Altered DNA methylation profile in Norwegian patients with Autoimmune Addison’s Disease

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Molecular Immunology

jo u r n al h om ep age:w w w . e l s e v i e r . c o m / l o c a t e / m o l i m m

Altered DNA methylation profile in Norwegian patients with Autoimmune Addison’s Disease

Trine E. Bjanesoy

, Bettina Kulle Andreassen

, Eirik Bratland

, Andrew Reiner

, Shahinul Islam

, Eystein S. Husebye

, Marit Bakke

aDepartmentofBiomedicine,UniversityofBergen,JonasLiesvei91,5009Bergen,Norway

bDepartmentofMolecularBiology,InstituteofClinicalMedicine,UniversityofOslo,P.OBox1171,0318Oslo,Norway

cDepartmentofBiostatistics,InstituteofBasicMedicalSciences,UniversityofOslo,P.OBox1110,0317Oslo,Norway

dDepartmentofClinicalScience,UniversityofBergen,P.OBox7804,5020Bergen,Norway

eStemCellEpigeneticsLaboratory,InstituteofBasicMedicalSciences,FacultyofMedicine,UniversityofOslo,P.OBox1110,0317Oslo,Norway

fDepartmentofMedicine,HaukelandUniversityHospital,P.OBox7804,5020Bergen,Norway

a r t i c l e i n f o

Articlehistory:

Received10January2014

Receivedinrevisedform21February2014 Accepted25February2014

Availableonline22March2014

Keywords:

DNAmethylation Epigenetics Autoimmunity

AutoimmuneAddison’sDisease Adrenalgland

a b s t r a c t

AutoimmuneAddison’sDisease(AAD)isanendocrineandimmunologicaldiseaseofuncertainpatho- genesisresultingfromtheimmunesystem’sdestructionofthehormoneproducingcellsoftheadrenal cortex.Theunderlyingmolecularmechanismsarelargelyunknown,butitiscommonlyacceptedthat acombinationofgeneticsusceptibilityandenvironmentalimpactiscritical.Inthepresentstudy,we identifiedmultiplehypomethylatedgenepromoterregionsinpatientswithisolatedAADusingDNAiso- latedfromCD4+Tcells.Theidentifieddifferentiallymethylatedregionsweredistributedevenlyacross the10.5-kb-promoterregionscoveredbythearray,andasubstantialnumberlocalizedtopromotersof genesinvolvedinimmuneregulationandautoimmunity.Thisstudyrevealsahypomethylatedstatusin CD4+TcellsfromAADpatientsandindicatesdifferentialmethylationofpromotersofkeygenesinvolved inimmuneresponses.

©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/3.0/).

1. Introduction

Autoimmunedisordersconstituteagroupofdiseasesthataffect 5–10%ofthepopulationinwesterncountriesandaround3%world- wide (Shapira et al., 2010; Youinou et al.,2010).Apart from a few autoimmunediseases caused by mutationsin singlegenes (Michelsand Gottlieb,2010), theetiologyof mostautoimmune diseasesisstillunclear.However,itiscommonlyacceptedthata combinationofgeneticsusceptibilityandenvironmentalfactors areat play, causing breakdown of thenatural tolerancemech- anisms (Costenbader et al., 2012; Gonzalez et al., 2011; Wing andSakaguchi,2010).Therelativeimportanceofenvironmental factorsis illustratedby thefacts that monozygotictwins show moderateratesofconcordanceforautoimmunedisordersandthat drug-induced autoimmunity is observed after medicationwith demethylatingagents(Javierreetal.,2011).Astheenvironment

∗Correspondingauthor.Tel.:+4755586196.

E-mailaddress:Marit.Bakke@biomed.uib.no(M.Bakke).

1 Theseauthorscontributedequally.

directlyimpacts theepigenetic code,thesurroundingmilieuof anindividualwilldirectlyaffectgeneregulatorymechanismsand diseasedevelopment.

