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Thyroiddysfunctioninchildrenwithautismspectrumdisorderisassociatedwithfolatereceptoralpha...

ArticleinJournalofNeuroendocrinology·February2017

DOI:10.1111/jne.12461

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Received:14November2015  |  Revised:7February2017  |  Accepted:9February2017DOI: 10.1111/jne.12461

O R I G I N A L A R T I C L E

Thyroid dysfunction in children with autism spectrum disorder is associated with folate receptor α autoimmune disorder

R. E. Frye1  | R. Wynne1 | S. Rose1 | J. Slattery1 | L. Delhey1 | M. Tippett1 |  S. G. Kahler1 | S. C. Bennuri1 | S. Melnyk1 | J. M. Sequeira2 | E. V. Quadros2

1DepartmentofPediatrics,ArkansasChildren’sResearchInstitute,UniversityofArkansasforMedicalSciences,LittleRock,AR,USA2DepartmentofMedicine,StateUniversityofNewYork–DownstateMedicalCenter,Brooklyn,NY,USA

CorrespondenceRichardE.Frye,ArkansasChildren’sResearchInstitute,LittleRock,AR,USA.Email:refrye@uams.edu

Funding informationArkansasBiosciencesInstitute,AutismResearchInstitute;JaneBotsfordJohnsonFoundationandAutismSpeaks,Grant/AwardNumber:#8202;UniversityofMarylandBrainandTissueBank

Folatereceptorα(FRα)autoantibodies(FRAAs)areprevalentinautismspectrumdis-order(ASD).FRAAsdisruptfolatetransportacrosstheblood-brainbarrierbybindingtotheFRα.ThyroiddysfunctionisfrequentlyfoundinchildrenwithASD.Wemeas-uredblockingandbindingFRAAsandthyroid-stimulatinghormone(TSH),freethyrox-ine (T4) (FT4), total triiodothyronine (T3) (TT3), reverse T3 (rT3), thyroid-releasinghormone(TRH)andothermetabolitesin87childrenwithASD,84ofwhomalsoun-derwentbehaviourandcognition testingand in42ofwhomFRAAs,TSHandFT4weremeasuredattwotimepoints.TobetterunderstandthesignificanceoftheFRα in relationtothyroiddevelopment,weexaminedFRαexpressiononprenatalandpost-natalthyroid.TSH,TT3andrT3wereabovethenormalrangein7%,33%and51%oftheparticipantsandTRHwasbelowthenormalrangein13%oftheparticipants.FT4was rarely outside the normal range. TSH concentration was positively and theFT4/TSH,TT3/TSHandrT3/TSHratioswereinverselyrelatedtoblockingFRAAti-tres.Onrepeatedmeasurements,changesinTSHandFT4/TSHratiowerefoundtocorrespondtochangesinblockingFRAAtitres.TSHandtheFT4/TSH,TT3/TSHandrT3/TSHratioswererelatedtoirritabilityontheAberrantBehaviorChecklistandsev-eralscalesoftheSocialResponsivenessScale(SRS),whereasTT3wasassociatedwithSRSsubscalesandTRHwasrelatedtoVinelandAdaptiveBehaviorScalesubscales.The thyroid showed significant FRα expression during the early prenatal period,althoughexpressiondecreased significantly in latergestationandpostnatal thyroidtissue.TheresultsofthepresentstudysuggestthatthyroiddysfunctioninASDmayberelatedtoblockingFRAA.ThehighexpressionofFRα intheearlyfoetalthyroidsuggeststhatfoetalandneonatalexposuretomaternalFRAAscouldaffectthedevel-opmentofthethyroidandmaycontributetothepathologyinASD.

K E Y W O R D S

autismspectrumdisorders,folatereceptorautoantibody,freeT4,thyroidfunction,thyroid-stimulatinghormone

1  | INTRODUCTION

Thereisincreasingrecognitionthatthyroiddysfunctionisassociatedwith neurological and psychiatric disease, including neurodevelop-mentaldisorderssuchasintellectualdisability1andautismspectrum

disorder (ASD),2-7 focal and generalised neurological abnormalities,8 aswellaspsychiatricmanifestationssuchaspsychosis,9,10panicat-tacks,11 anxiety disorders,11,12 bipolar disorder,13-15 depression12,14 andschizophrenia.16,17Inaddition,normalthyroidfunctionisessentialfornormalcognition.18However,theexactnatureoftherelationship

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betweenthyroiddysfunctionandneurologicalandpsychiatricdiseaseisunclear.

EarlyuncontrolledtreatmentstudiessuggestedthatchildrenwithASDshowedapositiveclinicalresponsetotriiodothyronine(T3).2,19 Asmallcontrolledcross-overstudyfoundthatT3resultedinlimitedsymptomatic improvement in childrenwithASD, especially in thosewithalowerintellectualquotient.20Otherstudiesexaminingthyroidhormones have been inconsistent. A letter to editor in 1970 indi-cated a high rateof hypothyroidism (73%) in 62 childrenwithASDusingT3uptaketesting,21althoughtwoothersmallstudiescouldnotconfirmthyroiddysfunctioninASD,findingnodifferenceinthyroid-stimulating hormone (TSH), thyroxin (T4) or T3 between ASD andcontrolchildren.22,23OtherstudiessuggestthatASDhasbeenassoci-atedwithafamilyhistoryofautoimmunethyroiddisorders24andthatdeficiencies inthyroidfunctionmaybepresentbeforeoratbirth inchildrenwithASD.5,6Morerecentstudieshaveexaminedthyroidreg-ulationinthecontextofthehypothalamic-pituitary-axis(HPA)inASD.TSHatbaselineandfollowingthyrotrophin-releasinghormone(TRH)stimulationwaslowerin41Japaneseautisticboyscomparedtocon-trolswithmental retardation,minimal brain dysfunction and typicaldevelopment,suggestingabluntedHPAresponse.3DiurnalvariationinTSHlevelswasfoundtobelargerinyoungadultswithASDcom-paredtocontrols,againindicatingdifferencesinHPAfunction.4

