igg4 subclass glutamic acid decarboxylase antibodies (gada...
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IgG4 subclass glutamic acid decarboxylase antibodies (GADA) are associated with areduced risk of developing type 1 diabetes as well as increased C-peptide levels inGADA positive gestational diabetes.
Dereke, Jonatan; Nilsson, Charlotta; Strevens, Helena; Landin-Olsson, Mona; Hillman,MagnusPublished in:Clinical Immunology
DOI:10.1016/j.clim.2015.11.001
2016
Document Version:Peer reviewed version (aka post-print)
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Citation for published version (APA):Dereke, J., Nilsson, C., Strevens, H., Landin-Olsson, M., & Hillman, M. (2016). IgG4 subclass glutamic aciddecarboxylase antibodies (GADA) are associated with a reduced risk of developing type 1 diabetes as well asincreased C-peptide levels in GADA positive gestational diabetes. Clinical Immunology, 162, 45-48.https://doi.org/10.1016/j.clim.2015.11.001
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IgG4subclassGlutamicaciddecarboxylaseantibodies(GADA)areassociatedwithareducedriskofdevelopingtype1diabetesaswellasincreasedC-peptidelevelsinGADApositivegestationaldiabetesJonatanDerekeMSc1,CharlottaNilssonMDPhD1,2,HelenaStrevens,MD,PhD4,MonaLandin-OlssonMDPhD1,3andMagnusHillmanPhD1*.
1.LundUniversity,DepartmentofClinicalSciences,DiabetesResearchLaboratory,Lund,Sweden
2.HelsingborgHospital,DepartmentofPediatrics,Helsingborg,Sweden
3.SkåneUniversityHospitalLund,DepartmentofEndocrinology,Lund,Sweden
4.SkåneUniversityHospitalLund,DepartmentofObstetrics,LundSweden.
*Correspondingauthor:MagnusHillman,B11,BMC,22184Lund,Sweden,[email protected],Phone:+46462220704.
Keywords:Gestationaldiabetesmellitus,Glutamicaciddecarboxylaseantibodies,Type1diabetes,IgGsubclasses.Shorttitle:GADAIgG4ingestationaldiabetesmellitus.Wordsinabstract:149Wordsinmanuscript:2618
Authorse-mailaddresses:[email protected];[email protected];[email protected];[email protected];[email protected]
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AbstractSomewomenwithgestationaldiabetes(GDM)presentwithautoantibodiesassociatedwithtype1diabetes.Theseareusuallydirectedagainstglutamicaciddecarboxylase(GADA)andsuggestedtopredictdevelopmentoftype1diabetes.TheprimaryaimofthisstudywastoinvestigateifGADAIgGsubclassesatonsetofGDMcouldassistinpredictingpostpartumdevelopment.Of1225womendiagnosedwithfirst-timeGDMonly51wereGADA-positive.TotalGADAwasdeterminedusingELISA.GADAsubclassesweredeterminedwithradioimmunoassay.Approximately25%ofGADA-positivewomendevelopedtype1diabetespostpartum.TitersoftotalGADAwerehigherinwomenthatdevelopedtype1diabetes(142.1vs74.2u/mL;p=0.04)andtheyalsohadlowertitersofGADAIgG4(index=0.01vs0.04;p=0.03).InconclusionwefoundthatthatwomenwithhightitersoftotalGADAbutlowtitersofGADAIgG4weremorepronetodeveloptype1diabetespostpartum.
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IntroductionGestationaldiabetesmellitus(GDM)isaheterogeneousdisorderaffectingapproximately2.5%ofpregnantwomeninsouthernSweden.Afewpercentofthesewomeninturnpresentwithantibodiestargetingisletspecificantigens,glutamicaciddecarboxylaseantibodies(GADA)inparticular[1-3].ThepresenceofGADAatonsetofGDM[4]oratdelivery[1]hasbeensuggestedtopredictfuturedevelopmentoftype1diabetes.