Inautoimmunedisorders,themoststudiedepigeneticmarkis DNAmethylation,whichinvolvesadditionofamethylgroupto cytosinesbyDNAmethyltransferases(Dnmts).In differentiated mammaliancells>98%ofallDNAmethylationoccursoncytosines thatare followedby guanines(i.e.,a CpGsite;Jinet al.,2011).

TheDNAmethylationstatus ofagenomicregionwillaffectthe expressionofgenes,buttheoutcomewilldependonthelocation ofthemethylatedcytosines.Ingeneral,wholegenomesequencing studiessuggestthathypermethylationofpromoterregionsisasso- ciatedwithgenesilencing,whereasintragenichypermethylationis linkedtogeneactivation(Aranetal.,2011;Honetal.,2012).How- ever,manyCpG-richpromoters(i.e.,containingCpGislands)are keptdemethylatedregardlessofthetranscriptionalactivityofthe correspondinggene(Hawkinsetal.,2010).TheDNAmethylation signatureischangedinmanyautoimmunediseases,asexemplified byglobalhypomethylationaswellashypo-orhypermethylationof specificloci(Lu,2013;Selmietal.,2012).Moreover,DNAmethyl- ationplaysessentialrolesinnormaltranscriptionalcontrolofkey http://dx.doi.org/10.1016/j.molimm.2014.02.018

0161-5890/©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/3.0/).

factorsinTcelldifferentiationandfunction,suchastheinterferon gammalocus(Sawalha,2008),andthetranscriptionfactorsFoxP3 (forkheadboxP3(Laletal.,2009)andAIRE(Kontetal.,2011).

Primary adrenal insufficiency (Addison’s disease) is a rare diseasewithaprevalenceof around100–140per millioninha- bitantsinEuropeancountries(Betterleetal.,2002;Lauretietal., 1999;LovasandHusebye,2002; Myhreetal.,2002).Themajor cause(80–90% ofthecases)isanautoimmune reactiontoward theadrenalgland,resultingfromselectivedestructionofsteroid hormone-secretingcellsinthecortex.AutoimmuneAddison’sdis- ease(AAD)mayoccurisolated,but in50%ofthecasesanother autoimmunediseases suchasautoimmune thyroid diseaseand type1diabetes(T1D)ispresent(EisenbarthandGottlieb,2004).

Themechanismsunderlyingtheinitiationoftheimmune-mediated inflammationinAADremainenigmatic,andasforotherautoim- munediseases,it isbelieved thata combination ofgeneticand environmentalfactorsaffectdiseasedevelopment(Husebyeand Lovas,2009;Selmietal.,2012).ThevastmajorityofAADpatients (around80–90%indifferentcohorts,(BetterleandZanchetta,2003;

Erichsenetal.,2009))displayautoantibodiesagainstthesteroido- genicenzymecytochromeP45021-hydroxylase(CYP21),butthe preciseroleofthisimmunoreactioninthepathogenesisofAAD isnot wellunderstood.It hasbeensuggested thatthedestruc- tionoftheendocrinecellsof theadrenal cortexismediatedby antigen specific CD4+T-helper cells, and executed by cytotoxic T-lymphocytesandmacrophages(BratlandandHusebye, 2011).

Atthe geneticlevel, several reports from differentpopulations haveconfirmedastronglinktoHLA-DRB*03andHLA-DRB1*0404.