OneofthereasonsforinconsistentfindingscouldbeexplainedifonlysubgroupsofchildrenwithASDhadabnormalthyroidfunction.A recent study reported that folate receptorα (FRα) autoantibodies(FRAA),whicharepresentinupto75%ofchildrenwithASD,25 may contribute to thyroiddysfunction inASD.7 In32childrenwithASD,thosepositivefortheblockingFRAAshadahigherTSHcomparedtothosenegative for theblockingFRAAs,and theblockingFRAAwasfoundtocorrelatepositivelywithTSH.7

TherelationshipbetweenthyroidfunctionandFRAAtitreswasin-vestigatedbecauseofthelikelihoodthatautoantibodiescouldbindtotheFRαonthethyroidandaffectitsfunctionasaresultofmonoclonalanti-bodiestothetwodifferentepitopesoftheglycosyl-phosphatidylinositollinked folate-bindingmembranegp38proteinbeing foundonnormalthyroidtissue26withlimitedfocalreactivityinsomestudies.27,28BindingofFRAAstothyroidcellscouldaffectthyroidfunctioninseveralways.WhenFRAAsbindtoepithelialcellsofthechoroidplexus29 theycandisruptFRαfunction,resultinginreducedtransportoffolateacrosstheblood-brainbarrierandcausingadisorderknownascerebralfolatedefi-ciencysyndrome.30Thus,itispossiblethatFRAAbindingonthethyroidcoulddecreasefolateentryintothyroidcells.Folateisnotaprominentcofactorinthesynthesisofthyroidhormones.However,tyrosine,whichisderivedfromphenylalanineusingphenylalaninehydroxylase,isessen-tial for theproductionof thyroidhormones. Importantly, anessentialcofactorforphenylalaninehydroxylaseistetrahydrobiopterin,whichisultimatelyderivedfromthefolatecycle.31-33

Alternatively, FRAAs binding to thyroid cells could activate theimmune system, resulting in inflammation. Additionally, the folatepathwayisintimatelyinterconnectedwithmethylationandredoxreg-ulationpathways,bothofwhichareabnormalinchildrenwithASD.34 Methylationregulatesgeneexpressionandenzymefunction,andso

alterations in methylation can affect cellular function. The reducedformofglutathione(GSH) isthemajor intracellularanti-oxidantthatnot only protects the cell from damage, but also regulates enzymefunction;thus,decreasedGSHcanresultinbothcellulardamageanddeficitsinenzymeactivity.

Althoughthepreviousstudy7onlyexaminedtherelationshipbe-tweenFRAAsandTSH, inthepresentstudy,freeT4(FT4), totalT3(TT3),reverseT3(rT3)andTRHwerealsoexamined.WehypothesisethatFRAAsmaybedisruptingtheHPAbybluntingthesensitivityofthethyroidtoTSHor,inotherwords,makingthethyroidresistanttotheeffectsofTSH.ThiswouldresultinahigherTSHconcentrationtoproduce aparticular thyroidhormone concentration.Quantitatively,suchaneffectshouldbedetectablebymeasuringtheratioofathy-roidhormonetoTSHanddeterminingwhetherthisratioisalteredbyFRAAs.WeinvestigatedthisbyexaminingtherelationshipsbetweenFRAAsandTSH,FT4,TT3,rT3,TRHandtheratioofthyroidhormones(FT4/TT3/rT3) toTSH. Inaddition,wedeterminedwhethermarkersofglutathioneandmethylationandbloodconcentrationsoffolatearerelatedtothyroidfunctionaimingtoensurethatthesephysiologicalprocessesarenotconfoundersinthethyroidfunction-FRAAsrelation-ship.Wealsoexaminedtherelationshipbetweenthyroidfunctionandbehaviourandcognition.

Lastly,wealso investigatedthedevelopmental timingofthepo-tential relationship between FRAAs and thyroid function. Becausethyroid metabolism is critical prenatally35,36 and some mothers ofchildrenwithASDarepositiveforFRAAs,25,37weexaminedthede-velopmentalaspectoftheFRαonbothfoetalandpost-natalthyroidtissuetodeterminewhethertheFRαismoresignificantlyrepresentedduringa specificdevelopmental timeperiod.Wealsoexamined thechangeinFRAAs,TSHandFT4overa12-weekperiodduringaclinicaltrial.38Theaimwas todeterminewhetherFRAAsandthyroid func-tionchangetogether,insupportofthenotionthatFRAAsandthyroidfunctionaredynamicallylinked.

2  | MATERIALS AND METHODS

Eighty-seven children with ASD (mean±SD age 6years10months±3years1month)andtwelvetypicallydevelopingcontrols(mean±SD age 8years 2months±5years 2months) participated inthepresentstudy.ControlswereusedtoobtainmeasuresofnormalFT4andTSHinthesamepopulation,independentofestablishedcon-trolranges.ThesamplesusedinthepresentstudywereobtainedfromtworesearchprotocolsapprovedbytheInstitutionalReviewBoardattheUniversityofArkansasforMedicalScience(LittleRock,AR,USA).Forbothstudies,written informedconsentwasobtained frompar-entsofparticipants;participantassentwaswaived.Forbothstudies,fastingbloodsamplesbeforebreakfastwererequiredasoneofthefirstproceduresafterconsent.Theinclusionandexclusioncriteriare-mainedbroad,aimingtoincludearepresentativesampleofchildrenwithidiopathicASD.

AdiagnosisofASDwasrequiredforentryintothestudy.Adiagno-siswasdefinedbyoneofthefollowing:(i)agold-standarddiagnostic

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instrument such as the Autism Diagnostic Observation Scheduleand/orAutismDiagnosticInterview-Revised(ADI-R); (ii)theStateofArkansas diagnostic standard, defined as agreement of a physician,psychologist and speech therapist; and/or (iii) Diagnostic StatisticalManual diagnosis by a physician along with standardised validatedquestionnairesanddiagnosisconfirmationbytheprincipalinvestiga-tor(R.E.Frye).ReconfirmationofthediagnosisusingtheADI-Rbyanindependentresearchreliableraterwasrequestedforaportionofpar-ticipantstoconfirmthatthecriteriausedforincludingtheparticipantswasequivalenttothisgold-standardinstrument.39

Excludedfromthestudywerethosechildrenonmedicationinter-feringwiththyroidfunction,includingthyroidsupplements,steroids,β-blockers, antipsychoticsand lithium,aswell aschildrenwithwell-definedgeneticsyndromes.