Type1diabetesisconsideredtobeacellularTh1/Th17mediateddisease.TheexactmechanismsaredebatedbutthetopicconcerningTcellsubsets,toleranceinterferenceandpost-translationalmodificationsofantigensiscomprehensivelyreviewedbyHaskinsandCook[5].Theadaptiveimmunesystemduringpregnancy,however,ispolarizedtowardsahumoralTh2response[6].InsettingdominatedbyTh1/Th17cells,thelocalcytokineprofilewillinduceanisotypeswitchtowardsthehumanIgG1subclass[7,8],whileTh2cellswillinduceanisotypeswitchtowardsthehumanIgG4subclass[9].Whetherthisactuallymaypreventtype1diabetesornotwillneedtobefurtherinvestigatedinfuturestudies,butpromisingresultshavebeenshowninNODmice[10,11].
Wehavepreviouslyshownthatpatientswithlatentautoimmunediabetesinadults(LADA)haveahigherfrequencyofGADAIgG4antibodiescomparedtopatientswithadultonsettype1diabetes[12].WehavesuggestedthattheTh1andTh2balancewithinpancreaticbetacellsmaybedifferentbetweenpatientswithtype1diabetesandLADAsincetheIgG4isotypeswitchismediatedmainlybyTh2cytokines.ThusitseemsreasonabletohypothesizethatalsoGADApositivewomenwithGDMcouldhavehighertitersofGADAIgG4antibodies.OnlyonestudyhaspreviouslyreportedGADAreactivity,includingGADAIgG1andIgG4,aswellasepitopebindingduringGDM[13].TheGADAIgG4positivitywasreportedtobe44%,whileGADAIgG1positivitywasashighas76%inthe34womenwithGDM.TheauthorscouldnotfindadifferenceincirculatingGADAIgGsubclassesbetweenpatientswithtype1diabetesandGDM,althoughareducedandmorerestrictedantibodyresponsewassuggestedduetolesserepitopespreadinginpatientswithGDM.
TheassociationbetweentheHLA-regionatchromosome6andautoimmunediabeteshasbeenknownformorethanthreedecades[14]andwhilecertainvariantsoftheDQbetachainencodessusceptibilitymotifs(DQB1*0201andDQB1*0302andalsoknownasDQ2andDQ8respectively),othersareprotective(DQB1*0602)[15].BothDQB1*0201andDQB1*0302arereportedinonestudytobeslightlymorecommoninScandinavianwomenwithGDMcomparedtoinpregnantcontrols[16].ThepresenceofGADAwasespeciallyassociatedwithDQB1riskgenotypesinthisstudy.AnotherstudysuggestedtheprotectivegenotypetobelessfrequentinGDMpatientsbutwithoutbeingabletoconfirmanincreasedfrequencyofhighriskgenotypes[17].AthirdstudyinvestigatedHLAriskallelesinautoantibodypositivewomenwithGDMandconcludedthattherewasanassociationwithautoantibodiesandthedevelopmentoftype1diabetespostpartum[18].
OuraimwiththisstudywastoinvestigateifGADAIgG1andIgG4subclassdistributioninwomenwithGDMisassociatedwiththeendogenousinsulinsecretionanddevelopmentoftype1diabetespostpartum.WewerealsointerestedinwhetherDQB1risktypes(DQ2andDQ8)couldbeassociatedwiththeGADAsubclassdistributionorcouldpredictthedevelopmentofmanifesttype1diabetespostpartuminourmaterial.
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Materialandmethods
ParticipantsWomendiagnosedwithnew-onsetGDMatSkåneUniversityHospitalinLund,Sweden,1996-2013(n=1225)wereaskedtoparticipateinthesestudies.Allwomenconsentedandwerescreenedforautoantibodies.CriteriaforGDMdiagnosiswerebloodglucosevaluesof≥10mmol/Laftera2hour75goralglucosetolerancetest(OGTT).PatientswerefolloweduppostpartumtomakesurethatthepathologicalbloodglucosevaluewasnormalizedoraclassificationofmanifestdiabeteswasusedinsteadofGDM.AllGDMwomenareofferedfollow-upat3-monthto1-yearintervalspostpartum.
TheStudywasconductedinaccordancewiththeHelsinkideclarationandapprovedbytheRegionalEthicalReviewBoardatLundUniversity(LU526/00;849/2005;244/2007,2009/307and2014/78).