TheDRB1*0404alleleisparticularlymorefrequentamong AAD patientsthanotherDRB1*04 alleles(Maclaren andRiley, 1986;

Skinningsrud et al., 2011; Yu et al., 1999). As the differences betweenDRB1*0404andtheotherDRB1*04allelesinfluencethe peptidebindingcapabilitiesofHLAmolecules,aroleofantigen- specificTcellresponsesinAADpathogenesisisplausible(Gombos et al., 2007). The fact that homozygosity for the 5.1 allele of MIC-A gene (locatedin the HLAcomplex)increases theproba- bilitytodevelopAADwhenpresentincombinationwithcertain HLA genotypes (Triolo et al., 2009) supports a strong role of Humanleukocyteantigen(HLA).OutsidetheHLAcomplex,poly- morphismsinthegenesencodingtheimmunomodulatoryfactors CTLA4(cytotoxicT-lymphocyteantigen4),CIITA(classII,major histocompatibilitycomplex,transactivator),PTPN22(proteintyro- sinephosphatase,non-receptortype22)andPD-L1(programmed celldeath1ligand1)arecommonlyfoundinbothAADandother autoimmunediseases(Blomhoffetal.,2004;Ghaderietal.,2006;

Mitchelletal.,2009;Skinningsrudetal.,2008).InAADpatients, disease-associatedpolymorphismsarealsofrequentlyfoundinthe genesencodingthevitamin Dreceptorand NLRP1(NLRfamily, pyrindomaincontaining1),importantforcytokinesecretionand inflammatoryresponses,respectively (Magitta etal.,2009;Pani etal.,2002).ThereisnopublishedGenome-wideassociationstudy (GWAS)customizedforAAD,andatpresent,wehaveverylimited understandingofthegeneticfactorsthatcontributetoAADdevel- opmentattheindividuallevel.

TheaimofthepresentstudywastoinvestigatewhetherDNA methylationabnormalitieswerelinkedtoAAD.Indeed,anumber ofdifferentiallymethylatedregions(DMRs)wereidentified,the majorityofwhichwerehypomethylatedinthepatients.Moreover, thepatientsexhibitedanoverallhypomethylatedstatus.Interest- ingly,asignificantnumberoftheDMRslocalizedtothepromoters ofgenesencodingfactorsthatareimplicatedinimmuneresponses andautoimmunity.Theresultspresentedinthisstudymightserve asa foundationforfuture experimentsdesigned tounravelthe molecularmechanisms underlyingAAD, as well as togenerate hypotheses regarding the importance of environment–genome interactionsandautoimmunity.

2. Materialsandmethods

2.1. AADpatientsandcontrols

Peripheralbloodwascollectedin Ethylenediaminetetraacetic acid(EDTA)tubesfrompatientswithisolatedAADandfromhealthy blooddonors.Allpatientshadpreviouslybeendiagnosedwithclin- icallyevidentprimaryadrenocorticalinsufficiencyusingstandard biochemical measurements(Arltand Allolio,2003).Allpatients werepositive forcytochrome CYP21-antibodies(CYP21-ab)and had beentreated withcortisonecombinedwithfludrocortisone sincediagnosis(15±14years).Allsubjectswerefemalesfrom20 to60yearofage(mean45±13yearsand43±8years,forcases andcontrols,respectively).Thepatientswerediagnosedintheage rangeof13–54years (meanage30years),anddiseaseduration rangedfrom4to37(meanduration15years).Allpatientswere born,raisedanddwellinWesternNorway(HordalandandRoga- landCounties).Allparticipantssignedtheinformedconsentform approvedbytheNorwegianHealthRegionWestethicscommittee (149/96–47.96).

2.2. IsolationofCD4+TcellsandDNA

CD4+Tcellswereisolatedfrom12to15mlEDTAbloodbythe DynabeadsCD4positiveIsolationKit(LifeTechnologies/Invitrogen) accordingtothemanufacturer’sprotocolwithin6hofbloodcol- lection.GenomicDNAfromCD4+Tcellswasextractedusingthe DNeasyBloodandTissueKit(Qiagen)accordingtothemanufac- turer’sprotocol.