2.1 | Folate autoantibody assay

Approximately 1mL of serumwas collected and sent to the labo-ratoryofDrEdwardQuadrosat theStateUniversityofNewYork,Downstate(Brooklyn,NY,USA).Theassayforboththeblockingandbinding FRAAs has been described previously.30,40 Blocking FRAAswereexpressedaspmoloffolicacidblockedfrombindingtoFRαpermLofserum,andbindingFRAAswereexpressedaspmolofimmuno-globulin(Ig)GantibodypermLofserum.

2.2 | Thyroid function assays

PlasmaTSH,FT4,TT3,TRH, rT3andcortisolweremeasuredusingenzyme-linkedimmunosorbentassay(ELISA)kitsinaccordancewiththe manufacturer’s instructions (ab1000660, ab108686, ab108685from Abcam, Cambridge, MA, USA; CEA839Hu from Cloud-CloneCorp, Houston, TX, USA; MBS2510365 from MyBioSource, SanDiego, CA, USA; 11-CORHU-E01 from ALPCO, Salem, NH, USA).TSH,TT3andFT4were interpretedusingtheNationalAcademyofClinical Biochemistry standard for children.41 TSH reference rangewas0.4-5.0mIU/L, FT4 reference rangewas9-20pmol/L andTT3referencerangewas80-180ng/dL.BecausetheFT4toTSHratioisknowntobelogdistributed,thethyroidhormone(FT4,TT3,rT3)toTSHratioswerelog-transformedbeforeanalysis.41NormativevaluesforTRH,rT3andcortisolwereobtainedfromthemanufacturersasbeingintherange35.7-167.8pg/mL,2.4-45.8ng/dLand7-25μg/dL,respectively.Cortisolwasmeasuredbecausethyroidhormonescanbemodulatedbycortisol.

2.2.1 | Immunohistochemical analysis of FRα expression in the thyroid

Foetalthyroidtissuefrom15,17,18,19and20weeksoldfoetusespreservedinformalinwereobtainedfromtheUniversityofMarylandBrainandTissueBank,whichisaBrainandTissueRepositoryoftheNIHNeuroBioBank.Thesewereembedded inparaffinandsections(6 μm thick)were cut. Thyroid sections from a 5-month-old infant,3-year-old child and a 27-year-old adult were purchased from US

Biomax Inc. (Rockville, MD, USA). All sections were deparaffinisedandincubatedwith100μLofgoatserumfor1hourfollowedbyin-cubationwith a 1:500 dilution of rabbit antihumanFRα in 100 μLof goat serumovernight. The sectionswerewashed and incubatedwith a1:600dilutionof goat anti rabbit IgG-peroxidase conjugate(VectorLaboratories, Inc.,Burlingame,CA,USA) for1hour,washedandincubatedwithdiaminobenzidineasthechromagen.Thesectionswerestainedwithhaematoxalin(VectorLaboratories,Inc.)tovisualisethenuclei.

Thepolyclonalantiserumusedwaspreparedby immunisingrab-bits with affinity purified FRα from human epidermoid carcinomaKBcellsconditionedtogrowinlowfolatethatup-regulatesFRαex-pression.42Thepurityoftheantigenwascheckedbysodiumdodecylsulphate-polyacrylamidegel electrophoresis.The titreof theantise-rumwascheckedbydirect immunoprecipitationof 3HPGA-FRα and byELISAandthespecificityoftheantiserumwascheckedbyblockingthetitrewithexcesspurifiedFRα.ThespecificityofimmunostainingwasestablishedbyabsorbingouttheantibodyontoFRα-sepharosematrixandbyblockingofimmunostainingwhentheantiserumispre-incubatedwithaten-foldmolarexcessofpurifiedFRα.

2.3 | Redox, methylation, immune and vitamin biomarkers

Redoxandmethylationpotentialwasmeasuredbythefreereduced-to-oxidised glutathione redox ratio (GSH/GSSG) and SAM to SAHratio(SAM/SAH),respectively.Fastingblood(4mL)wascollectedintoanethylenediaminetetraceticacid-Vacutainertube,chilledoniceandcentrifuged at 1500g for 15minutes at 4°C. Plasmawas stored at−80°Candanalysedbyhigh-performanceliquidchromatographywithelectrochemicaldetectionwithin2weeksofcollectionasdescribedpreviously.43PlasmatotalfolateandvitaminB12wasmeasuredusingaSimulTRAC-SNBRadioassayKit(cataloguenumber06B264806;MPDiagnostics,SantaAna,CA,USA).

2.4 | Cognitive and behavioural assessments

The Preschool Language Scale-4 and two versions of the ClinicalEvaluationsofLanguageFundamentalswereusedtoassesslanguageability.38 Both instruments provide a standardised core languagescore,thusprovidinganindexoflanguageabilityindependentoftheinstrument used. For each participant, themost ability appropriateinstrumentwasused toprevent floor and ceiling effects. Languagetestingwasavailableon84participants.

Adaptive behaviour was assessed using the Vineland AdaptiveBehaviorScales(2ndedition),InterviewEdition,SurveyForm(VABS),an instrument that has demonstrated good reliability andvalidity.44 Standardisedscoresforsummaryscalesexaminedwerecommunica-tion,dailylivingskills,socialskills,motorskillsandadaptivebehaviourcomposite.VABStestingwasavailableon84participants.