Bloodtests
BloodwasdrawninserumandEDTAplasmavialsandsenttothelaboratorybyhospitalpost.Plasmaandserumwasseparatedbycentrifugationat2000xgandstoredat-70°Cuntiluse.
DetectionofGADApositivity
GADAwasidentifiedusingcommerciallyavailableenzymelinkedimmunosorbentassay’s(ELISA)fromRSRLtdwithareportedspecificityandsensitivityof100%and90%respectively[19].AbsorbancewasmeasuredinaFLOUstarOptima(BMGLabtech).Thecut-offlevelforpositivitywassetat10u/ml.Atotalof53womenwerepositiveforGADA(4.3%)andwereincludedinthestudy.SerumorEDTAplasmawasavailablefrom51oftheGADApositivepatients
GADAIgG1andIgG4subclassesAliquidphasebindingradioimmunoprecipitationassay(LPBA)wasusedtodetectGADAIgG1andIgG4subclassesinGADApositivewomenasdescribedindetailelsewhere[20].Briefly,serumorplasmawasincubatedinduplicateswithrecombinant35S(Perkin-Elmer)-labeledGAD65,followedbyincubationwithanti-humanIgG1antibodies(15µg/ml,BDPharmingen)oranti-humanIgG4antibodies(25µg/ml,BDPharmingen)overnightat4°C,formingimmunecomplexesbetweenGADAandGAD65,aswellasbetweenhumanIgG-subclassesandmouseanti-humansubclassantibodies.Theimmunecomplexeswereprecipitatedontostreptavidinsepharose(40%dilution,GEhealthcare)in96-wellfilterplates(Millipore,France)pre-coatedwith1%BSAtoavoidunspecificbindingandpunchedoutintovialswith4mlscintillationfluid(UltimaGold,Perkin-Elmer).Countsperminute(cpm)weremeasuredinabeta-counter(PackardTri-carb2100TR)for2minutespersample.Quadraplicatesofin-housestandardswereusedandindexeswerecalculatedtoestimateIgG-subclasstiters.Anindexof0.04wasusedascut-offlevelforGADAIgG1positivityand0.03forGADAIgG4positivity[20].
C-peptideC-peptidelevelsweremeasuredwithcommerciallyavailableready-to-useELISAkits(Mercodia)accordingtomanufacturers’instructions.Internalcontrolswerealsopurchasedfromthesamemanufacturer.Thereporteddetectionlimitwas15pmol/landabsorbancewasmeasuredat450nminaFLOUstarOptima(BMGLabtech).
HLA-genotypingDNAwasextractedfromleukocyteswithasalt-outprecipitationmethod[21].Concentrationandpuritywasdeterminedbymeasuringtheratioofabsorbanceat260/280nm.HLA-DQβ1riskalleles(DQ2/DQ8)weredeterminedusingacommerciallyavailableMutaGEL®HLA-DQ2+8kit
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(immundiagnostikAG)withspecificprimersforHLADQ2(allelecombinationDQα1*05/DQβ1*0201)andHLA-DQ8(allelecombinationDQα1*03/DQβ1*0302).Allgenotypingwasperformedaccordingtothemanufacturers’instructions.
StatisticalanalysesDistributionofdatawasestimatedusingD’Agostino-Pearsontest[22].Resultsarereportedasmean±standarddeviationfordatathatfollowedanormaldistributionandmedianfollowedbyinterquartilerange(IQR)inbracketswhennormaldistributionwasrejected.The2-tailedMann-WhitneyU-testwasusedtoanalyzedifferencesinIgG-subclasstitersbetweenthetwogroupsandtheWilcoxonsignedranktesttocompareIgG-subclasstitersbetweenthefirstandsecondpregnancywithGDM.TheSpearmanrankcorrelationwasusedtodetectassociationsbetweenantibodiesand/orC-peptidelevels.Fischer’sexacttestwasusedtotestfordifferencesinfrequencyofGADAIgG-subclasspositivityinthegroups.P<0.05wasconsideredtobestatisticallysignificant.ThestatisticalsoftwareMedCalcforWindows®v.14.12.0wasusedtoanalyzethedata.