2.3. Globalmethylationanalyses

TheglobalDNA methylationstatus wasdeterminedonDNA isolatedfromCD4+Tcellsfrom16AADpatientsand16healthy controlsbyanELISAassay(5-mCDNAELISAkit,ZymoResearch, Irvine, CA,USA).Five ofthesepatientsandfourof thecontrols contributedtotheMeDIPanalysis.Thiskitisbasedontherecog- nition and quantificationof themethylated DNAfractionusing a5-methylcytosineantibodyanda standardcurvegeneratedby methylated/demethylated E. coli DNA. The output values were multipliedbythehumanCpGdensity/genomefactorprovidedby themanufacturer(accordingtothemanufacturer,theE.coliCpG density/genomelengthis0.075andhumanCpGdensity/genome lengthis0.009(28,700,086/3,137,161,264),andthefolddifference betweenE.coliandhumanCpGdensityis8.3).

2.4. MeDIP

GenomicDNA wasisolatedfromnine AADpatientsandfive healthy controlindividuals.The DNAwasshearedtofragments withanaverageof400bp.MethylatedDNAwasprecipitatedusing theMagMeDIPkit(Diagnode,Belgium),whichisbasedonmag- neticbeadsanda5-methylcytidineantibody.TheprecipitatedDNA waspurifiedandelutedfromthebeadsbytheIPurekit(Diagnode, Belgium)accordingtothemanufacturer’sprotocol.Toobtainsuf- ficientamountofDNAformicroarrayhybridization,theimmuno- precipitatedDNAandinputDNAwereamplifiedusingtheWGA2 kit(Sigma,USA).AmplifiedproductswerecontrolledbyqPCRusing primerpairsspecificforamethylatedregion(TSH2B)andunmethy- latedregion(GAPDH)accordingtothemanufacturer’sprotocol.

2.5. Arrayhybridization

TheprecipitatedDNAwashybridizedtotheNimbleGenHuman DNAMethylation2.1MDeluxePromoterArray(NimbleGen-Roche, USA)byRocheNimbleGenServices(Iceland).Thisarraydesignis

basedontheHG18 genomebuild,and coversalltranscript and microRNApromotersaswellasall28,226annotatedCpGislands withspacingthroughoutalltiledregions.Tilingofeachtranscript promoterbegins7.25kbupstreamofthetranscriptionstartsite (TSS)andextendsdownstream3.25kbforatotalof10.5kbofpro- motercoveragepertranscript.EachmiRNApromoter(475total)is tiledfromthematuremiRNAsequenceto15kbupstream.

2.6. Statistics

Preprocessing and quality control: The raw data included 2,129,040annotatedprobesforeachoftheninepatientsandfive controls.2.19% of theprobeidsare matchedtomorethan one probesequenceandwereexcludedfromfurtheranalysisleading to2,082,409probeidsleft.Afterremovingprobeswhosepositions have not beenlocalized onthechromosome, thefinal number fortheanalysis became2,000,078 probeids.We used median- centeredwithin-arraynormalizationandquantilenormalization forbetweenarraynormalization.

Differentiallymethylatedregions:Weperformedattestforeach ofthe2,000,078probesanddefinedthe1%percentileofthenom- inalpvaluesastobethethresholdforaninterestingprobe.These interestingprobeswerethenmergedintoDMRwithneighboring probesifthedistancetothenextinterestingprobewaslessthan 500bp.Theregion-wiseteststatisticwasdefinedtobethesum ofall(absolutevaluesofthe)tteststatisticswithintheDMR.We endedupwith3373DMRs(SupplementaryTableS1DMRs.xlsx).

2.7. GenomicannotationofDMRs

DMRswereassociatedwithTSSsbyidentifyingDMRswhose genomic coordinatesintersected with theTSS coordinates. The 10.5Kgenepromoterregionsweredividedintofoursubregions tofurthercharacterizethepositionsandpropertiesoftheiden- tified DMRs(start:end pairs relative to theTSS: −7250/−4625,

−4625/−2000,−2000/+625and+625/+3250).Thestartcoordinate isthefirstbpinthesubregion,andtheendcoordinateisonepast thefinalbp.WhichDMRsoverlapwithwhichpromotersubregions ispresentedinSupplementaryTableS2regionsoverlap.xlsx.Ifa DMRoverlapswithtwoormoresubregionsinapromoterregion, itisassociatedwitheach.