TheAberrantBehaviorChecklist(ABC)wasdesignedtomeasuredisruptivebehavioursinindividualswithdevelopmentaldisabilities.45 TheABChasbeenshowntohaveconvergentanddivergentvalidity

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inASD46 andhasbeenused inmultipleautismclinical trials.47ABCscoreswereavailableon82participants.

The Social Responsiveness Scale (SRS)measures the severity ofsocialskilldeficits.48Ithasbeenvalidatedandshowntobereliableandtohavegoodcorrespondencetothegold-standardADI-R,atthesametimeasbeingmoretimeefficientandcosteffective.49SRSscoreswereavailableon81participants.

2.5 | Folinic acid treatment

Forty-twoparticipantswerepartofadouble-blind,placebo-controlledtrialontheeffectoffolinicacidonchildrenwithASD.38Inthesepar-ticipants, FRAA titres,TSHandT4weremeasuredprior to startingtreatmentandattheendof12weeksoftreatmentwitheitherhigh-dosefolinicacid(2mg/kg/dayintwodivideddoses;maximum50mg/day)orplaceboinmostoftheparticipantsinthetrial.

2.6 | Statistical analysis

The ‘glm’and ‘glimmix’procedureofSAS,version9.1 (SASInstituteInc.,Cary,NC,USA)wereused for the cross-sectional analysis andrepeated-measuresanalysis,respectively,andatwo-tailedαof0.05wasused.WedichotomisedblockingandbindingFRAAstatussepa-rately,aswellas theoverallFRAAstatus (positivevsnegative).Wealsoexaminedtherelationshipbetweenthyroidhormonesandblock-ingandbindingFRAAtitresseparately.Amixed-modelwasusedforthe repeatedmeasures analysiswith theparticipant variable as therandom-effect.

3  | RESULTS

3.1 | Participants

Participantswererecruitedfromourresearchregistry(40%),autismclinic(23%),communityadvertisementandsocialmedia(10%),word-of-mouth (15%)andphysician referrals (13%).Thebasicparticipantcharacteristics did not differ across the FRAA status (Table1). Allparticipantsevaluatedbyan independentresearchreliableraterex-ceededthethresholdforautismdiagnosis.

3.2 | Overall thyroid function

FortheoverallASDpopulation,themean±SDTSHconcentrationwas2.76±1.82mIU/L(range0.63-11.55mIU/L)andwasbelowandabovethestandardreferencerangein0%and7%ofASDparticipants,re-spectively,andnodifferentfromcontrols(4.14±2.13mIU/L).

For the overall ASD population, FT4 concentration was14.38±2.27pmol/L (range 8.66-21.12pmol/L) and was below andabove thestandard reference range in1%and2%ofASDchildren,respectively, and slightly higher than controls (13.77±3.00pmol/L)(F1,94=5.19,P=.03).

FortheoverallASDpopulation,theFT4/TSHratiowas0.78±0.26andwasnotsignificantlydifferentfromcontrols(0.56±0.29).

TT3 concentration was 170.5±24.4ng/dL (range 117.4-224.6 ng/dL)andwasabnormallylowandhighin0%and33%oftheASDchildren, respectively,using thestandard reference range.TheTT3/TSHratiowas1.86±0.28(range1.12-2.50).

rT3 concentrationwas 49.8±13.5ng/dL (range 28.8-95.3ng/dL)andwasabnormallylowandhighin0%and51%oftheASDchildren,respectively,usingthemanufacturer’sreferencerange.TherT3/TSHratiowas1.32±0.28(range0.65-1.92).

Thyroid-releasing hormone concentrationwas 61.0±24.0pg/mL(range19.7-127.9pg/mL) andwasabnormally lowandhigh in13%and 0%of theASD children, respectively, using themanufacturer’sreferencerange

Thyroidhormoneswerenotsignificantlycorrelatedwithcortisollevels.

3.3 | Thyroid function and folate- related metabolism

FT4wasrelatedtotheSAM/SAHratio,anindexofmethylationab-normalities, such that a higher SAM/SAH ratio (bettermethylation)wasrelatedtoalowerFT4(F1,84=8.30,P=.005).TSHwasfoundtobepositivelyrelatedtotheglutathioneredoxratio,suchthathigher(bet-ter)glutathioneredoxratiowasrelatedtohigherTSHconcentrations(F1,84=4.48,P=.05).

Noneoftheotherthyroidfunctionmeasureswerelinearlyrelatedtoglutathione,SAM/SAH,folateorB12.

3.4 | Thyroid function and folate receptor α autoantibodies

Thyroid-stimulatinghormonewassignificantlyhigher inparticipantspositive(3.94±2.94mIU/L)fortheblockingFRAAcomparedtothosenegative (2.49±1.38mIU/L) for the blocking FRAA (F1,83=10.85,P=.001) and the TSH concentration was found to increase as theblockingFRAAtitresincreased(F1,84=7.85,P=.006)(Figure1A).

Participants who were positive for the blocking FRAA demon-strateda lowerFT4/TSHratio(0.66±0.30)comparedtoparticipantsnegativefortheblockingFRAA(0.82±0.25)(F1,83=6.12,P=.02). In ad-dition,theblockingFRAAtitredemonstratedaninverserelationshipwiththeT4/TSHratio(F1,84=5.27,P=.02)(Figure1B).

Participants who were positive for the blocking FRAA demon-strateda lowerTT3/TSHratio(1.71±0.32)comparedtoparticipantsnegativefortheblockingFRAA(1.90±0.27)(F1,83=6.09,P=.02). In ad-dition,theblockingFRAAtitredemonstratedaninverserelationshipwiththeTT3/TSHratio(F1,84=7.58,P<0.01(Figure1C).

Participants who were positive for the blocking FRAA demon-strateda lower rT3/TSH ratio (1.36±0.27) compared toparticipantsnegativefortheblockingFRAA(1.67±0.28)(F1,83=6.09,P=.02). In ad-dition,theblockingFRAAtitredemonstratedaninverserelationshipwiththerT3/TSHratio(F1,84=4.95,P=.03)(Figure1D).