ResultsClinicalandlaboratorydataarepresentedintable1.Atotalof53ofthe1225GDMwomenwerepositiveforGADA(4.3%)andserumorEDTAplasmawasavailablefrom51oftheGADApositiveGDMwomenforIgGsubclassanalyses.ThetwowomenwithoutavailableserumorEDTAplasmasampleswereexcludedfromthestudy.Neitheroneofthesetwowomenwerereportedtohavedevelopedmanifestdiabetesduringfollow-up.AllGADApositiveGDMwomenparticipatedinfollow-upatregular3-monthto1-yearintervals.Twelveofthe51GDMwomenwithGADApositivityincludedinthestudydevelopedtype1diabeteswithin0-5yearsafteronsetofGDM(1.4±1.1years).SomeoftheGADApositivewomen(n=15)returned,duringtheirsecondpregnancywithGDM,1-9yearsafterpostpartum(4.5±2.8years).TwoofthemalsohadathirdpregnancywithGDMwithintwoyearsafterthesecond.
TotalGADAtitersweresignificantlyhigherinpatientsthatdevelopedtype1diabetes;142.1(61.6-821.5)u/ml,comparedtoinpatientsthatdidnot;74.2(30.1-227.8)u/ml(p=0.04).
TheGADAIgG4titers,tothecontrary,weresignificantlylowerinwomenthatdevelopedtype1diabetes(p=0.03)andthefrequencyofGADAIgG4positivitywassignificantlydecreasedinthisgroup(Fig1,p=0.04).NosignificantdifferencesoftheGADAIgG1levelswerefoundbetweenthegroups.
AllpatientspositiveforGADAIgG4werealsopositiveforGADAIgG1.However,positivityforGADAIgG1wastwiceasfrequentasGADAIgG4inourmaterialwithfrequenciesof82%(42/51)and41%(21/51)respectively.TotalGADAcorrelatedsignificantlywithGADAIgG1(rs=0.65;p<0.0001)butnotwithGADAIgG4(rs=0.11;p=0.47).
C-peptidelevelswerenotsignificantlydifferentbetweenwomenwhodevelopedtype1diabetes;1.09(0.42-1.40)nmol/Landwomenwhodidnot;1.35(0.64-1.90)nmol/L.However,apositivecorrelationwasfoundbetweenC-peptidelevelsandGADAIgG4(Fig2;rs=0.32;p=0.04).TherewasnosuchcorrelationbetweenC-peptideandGADAIgG1(rs=-0.03;p=0.86).
WomenwithGDMduringtheirsecondpregnancy(n=15),andinafewcasesalsotheirthirdpregnancy(n=2),retainedtheirGADAlevelsaswellastheirIgGsubclassprofile.Onlyoneofthewomen,withverylowlevelsoftotalGADA,IgG1andIgG4atclinicalonset,inherfirstpregnancy,turnedouttobeGADAnegativeduringhersecondpregnancywithGDM.
Inourmaterial15womenwerepositivefortheinvestigatedHLAriskallelesDQ2(n=7),DQ8(n=6)orboth(n=2).NostatisticallysignificantincreaseoftheanalyzedHLAriskalleleswasfoundinwomen
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whodevelopedtype1diabetes.However,GADAIgG4positivewomenhadasignificantlylowerfrequencyofDQ2or8(2/21;11%,bothbeingDQ8)comparedtoGADAIgG4negativepatients(13/30;43%;p=0.012).Also,womenwiththeDQ2genotype(n=9)hadhigherlevelsofGADA;240.6(132.1-816.7)u/mLcomparedtoinwomenwithoutDQ2(n=42);74.3(48.7-140.5)u/mL,althoughthiswasnotstatisticallysignificant(p=0.07).
DiscussionOurmainfindingwasthatnotonlyGADApositivityinitself,butalsoGADAsubclassdistributioncouldinfluencetheriskofdevelopingtype1diabetespostpartum.GADApositivewomenwithGDMandhighertitersofGADAIgG4werelesspronetodeveloptype1diabetes,regardlessofGADAtiters.WealsofoundapositivecorrelationbetweenGADAIgG4andendogenousinsulinsecretion,asreflectedbytheC-peptidelevels,whichseemedtobehigherinIgG4positivewomen.