2.8. CpGclassassignment

Eachpromoter regionorsubregionwasscannedbysliding a 500-bpwindowacrosstheregion,andcalculatingtheCpGratioand CGcontentateachposition.CpGratiowascalculatedasfollowsfor each500bpwindow:Obs/ExpCpG=N×numberofCpG/(number ofC×numberofG)N:totalnumberofnucleotides.Theregionis highifthereisa500-bpwindowwheretheCpGratiois>0.75and theC+Gcontentis>0.55. Theregionis lowifthereisnowin- dowwithaCpGratio>0.48.Theregionisintermediateotherwise (Weberetal.,2007).

2.9. Geneontologyandpathwayanalysis

Geneontology(GO)analysiswasperformedusingtheDAVID BioinformaticsResource(Huangdaetal.,2009)withapvaluecut- offof0.05.Pathwayanalysisandenrichmentwasdoneusingthe KEGGpathwaycomponentoftheDAVIDBioinformaticsResource.

Pathwayenrichmentwasrunusingthedefaultthreshold=2and EASEscore=0.1.

Fig.1.GlobalDNAmethylationlevels(%)inCD4+Tcellsfromhealthycontrols (n=16)andfromAADpatients(cases)(n=16)weredeterminedbya5-mCDNA ELISAkit.Studentsttest;p=0.0438.

3. Resultsanddiscussion

3.1. Globalandpromoter-specifichypomethylationinpatients withAAD

AADisprimarilyassociatedwithclassIIHLAalleles,suggesting thatactivationofCD4+Thelpercellsisanecessityforthedisease todevelop.CD4+TcellswerethereforeusedasthesourceforDNA infurtheranalysesonglobalDNAmethylationandfortheiden- tificationofpotentialgenomicregionswithalteredmethylation statusinAADpatients.ByMeDIPcombinedwitharrayhybridiza- tion,weidentified3373regionsthatweredifferentiallymethylated betweenAADpatientsandcontrols(SupplementaryTableS1).The majorityoftheDMRswerelocalizedinpromoterregions,andvery fewnon-promoterCGIsweredifferentiallymethylated(i.e.,<0.2%).

Of the identified DMRs, 89% were hypomethylated in patients compared to controls (Supplementary Data, TableS1). Also, as determinedbya5-mCDNAELISAassay,CD4+TcellDNAfromAAD patientswashypomethylatedcomparedtocontrols(Fig.1).The observedglobalmethylationlevelinCD4+Tcellderivedfromcon- trolsiscomparabletowhathasbeenpreviouslyreported(Ziller et al., 2013), and the relative decrease in methylation in AAD patientsisanalogoustowhathasbeenreportedforotherautoim- munediseases(Dongetal.,2011;Javierreetal.,2011;Leietal., 2009).Arecent comprehensive wholegenomebisulfite dataset demonstratesthatonlyaround20%ofautosomalCpGsaredynam- icallyregulatedwithregardtomethylation.However,theseCpG sitesfrequentlylocalizetogeneregulatoryelements,explaining whyrelativelyminorchangesinglobalmethylationcanhavemajor effectsoncellfunction(Zilleretal.,2013).Cancer-derivedcellsare alsogenerallyhypomethylated,andwholegenomeDNAmethyla- tionanalyseshaverevealedhypomethylatedregionsthatclusterin theso-calledpartiallymethylateddomains(PMDs;Hansenetal., 2011;Honetal.,2012).PMDsareassociatedwithgeneexpression hypervariability,presumablyasaresultofthemethylationchanges (Hansenetal.,2011;Honetal.,2012).Itremainstobedetermined whetherasimilarorganizationofhypomethylatedregionsexists indiseasedcellsfromautoimmunepatients.

