyskinesia-associatedglutamatereceptorgenotypes
SAIvanovaetal
4
5. EstradaSanchezAM,Mejia-ToiberJ,MassieuL.Excitotoxicneuronaldeathandthe 18. KooperbergC,RuczinskiI.IdentifyinginteractingSNPsusingmontecarlologicregression.
pathogenesisofhuntington’sdisease.ArchMedRes2008;39:265–276. GenetEpidemiol2005;28:157–170.
6. FanMM,RaymondLA.N-methyl-D-aspartate(NMDA)receptorfunctionandexcitotoxicity 19. GonzalezJR,ArmengolL,SoleX,GuinoE,MercaderJM,EstivillXetal.SNPassoc:an
inhuntington’sdisease.ProgNeurobiol2007;81:272–293. Rpackagetoperformwholegenomeassociationstudies.Bioinformatics2007;23:
7. KumarP,KaloniaH,KumarA.Huntington’sdisease:Pathogenesistoanimalmodels. 644–645.
PharmacolRep2010;62:1–14. 20. CherlynSY,WoonPS,LiuJJ,OngWY,TsaiGC,SimK.Geneticassociationstudiesof
8. GroenewegenHJ.Thebasalgangliaandmotorcontrol.NeuralPlast2003;10:107–120. glutamate,GABAandrelatedgenesinschizophreniaandbipolardisorder:Adecadeof
9. LohrJB,KuczenskiR,NiculescuAB.Oxidativemechanismsandtardivedyskinesia.CNS advance.NeurosciBiobehavRev2010;34:958–977.
Drugs2003;17:47–62. 21. LiouYJ,WangYC,ChenJY,BaiYM,LinCC,LiaoDLetal.Associationanalysisof
10. Konitsiotis S, Tsironis C, Kiortsis DN, Evangelou A. Effects of N-methyl-D-aspartate polymorphismsintheN-methyl-D-aspartate(NMDA)receptorsubunit2B(GRIN2B)gene
receptorantagonismonneuroleptic-inducedorofacialdyskinesias.Psychopharmacology andtardivedyskinesiainschizophrenia.PsychiatryRes2007;153:271–275.
(Berl)2006;185:369–377. 22. LeeJY,ChoJ,LeeEK,ParkSS,JeonBS.Differentialgeneticsusceptibilityindiphasicand
11. ArningL,KrausPH,ValentinS,SaftC,AndrichJ,EpplenJT.NR2AandNR2Breceptor peak-dosedyskinesiasinparkinson’sdisease.MovDisord2011;26:73–79.
genevariationsmodifyageatonsetinhuntingtondisease.Neurogenetics2005;6:25–28. 23. ShadrinaMI,SlominskyPA,LimborskaSA.Molecularmechanismsofpathogenesisof
12. AndresenJM,GayanJ,ChernySS,BrocklebankD,Alkorta-AranburuG,AddisEAetal. parkinson’sdisease.IntRevCellMolBiol2010;281:229–266.
Replicationoftwelveassociationstudiesforhuntington’sdiseaseresidualageofonsetin 24. Del Dotto P, Pavese N, Gambaccini G, Bernardini S, Metman LV, Chase TN et al.
largevenezuelankindreds.JMedGenet2007;44:44–50. Intravenousamantadineimproveslevadopa-induceddyskinesias:anacutedouble-blind
13. ArningL,SaftC,WieczorekS,AndrichJ,KrausPH,EpplenJT.NR2AandNR2Breceptor placebo-controlledstudy.MovDisord2001;16:515–520.
genevariationsmodifyage atonsetinhuntington disease inasex-specific manner. 25. Merello M, Nouzeilles MI, Cammarota A, Leiguarda R. Effect of memantine (NMDA
HumGenet2007;122:175–182. antagonist) on parkinson’s disease: a double-blind crossover randomized study. Clin
14. AndreasenNC,PresslerM,NopoulosP,MillerD,HoBC.Antipsychoticdoseequivalents Neuropharmacol1999;22:273–276.
anddose-years:astandardizedmethodforcomparingexposuretodifferentdrugs.Biol
Psychiatry2010;67:255–262.
15. LoonenAJ,vanPraagHM.Measuringmovementdisordersinantipsychoticdrugtrials:the
Translational Psychiatry is an open-access journal
needtodefineanewstandard.JClinPsychopharmacol2007;27:423–430.
16. SchoolerNR,KaneJM.Researchdiagnosesfortardivedyskinesia.ArchGenPsychiatry published by Nature Publishing Group. This work is
1982;39:486–487. licensedundertheCreativeCommonsAttribution-Noncommercial-Share
17. Xu Z, Taylor JA. SNPinfo: Integrating GWAS and candidate gene information into
Alike3.0UnportedLicense.Toviewacopyofthislicense,visithttp://
functionalSNPselectionforgeneticassociationstudies.NucleicAcidsRes2009;37:
W600–W605. creativecommons.org/licenses/by-nc-sa/3.0/
SupplementaryInformationaccompaniesthepaperontheTranslationalPsychiatrywebsite(http://www.nature.com/tp)
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