OnestrengthofthisstudyisthatitwasconductedinaregionofSweden,whereascreeningprogramforGDMincludesallpregnantwomen.AnOGTTisofferedtoallpregnantwomenat28weeksofgestationandalsoasearlyasthe12thweekofgestationinthecaseofknownriskfactors.TheseincludeapreviousGDM,1stdegreerelativewithdiabetesorBMI>30.
VirtuallyallwomenintheregionaccepttheofferofOGTT.ThereforepracticallyallpregnancieswithGDMaredetected.AlimitationofthestudyistherelativelysmallnumberofparticipantsduetothelowamountofGADApositivewomenwithGDM.TheassociationbetweenC-peptideandGADAIgG4
antibodiesmustbeinterpretedwithcautionduetothesmallnumberofpatients.
InourmaterialGADAIgG1positivitywastwiceasfrequentasGADAIgG4positivitywithfrequenciesof82%and41%respectively.ThesefiguresarewellinaccordancewithanotherGDMstudyreportingfrequenciesof76%and44%respectively[13].However,theauthorsalsoreportedpresenceofGADAIgG4inpatientswithtype1diabetes,whichwehavenotyetourselvesobservedintwopreviousGADAstudiesinadultpatients[12,23].Thisdiscrepancycouldbeduetodifferencesinmethodologyanddeterminationofthecut-offlevelsforpositivityineachIgG-subclass.
TherewasnodifferenceinGADAIgG1levelsbetweenpatientsthatdevelopedtype1diabetespostpartumandthosewhodidnot.ThissuggeststhatthecytotoxicTcellresponseinTh1/Th17cellsaresimilarinbothgroupsofwomen,butmightbebetterregulatedwithTh2cellspresentinthepatientswithoutdevelopmentoftype1diabetes.Mostofthewomenwhodevelopedtype1diabetespresentedwithinthefirsttwoyearspostpartum.OneGADAIgG4positivewomanwithGDMdidactuallydeveloptype1diabetes(Fig2).Although,notuntilfiveyearspostpartum,whichsuggeststhatthepresenceofTh2cellcytokines-asreflectedbytheIgG4subclass-inpancreaticlymphoidtissuesmightslowdownthebetacelldestruction.
Autoantibodypositivity,especiallyGADApositivity,hasbeenimpliedasariskfactorforthedevelopmentoftype1diabetespostpartuminseveralstudies[1-4]GADAtitersinwomenwithGDMhavenotyetassembledsuchinterest.However,inLADApatientsGADAtitershavebeenreportedtobeofprognosticvalueforbetacellfunction[24-26],whichmakesitreasonabletoassumethatthiswouldbethecasealsoinGADApositivewomenwithGDM.InmostcasestherewasagoodcorrelationbetweentotalGADAandGADAIgG1levels,suggestingthatIgG1isthemostabundantheavychaininthecirculationinGADApositivewomen.InsomewomenwithhightotalGADAbothGADAIgG1andIgG4werelowerthanexpected.ThiscouldbeduetohigherGADAIgMlevelsinthesesubjectsorthattheepitopesrecognizedbytheassaysaredifferent.
WhencomparingtitersoftotalGADAbetweenwomenwithandwithoutDQ2orDQ8nodifferenceswerefound.However,wedidfindatrendtowardshigherlevelsofGADAinwomenwiththeDQ2
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allelealone,whichalsoisinaccordancewiththeNIRADstudygroup’sreportonLADApatients.DQ2wasreportedtobemoreprevalentinthehigh-titerGADAgroupwhileDQ8wasequallydistributedbetweenhigh-andlow-titerGADApatientswithLADA[25].InourmaterialonlyonewomanwithDQ2andonlytwowomenwithDQ8werepositiveforGADAIgG4.Similarly,only11%(2/21)ofGADAIgG4positivewomenhadriskallelescomparedto43%(13/30)oftheGADAIgG4negativewomen.