Fig.2. (a)Thedistributionofhigh,intermediate(med),andlowCpGdensityregionsacrossalltiledpromoterscoveredbythearrayisshownintheupperpanel.The distributionacrosspromoterregionsofhigh,intermediate(med),andlowCpGdensityDMRsidentifiedinthisstudyisshowninthelowerpanel.Differentdistributionof thevariousCpGcategoriesbetweentheidentifiedDMRsandalltiledpromoterregionswasdeterminedwithPearson’schi-squaredtestofassociation:Forpromoterregion +625/+3250:²=46.5,ds=2,p=8.0×10⁻¹¹;forpromoterregion−2000/+625:²=46.5,ds=2,p=1.8⁻⁶;forpromoterregion−4625/−2000:²=109.5,ds=2,p<2.2×10⁻¹⁶; forpromoterregion−7250/−462:²=109.5,ds=2,p<2.2×10⁻¹⁶.(b)Relativelevelsofdifferentialmethylationlevelsinhypo-andhypermethylatedDMRscalculatedas theabsolutetstatisticvalue.Studentsttest,p=2.7×10⁻²⁷(n=2280forhypomethylatedDMRs,n=275forhypermethylatedDMRs).Thedataarepresentedasmeanvalues

±SE.

AsdescribedinSection2.7,thepromoterregionscoveredby thearrayweredividedintofoursubregionstofurthercharacterize thepositionsandpropertiesoftheidentifiedDMRs.Theidentified DMRsweredistributeduniformlyacrossthefourpromotersub- regions(SupplementaryTableS2).WecategorizedtheDMRsinto high,intermediate,andlowCpGcontent,accordingtothecriteria postedby(Weberetal.,2007;seeSection2.8).Asexpected,based onthedocumentedenrichmentofCGIsaroundTSSs(Deatonand Bird,2011),thelargestproportionoftheDMRsclassifiedas“high”

wasfoundinthe−2000/+625promoterregions(Fig.2a).Thelargest proportionofDMRsinallotherpromoterregionswasthoseDMRs withintermediateCpGcontent,whichmightreflectthefactthat mediumCpG-containinggenomicregionsaremorefrequentlysub- jectedtodifferentialmethylation(Irizarryetal.,2009).Therelative levelofdifferentialmethylation(calculatedastheabsolutetstatis- ticvalue,SupplementaryData,TableS1)wasslightly higherfor hypomethylatedthanforhypermethylatedDMRs(Fig.2b).Therel- ativedifferencesinmethylationdidnotdifferbetweenthedifferent promoterregions,orbetweenregionsclassifiedashigh,interme- diate,orlowwithregardtoCpGcontent(SupplementaryTable S2).

3.2. DMRslocalizetopromotersofgeneswithessentialrolesin immunity

AconsiderablenumberoftheidentifiedDMRslocalizedtopro- moters of genes withimmunological functions,some of which

have already been associated with AAD. The majority of the genesdiscussedhereweredifferentiallymethylatedintheprox- imalpromoterregion(−2000/+625).However,severalpotentially interestinggenescarriedabnormalmethylationmarksinotherpro- motersubregions,demonstratingtheimportanceofexaminingthe wholetiledpromoter(SupplementaryTableS2).Aswedetected relativelymilddifferencesinMeDIPprofilesbetweenpatientsand controls,thesemethylationchangescouldnotbevalidatedbybisul- fitesequencing(datanotshown).IntheremainingofSection3.2,

“hypomethylated”and“hypermethylated”refertothattheDMR washypoorhypermethylatedinpatientscomparedtocontrols.