Thyroid-stimulatinghormoneandtheratiosofthyroidhormones(FT4,TT3,rT3)toTSHwerenotsignificantlydifferentacrosspositivevsnegativebindingFRAAparticipants,norweretheysignificantlylin-earlyrelatedtobindingFRAAtitres.

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TABLE  1 Demographicandclinicalcharacteristicsbyfolatereceptorαautoantibodygroups

Variable FRAA negative (n=35) FRAA positive (n=52)

Age(years,months),mean±SD 6years10months±3years6months 6years11months±2years11months

Males,n(%) 28(80%) 42(81%)

Vinelandadaptivebehaviourcomposite,mean(SD) 64.2 (11.7) 64.8 (9.8)

TSH(mIU/L) 2.44(1.36) 3.00(2.06)

HighTSH(<0.4mIU/L) 2(5%) 5(9%)

LowTSH(>5.0mIU/L) 0(0%) 0(0%)

FreeT4(pmol/L) 14.47 (1.92) 14.33(2.51)

HighT4(<9pmol/L) 0(0%) 2(4%)

LowT4(>20pmol/L) 0(0%) 1(2%)

FreeT4toTSHratio(log-transformed) 0.84 (0.26) 0.75 (0.27)

TotalT3(ng/dL) 170.41 (25.1) 170.5 (24.2)

HighT3(>180ng/dL) 0(0%) 0(0%)

LowT3(<80ng/dL) 13(37%) 16(31%)

TotalT3toTSHratio(log-transformed) 1.91 (0.28) 1.73(0.23)

ReverseT3(ng/dL) 48.9(8.3) 57.9(23.4)

HighrT3(>45.77ng/dL) 0(0%) 0(0%)

LowrT3(<2.39ng/dL) 20(57%) 24(46%)

ReserveT4toTSHratio(logtransformed) 1.36(0.27) 1.18 (0.22)

Thyroid-releasinghormone(pg/mL) 65.6 (24.6) 50.4 (16.2)

HighTRH(>167.8pg/mL) 3(9%) 8(15%)

LowTRH(<35.7pg/mL) 0% 0%

Cortisol(μg/dL) 18.45 (8.9) 20.4(13.0)

Folate(ng/mL)(normal5-21) 16.9(3.9) 18.0 (4.5)

B12(pg/mL)(normal200-900) 786(395) 1313(1391)

Languagetesting,n(%)

PreschoolLanguageScales 13(37%) 16(30%)

Clinicalevaluationoflanguagefundamentals2 14(40%) 17(32%)

Clinicalevaluationoflanguagefundamentals4 7(20%) 20(38%)

Diagnosticdocumentation,n(%)

Autismdiagnosticobservationschedule 18(51%) 25(47%)

3Practitioneragreement 27(77%) 35(66%)

Singlepractitionerwithstandardisedquestionnaires 5(14%) 13(25%)

Autismdiagnosticinterview-revised

Participatedinconfirmationtesting,n(%) 28(80%) 41(77%)

Socialinteractionscore,mean±SD(range) 21.57±5.41(10-30) 22.68±4.85(11-30)

Communicationscore:verbal,mean±SD(range) 16.67±4.24(10-24) 19.38±3.67(7-25)

Communicationscore:nonverbal,mean±SD(range) 12.69±2.36(7-14) 13.07±1.53(9-14)

Restricted&repetitiveplayscore,mean±SD(range) 5.79±1.83(2-10) 5.95±2.47(2-12)

Summaryscore,mean±SD(range) 4.43±0.92(2-5) 4.39±0.89(2-5)

Medications(concurrenttreatments),n(%)

Melatonin 15(43%) 11(21%)

Allergy/asthmamedications 11(31%) 11(21%)

Gastrointestinalmedications 11(31%) 9(17%)

α-Adrenergicagonists 6(17%) 10(19%)

Stimulant 6(17%) 8(15%)

(Continues)

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Thyroid-releasinghormone,FT4,T3andrT3werenotsignificantlydifferentacrossFRAAgroups,norweretheysignificantlylinearlyre-latedtoblockingorblindingFRAAtitres.

3.5 | Relationship between thyroid function and behavior and cognition

HigherTSHwasrelatedtolower(better)ABCIrritability(F1,80=5.94,P=.02)(Figure2A)andSRSAwareness(F1,79=5.01,P=.03)(Figure2B),Cognition (F1,79=5.97, P=.03) (Figure2C), Motivation (F1,79=4.46,P=.04) (Figure2D), Mannerisms (F1,79=6.87, P=.01) (Figure2E) andTotalscore(F1,79=5.72,P=.02)(Figure2F).

A higher FT4/TSH ratio was related to higher (worse) ABCIrritability(F1,80=7.56,P=.01)(Figure2G)andHyperactivity(F1,80=5.29,P=.02) (Figure2H)andSRSAwareness(F1,79=4.70,P=.03) (Figure2I),Cognition (F1,79=5.82,P=.02) (Figure2J),Communication (F1,79=4.00,P=.05) (Figure2K), Motivation (F1,79=4.89, P=.03) (Figure2l),Mannerisms(F1,79=5.35,P=.02)(Figure2M)andTotalscore(F1,79=5.61,P=.02)(Figure2N).

HigherT3wasrelatedtohigher(worse)SRSCognition(F1,79=4.83,P=.03) (Figure2O), Motivation (F1,79=6.98, P=.01) (Figure2P),

Mannerisms(F1,79=4.55,P=.04)(Figure2q)andTotalscore(F1,79=5.14,P=.03)(Figure2R).

AhigherTT3/TSHratiowasrelatedtohigher(worse)ABCIrritability(F1,80=8.40,P=.005)(Figure2S)andSRSAwareness(F1,79=6.45,P=.01) (Figure2T),Cognition(F1,79=6.87,P=.01)(Figure2U),Communication(F1,79=3.83, P=.05) (Figure2V), Motivation (F1,79=4.90, P=.03)(Figure2W), Mannerisms (F1,79=6.35, P=.01) (Figure2X) and Totalscore(F1,79=7.50,P<.01)(Figure2Y).