PreviousstudiesonHLAriskallelesinGDMhavereportedthepresenceofGADAtobeassociatedwithDQriskgenotypesinScandinavianwomen[16].AfurtherstudyfoundnostatisticallysignificantassociationbetweenDQrisktypesandGDMbutinsteadthattheprotectivealleleDQB1*0602(DQ6)waslessfrequentinSwedishwomenwithGDM[17].Anexplanationtothesedifferentfindingscouldbethevaryinginclusioncriteriainthestudies.InourstudyonlyGADApositiveGDMwomenwereincluded,whichwouldexplainwhyDQrisktypesweremorecommoninthematerial.
GADApositivewomenwithmorethanoneGDMpregnancyretainedtheirGADApositiveandIgGsubclassprofileduringtheirsecondandthirdpregnancy,suggestingaremainingriskfordevelopmentoftype1diabetespostpartum,oftenwithintwoyearsafterclinicalonsetoftheirfirstGDM.
FuturestudiesshouldbeconductedinlargerpopulationsofGADApositivewomenwithGDMtofurtherinvestigatetheassociationbetweenC-peptideandIgGsubclasses.Moreinvestigationisrequiredbeforethisinformationmaybeusedforprognosticevaluationofresidualbeta-cellfunction.
InconclusionwefoundthatthatwomenwithGDMthathadhightitersofGADAweremorepronetodeveloptype1diabetespostpartum.However,presenceofGADAIgG4waspositivelycorrelatedwithC-peptidelevelsinourstudy,associatedwithalowerfrequencyofDQβ1riskallelesandreducedriskoftype1diabetes.
AcknowledgementsFundingfromTheSwedishDiabetesFoundationforDrMagnusHillmanandfromSkåneUniversityHospitalFundingandDonationsforDrMonaLandin-Olssonwereusedtosupportthestudy.MrsBirgitteEkholmisthankedforexcellenttechnicalassistanceintheresearchlaboratory.
ConflictofinterestTheauthorsdeclarenoconflictofinterest.
AuthorcontributionsJD:Laboratoryanalyses,compilationofdata,statisticalanalysesandwritingpartsofthemanuscript.CN:Reviewofmedicalrecordsforfollowupofmanifestdiabetesmellituspostpartum,ethicalapplication,criticallyreviewofthemanuscript.HS:Criticalreviewofthemanuscript,proofreading,languageandcollectionofbloodsamples.MLO:Partsofthestudydesign,ethicalapplicationandcollectionofbloodsamples.MH:Studydesign,ethicalapplication,methoddevelopment,compilationandinterpretationofdata,statisticalanalysesandwritingpartsofthemanuscript.
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Table1.Serumorplasmawasavailablefor51GADApositivewomenatclinicalonsetofGDM.Twelvewomendevelopedtype1diabeteswithin5yearspostpartum.Ageisreportedasmean±standarddeviation.AntibodytitersandC-peptidelevelsarereportedasmedianfollowedbyinterquartilerange.PresenceofHLA-DQ2(DQB1*0201)and/orHLA-DQ8(DQB1*0302)isgivenintotal.
Nopostpartumtype1diabetes
(n=38)
Type1diabetespostpartum
(n=13)P-value
Age(years) 31.8±5.3 31.4±5.0 0.80
TotalGADA(u/ml) 74.2(30.1-227.8) 142.1(61.6-821.5) 0.04
GADAIgG1(index) 0.16(0.04-0.41) 0.41(0.12-0.54) 0.19
GADAIgG4(index) 0.04(0.02-0.06) 0.01(0.00-0.03) 0.03
C-peptide(nmol/L) 1.35(0.64-1.90) 1.09(0.42-1.40) 0.23
HLADQ2/8(yes/no) 10/28(26.3%) 5/8(38.5%) 0.48
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Figure1.GADAIgG4titersinwomenthatdevelopedtype1diabetes(n=13)comparedtoinwomenwithoutmanifesttype1diabetes(n=38;p=0.03).
Figure2.AsignificantcorrelationwasfoundbetweenendogenousinsulinsecretionandGADAIgG4subclassofantibodies(rs=0.32,p=0.04)whichsuggestsaprotectiveroleofGADAIgG4.