HypomethylatedDMRslocalizedtoseveralinterferonregulated genes.Among201genesrecentlyshowntobeupregulatedbytype IinterferonsinPBMCsofhealthyhumans(Waddelletal.,2010), 13%weredifferentiallymethylatedinAADpatientscomparedto controlsinourdataset.Theseincludedwell-describedtargetgenes ofinterferons suchasIRF2(interferonregulatoryfactor 2),IRF7 (interferonregulatoryfactor7),EIF2AK2(eukaryotictranslationini- tiationfactor2-alphakinase2),USP18(ubiquitinspecificpeptidase 18),OAS2 (2’-5’-oligoadenylatesynthetase2),and CXCL10(C-X- Cmotifchemokine10/interferongamma-inducedprotein10).A numberofthesegenesarealsohypomethylatedinnaïveCD4+T cellsofpatientswithSLE,asystemicautoimmunedisorderwith astronginterferonsignature(Coitetal.,2013).WhilethetypeI interferonpathwayisconsideredtoplayadominantroleinsys- temicautoimmunedisorders,limiteddataareavailableonitsrole inorgan-specificautoimmunedisorderssuchasAAD.Yet,IFN␣is

elevatedinserumofpatientswithautoimmuneendocrinopathies, includingchildren withnewly diagnosed T1D(Chehadehet al., 2000;Mavraganietal.,2013).Furthermore,IFN␣hasbeensug- gestedasapossibleendogenouspredisposingfactor foradrenal inflammation,astreatmentofchronichepatitisCinfectionwith recombinantIFN␣inducesthelevelofCYP21-Ab(Wescheetal., 2001),causestransientprimaryadrenalinsufficiency(Krysiaketal., 2011;Tranetal.,2008),and worsenclinicalfeaturesofalready existingAAD(Oshimotoetal.,1994).WithregardtoCXCL10,itis ofinterestthatseveralindependentstudiesdemonstratethatthis chemokineiselevatedinseraofAADpatients(Bratlandetal.,2013;

Ekmanetal.,2013;Rotondietal.,2005).Consideringtheidentifi- cationofDMRsinthesegenepromoters,itmightbehypothesized thatthetypeIinterferonpathwayisinvolvedinthedevelopmentof AAD.Whetherthisrepresentsanepigenetic“poising”ofthetypeI interferonsystem(ashasbeendescribedforSLE(Coitetal.,2013)), ormerelyareflectionoftheongoingadrenalitisinAAD,remainsto beestablished.

Genes encoding members of the NLRP family of proteins (nucleotide-bindingoligomerizationdomain,leucine-richrepeat and pyrindomaincontaining; also calledNALP) alsocontained DMRsintheirpromoterregions.Specifically,NLRP-1,-5,-7,-8,- 12,and-13werefoundtocontainhypomethylatedDMRsintheir promoterregionsinourgroupofpatients.TheNLRPsaremembers of the nucleotideoligomerization domain (NOD)-like receptors (NLRs)thatplaycriticalrolesininnateimmunefunction(Lupfer andKanneganti,2013).SuchfunctionshavebeenascribedNLRPs 1and12,andtheidentificationofNLRP1inourdatasetisofpar- ticularinterestsinceSNPsinthisgenehavebeenassociatedwith AAD(Magittaetal.,2009;Zuraweketal.,2010).However,theDMRs identifiedheredonotoverlapwiththedescribedSNPs.Uponrecog- nitionofmicrobialligandsordanger-associatedmolecularpatterns NLRP1formspartoftheinflammasome,whichcleavesthezymogen pro-caspase 1 into enzymatically active and highly proinflam- matorycaspase1(Franchietal.,2012).Intriguingly,anothercentral componentoftheinflammasome,PYCARD,wasalsofoundtobe hypomethylatedinAADpatients.PYCARD(PYDandCARDdomain containing,also knownas ASC;apoptosis-associated speck-like proteincontainingaCARD)is akey adaptorproteinthat medi- atestheinflammatoryandpro-apoptoticeffectsoftheNLRP1.Once theinflammasomecontainingNLRP1,PYCARD,andpro-caspase1 hasbeenestablished,theproinflammatorycytokinesIL1␤andIL18 areproducedfrominactiveprecursorsbytheactivatedcaspase1.