AhigherrT3/TSHratiowasrelatedtohigher(worse)ABCIrritability(F1,80=10.26, P=.002) (Figure2AA) and SRS Awareness (F1,79=5.14,P=.03) (Figure2AB), Cognition (F1,79=4.35, P=.04) (Figure2AC) andTotalscore(F1,79=5.30,P=.02)(Figure2AD).

HigherTRHwasrelatedtolower(worse)VABSDailyLivingSkills(F1,82=5.36, P=.02) (Figure2AE) and Social Skills (F1,82=4.25, P=.04) (Figure2AF).

3.6 | Repeated thyroid and autoantibody measurements

Forty-twoparticipantswerepartofaclinicaltrialinwhichtheywereblindlyrandomisedtoreceiveeitherplaceboorhigh-dosefolinicacid

Variable FRAA negative (n=35) FRAA positive (n=52)

Anti-epilepticmedication 1(3%) 7(13%)

Selectiveserotoninreuptakeinhibitors 0(0%) 7(13%)

Antimicrobialmedications 2(6%) 3(6%)

Immunomodulatorymedications 1(3%) 2(4%)

Otherpsychotropicmedications 0(0%) 1(2%)

Supplements(concurrenttreatments),n(%)

Multivitamin 14(40%) 16(30%)

Minerals 5(14%) 11(21%)

Fattyacids 4(11%) 9(17%)

VitaminB12 1(3%) 10(19%)

OtherBvitamins 2(6%) 8(15%)

Folate 4(11%) 6(11%)

Otherantioxidants 2(6%) 7(13%)

Carnitine 2(6%) 6(11%)

Othervitamins 4(11%) 4(8%)

CoenzymeQ10 2(6%) 3(6%)

Othersupplements 0(0%) 2(4%)

Aminoacids 0(0%) 1(2%)

Comorbidmedicalconditions,n(%)

Allergicdisorders 15(43%) 21(40%)

Gastrointestinaldisorders 14(40%) 22(42%)

Neurologicaldisorders 6(17%) 17(32%)

Nondiagnosticcopynumbervariants 8(23%) 15(28%)

Psychiatricdisorders 2(6%) 15(28%)

Immuneabnormality 6(17%) 9(17%)

FRAA,folatereceptorαautoantibody;T3,triiodothyronine;T4,thyroxine;TRH,thyroid-releasinghormone;TSH,thyroid-stimulatinghormone.

TABLE  1  (Continued)

     |  7 of 12FRYE YEet  al

andhadboththyroidfunctionandFRAAmeasuredatthebeginningandendofthetrial.Thisallowedustoexaminethecorrespondencebetweenthechange inthyroidfunctionandFRAAtitresandtode-terminewhetherhigh-dosefolinicacidhadanysystematiceffectonthyroidfunction.

ThechangeinTSHandFT4/TSHratio(butnotFT4)wassignifi-cantly related to the change in blockingFRAA, but not thebindingFRAA.ThechangeinTSHhadapositiverelationshipwiththeblockingFRAAtitres(F1,40=6.22,P=.02)(Figure3A),whereasthechangeintheFT4/TSHratiohadaninverserelationshiptotheblockingFRAAtitres(F1,40=4.39,P=.04) (Figure3B). NeitherTSH and FT4, nor FT4/TSHratioswererelatedtowhetherornotthepatientreceivedhigh-dosefolinicacidorplacebo.

3.7 | FRα expression in the thyroid

ToexaminethedevelopmentalaspectoftheFRαwithrespecttothy-roiddevelopment,FRαexpressionwasmeasuredonthethyroidglandduringvariousagesoflife.Figure4showsthatFRαishighlyexpressedat15and17weeksofgestation,whereasFRαisnotexpressedinthefoetaltissueat18,19or20weeksofgestation,norwasitexpressedinthe5-monthpostpartumthyroidtissue,suggestingthatFRαexpressionisonlypresentinearlygestationandlostinlatergestationasthethy-roidmatures.Wehavealsoexaminedthyroidfroma3-year-oldchildanda27-year-oldadultandfoundnoFRαexpression(datanotshown).

4  | DISCUSSION

BothblockingandbindingFRAAsareconsideredtohavepathologicalconsequencesinASDbecausetheybindtotheFRαwheretheycaninterferewithfunctionoftheFRαandreducethetransportationoffolate intothecentralnervoussystem25andtheymayalsoactivatetheimmunesystem.50TherearereportsoftheFRαbeingexpressedonnormalthyroidtissue,26althoughsometimesonlytoalimitedex-tent.27,28 In the present study, we investigated the significance ofFRAAsinconjunctionwiththyroidfunction inchildrenwithASDtofollow-up on our previous report.7We also examined FRα expres-siononthethyroidtissueatdifferentstagesofhumandevelopment.Severalinterestingrelationshipswereidentified.

AutismspectrumdisorderchildrenpositiveforblockingFRAAshadhigherTSHand lowerFT4/TSH,TT3/TSHand rT3/TSH ratios thanthosenegativeforblockingFRAAs.Additionally,blockingFRAAtitreswerelinearlyrelatedtoTSHconcentrationsandFT4/TSH,TT3/TSHandrT3/TSHratios.Furthermore,whenweexaminedthechangesinFRAAtitresandthyroidfunction(TSH,FT4)overa12-weekperiod,wefoundastrongrelationshipbetweenachangeinblocking(butnotbinding)FRAAtitresandachangeintheTSHandFT4/TSHratio,con-sistentwiththecross-sectiondata.TheblockingFRAAdidnotappeartohavesignificantinfluenceonFT4,TT3orrT3specifically.

ItappearsthattheblockingFRAAhasaninfluenceonTSHandtheFT4/TSH,TT3/TSHandrT3/TSHratiosbyincreasingtheTSHrelative

F IGURE  1  Inchildrenwithautismspectrumdisorderhigherblockingfolatereceptorαautoantibody(FRAA)titresareassociatedwith(A)higherthyroid-stimulatinghormone(TSH),(B)lowerthyroxine(T4)toTSHratio,(C)lowertotaltriiodothyronine(T3)toTSHratioand(D)lowerreserveT3toTSHratio

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totheproductionofthyroidhormones.ThissuggeststhattheeffectoftheblockingFRAAonthyroidfunctionmayoccuratthelevelofthethyroidbymakingthethyroidlesssensitivetoTSHoralteringthesen-sitivityoftheHPA.Specifically,alowerFT4/TSH,TT3/TSH,rT3/TSHratioswouldsuggestthatahigherTSHconcentrationisneededforthe

productionof thyroidhormones, implyingadecreasedsensitivityofthethyroidorHPA.TheideathatchildrenwithASDmayhavediffer-encesinHPAdynamicsand/orthyroidsensitivitytoTSHisconsistentwithbothresearchandclinicalstudies.Studieshaveshownabluntedpituitary response toTRHstimulation inASDboys3and thediurnal

F IGURE  2 Therelationshipbetweenthyroidfunctionandbehaviourinchildrenwithautismspectrumdisorder.Higherthyroid-stimulatinghormone(TSH)wasassociatedwithlower(better)(A)AberrantBehaviorChecklist(ABC)Irritability,(B)SocialResponsivenessScale(SRS)Awareness,(C)SRSCognition,(D)SRSMotivation,(E)SRSMannerisms(F)SRSTotalScore;Higherfreethyroxine(T4)toTSHratiowasassociatedwithhigher(worse)(G)ABCIrritability,(H)ABCHyperactivity,(I)SRSAwareness,(J)SRSCognition,(K)SRSCommunication,(I)SRSMotivation,(M)SRSMannerismsand(N)SRSTotalScore;HighertotalT3wasassociatedwithhigher(worse)(O)SRSCognition,(P)SRSMotivation,(Q)SRSMannerismsand(R)SRSTotalScore.Highertotaltriiodothyronine(T3)toTSHratiowasassociatedwithhigher(worse)(S)ABCIrritability,(T)SRSAwareness,(U)SRSCognition,(V)SRSCommunication,(W)SRSMotivation,(X)SRSMannerismsand(Y)SRSTotalScore;HigherreverseT3toTSHratiowasassociatedwithhigher(worse)(AA)ABCIrritability,(AB)SRSAwareness,(AD)SRSCognition,and(AD)SRSTotalScore;HigherThyroidReleasingHormonewasrelatedtoworse(lower)(AE)VABSDailyLivingSkillsand(AF)VABSSocialSkills

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F IGURE  3 Changein(A)thyroid-stimulatinghormone(TSH)and(B)thefreethyroxine(T4)toTSHratioisassociatedwithachangeintheblockingfolatereceptorαautoantibody

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     |  9 of 12FRYE YEet  al

variationofTSHappearstobelargerinyoungadultswithASD.4TRHconcentrationswerenotrelatedtoblockingFRAAtitres,whichsug-geststhattheeffectisatthelevelofthethyroidandnotthepituitary.

Theeffectofthyroidhormonesonthebrainiscomplex,especiallyduringfoetalandneonataldevelopment.35,36FRαishighlyexpressedinfoetalthyroidtissueonlyearlyingestation,withnoobservableex-pressioninlategestationorpostnatalthyroidtissue.Thisisbasedonalimitednumberofthyroidsamplesandthereforethisfindingshouldbe considered preliminary. Exposure tomaternal FRAAs and folatedeficiency in uteromaycontributetopoordevelopmentoftheglandanddysfunction in later life.Thereportedhistologicaldatasuggeststhatthe influenceofFRAAscouldoccurduringfoetaldevelopment.BecauseseveralstudieshavedemonstratedthatmothersofchildrenpositiveforFRAAsalsoarepositiveforFRAAs,25,37itispossiblethatmaternalFRAAspresentduringgestationcoulddisruptthyroiddevel-opmentand/oralterthedevelopmentalexpressionoftheFRαonthethyroid.

There isevidencethatmaternalFRAAscandisruptfoetaldevel-opment.MaternalFRAAsresultinneurodevelopmentalabnormalitiesinrodentoffspring51andmaternalFRAAshavebeenlinkedtoneuraltubedefectsandsubfertilityinhumans,52aswellaspretermbirth.53ItispossiblethatdisruptionoffoetalthyroiddevelopmentbymaternalFRAAscouldhaveresultedinalteredregulationofthethyroidduringchildhood.However,thechangeinTSHwithchangeinFRAAdemon-stratedon repeatedmeasurements suggest thatFRAAsmayhaveamoredirecteffectonregulationofthethyroidduringchildhood.Forexample,itispossiblethatfoetalexposuretoFRAAsmayhavealtereddevelopmentalFRαexpressionsothattheFRαhasincreasedexpres-siononthethyroidduringchildhood.Examiningthyroidtissuefrom

children exposed to FRAAs during gestationwould help clarify thispossibility.However,toourknowledge,nosuchtissueisavailableatpresent.

Behaviourappearstoberelatedtothyroidfunction inASDchil-drensuchthathigherlevelsofTSHisassociatedwithbetterASDbe-haviouralcharacteristicsusingtheABCaswellastheSRSscales,aninstrument thathashighcorrespondencewithagold-standardASDassessment, theADI-R.TheFT4/TSH,TT3/TSHandrT3/TSHratiosalsoappeartobesimilarlyrelatedtoABCandSRSscales,suchthatalowerratio isassociatedwithbetteroverallbehaviour.Therelation-ship between thyroid hormones and behaviour should not be sur-prisingbecausethyroidhormonescanbothpositivelyandnegativelymodulateneurotransmittersystemsas,wellashaveadirecteffectongeneexpression.54However, theexactmechanismbywhichthyroidhormones influencebehaviourandcognition inASDremains to theclarified.

Thyroid-stimulatinghormoneisrelatedtobehavioureventhough,forthemostpart,itiswithinthenormalrangeinoursample,andbothTSHandtheFT4/TSHarerelatedtobehavioureventhoughtheyarenotsignificantlydifferentthanthecontrolvaluesinoursample.Thissuggests that theeffect is not related to absolute thyroidhormonelevelsbutrathertoHPAsensitivity,suchthatsomechildrenwithASDmaybemoresensitivetoasmallvariationinthyroidhormonelevels.Thiswould suggest that the relationship betweenTSH and thyroidhormonesproducedbythethyroid(ie,FT4,TT3,rT3)ismoreimport-ant that theabsoluteconcentrationsof thesehormones.This couldexplainwhysomestudieshavedemonstratednormalthyroidhormonelevels in childrenwithASD,22,23whereas others have showndiffer-encesindynamicchangesinthyroidhormonesinchildrenwithASD,

F IGURE  4 Folatereceptorαexpressioninthethyroidtissueduringembryonicdevelopmentandfollowingbirth.(A)Folatereceptorα expressioninthethyroidgland.Counterstainedsampleswithhaematoxylintovisualisenuclei.(B)Tissuestainedwithhaematoxilinandeosin.

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suchasabluntedresponsetoTRHstimulation3anddifferencesinthediurnalvariationofthyroidhormonelevels.4Thesedatasuggestthatsimply using normal ranges to interpret levels of thyroid hormonesinchildrenwithASDmaynotcapturesubtleabnormalitiesinthyroidfunction.

Severalpreliminaryclinicalstudiesthathavesuggestedafavour-able response to thyroidhormonesupplementation inchildrenwithASD2,19,20andanimalmodelshavedemonstratedanimprovementinASD behaviours with T4 supplementation.55 Although thyroid hor-monesupplementationwouldappeartobecountertoourbehaviouraldataandthefactthatTT3andrT3wasfoundtobeabovenormalinahighpercentageofchildrenwithASD,itispossiblethatsuchsupple-mentationcouldreducevariationsintheHPA,leadingtoamorestableregulationofthyroidhormones. Interestingly,clinicalstudies inASDindividuals show a positive effect of propranolol, amedication thatblockstheconversionofT4toT3,therebypotentiallyprovidingben-efit inthosewithhighTT3orrT3concentrations.Indeed,inindivid-ualswithASD,propranololhasbeenshowntoimprovelanguage,56,57 cognitive flexibility,58 working memory,59 facial scanning60 and be-haviour.61,62Lithium,amedicationthat inhibitsthyroidfunction,hasbeenshowntohavepositiveeffectonASDbehavioursinseveralclin-ical63-65andanimalstudies,66therebyalsoindicatingarelativeoverac-tivityofthethyroidastheculprit.

Thefindingsofthepresentstudycouldguidefutureresearchstud-ies aiming to better understand the importance of thyroid functioninASD,suchthattheclinical implicationsofthethyroid inASDcanbebetterunderstood.First,itdoesappearthatthereisarelationshipbetweenFRAAsandthethyroid,withFRAAsmostlikelyaffectingthesensitivityoftheHPA,probablyatthelevelofthethyroid.TherealsoappearstobearelativelyincreasedexpressionoftheFRαduringpre-nataldevelopment,suggestingthattheeffectsofFRAAsmaybemostsignificant before birth.Additionally, data reportedwithin the pres-entstudysuggestthatvariationsinthyroidfunctiondoindeedhaveaneffectonthebehaviourofchildrenwithASD,althoughtheexactmechanismforthisinfluenceinnotclear.WeconsiderthatitmightbewiseforfurtherresearchtoconcentrateontheHPAwithrespecttothethyroidandfocusonthedynamicchangesinthyroidhormonesatbaseline,aswellasontreatmentsthataffectthethyroid.

Inconclusion,thepresentstudyhashelpeddefinetheimportanceof thyroid function inASD and the effect of the FRAA on thyroidfunction inASD. It appears that foetal exposure toblockingFRAAscouldaffect thedevelopmentof the thyroid,potentiallymaking thethyroid less sensitive toTSH. TSH and the ratio of hormones pro-ducedbythethyroidtoTSHappeartoberelatedtoASDbehaviour,implicatingdysregulationoftheHPA,althoughtheexactsignificanceof thesefindingswill require furtherclinicalstudywith largerpopu-lations. Overall the present study highlights the importance of theFRAAsandthyroidfunction,bothtogetherandseparately,inASDandtheneedforfurtherclinicalstudiestobetterunderstandhowtargetedtherapeutic interventions can help improve the lives of individualswithASDandtheirfamilies.ThesefindingsmayalsobeapplicabletootherdisordersassociatedwithFRAAs,suchasschizophrenia67 and subfertility.68

ACKNOWLEDGEMENTS

The present study was supported, in part, by funding from theArkansas Biosciences Institute, Autism Research Institute, the JaneBotsfordJohnsonFoundationandAutismSpeaksgrant#8202.HumanfoetalthyroidtissuewasprovidedbytheUniversityofMarylandBrainandTissueBank,whichisaBrainandTissueRepositoryoftheNIHNeuroBioBank.

CONFLICT OF INTERESTS

DrsFryeandQuadros areon theScientificAdvisoryBoardof IliadNeurosciencesInc.(PlymouthMeeting,PA).Twooftheauthors(JMSandEVQ)areinventorsonaUSpatentforthedetectionofFRautoan-tibodiesissuedtotheResearchFoundationoftheStateUniversityofNewYork.Theotherauthorsdeclarethattheyhavenoconflictsofinterest.

AUTHOR CONTRIBUTIONS

REF, RW, SR, JS, LD,MT, SCB, SM, JMS and EVQ performed theresearch.REF,SR,JS,LDandEVQdesignedtheresearchstudy.REF,LDandEVQanalysedthedata.REF,SR,JS,LD,SGK,JMSandEVQwrotethepaper.

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How to cite this article:FryeRE,WynneR,RoseS,etal.Thyroiddysfunctioninchildrenwithautismspectrumdisorderisassociatedwithfolatereceptorαautoimmunedisorder.J Neuroendocrinol.2017:29:1-12.https://doi.org/10.1111/jne.12461

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