Interestingly,thegenesencodingthereceptorsforIL1␤,IL1R1and IL1R2,werealsofoundtocontainhypomethylatedDMRs.Theacti- vatedinflammasomehaswidespreadeffects,includingincreased Tcell-dependentantibodyresponses,increasedIFN-␥production byT_H1cells,andincreasedrateofdifferentiationofnaïveTcells intoT_H17effectorcells(Shawetal.,2011).Thesefindingsmight indicatethatNLRP1andtheinflammasomeareimplicatedinthe onsetorperpetuationofAAD.Asimilarrolefortheinflammasome hasbeensuggestedforotherautoimmunediseases,includingRA, vitiligoandT1D(Jinetal.,2007;Kastbometal.,2008).

Another striking observation was the high number of hypomethylatedDMRsingenesthatareoftenmutatedinlymphoid malignancies. These DMRswere locatedtopromoters of genes encodingboth chromatinmodifiersandhistoneregulatorssuch asBRCA1(encodingbreastcancertype1 susceptibilityprotein), KAT6A/MYST3 (K (Lysine) Acetyltransferase 6A), WHSC1 (prob- able histone-lysine N-methyltransferase NSD2), DNMT3A (DNA (cytosine-5)-methyltransferase 3A) and BRD3 (bromodomain- containingprotein3),butalsocriticalcomponentsofactivatingand anti-apoptoticsignalingpathwayssuchasCARD11(caspaserecruit- mentdomain-containingprotein11),BIRC5(baculoviralinhibitor ofapoptosis repeat-containing5/surviving), and MCL1(induced myeloidleukemiacelldifferentiationprotein;KnoechelandLohr,

2013).Thesefindingsunderlinepossibleconvergingpathwaysand mechanismsofautoimmunityandlymphoidneoplasms,bothat geneticandepigeneticlevels.

HypomethylatedDMRsalsomappedtoastrikinglyhighnum- berofgenesencodingenzymesofthemitogen-activatedprotein kinase (MAPK) family. In particular, we identified members of the p38MAPK pathway, a signaling cascade involved in many autoimmune diseases (Cuenda and Rousseau, 2007). Also, this pathwayisimplicatedinmanyprocessesinCD4+Tcells(Dodeller etal.,2005),includingintegrationofextracellularsignals,Tcell activationanddifferentiationandproductionofproinflammatory cytokines(Mavropoulosetal.,2013).Specifically,wefoundDMRs inthepromoterregionofthegeneencodingMAPK13(mitogen- activatedproteinkinase13,alsocalledp38␦), andingenesup- anddownstreamofMAPK13inthep38MAPKsignalingcascade:

MAP3K4 (encoding MAPK kinase kinase4), MAP2K6 (encoding MAPKkinase6),LAT(linkerforactivationofTcells),andthep38 MAPKsubstrateATF2(activatingtranscriptionfactor2).TheJNK pathway,anotherMAPKsignalingpathwayconvergingwithp38 atATF2,wasalsorepresentedamongthehypomethylatedDMRs.

Similartothep38MAPKpathway, theJNK signalingcascade is activatedbyproinflammatorystimuli,butalsoinresponsetoTcR stimulation(Dongetal.,2002).Bothp38andJNKpathwaysare thereforeinvolvedintheactivationanddifferentiationofantigen- specificTcells,predominantlyoftheTH1type.Itispresumable, therefore,thatthesepathwaysareinvolvedinthedevelopment ofdisease-specificIFN␥producingTcellsrecognizingCYP21that havepreviouslybeendemonstratedinAADpatients(Bratlandetal., 2009;Rottembourgetal.,2010).HypomethylatedDMRswereiden- tifiedin a considerablenumber of genes encoding membersof thetumor necrosisfactor (TNF)superfamily. Among these was TNFRSF1AencodingTNF-R1,thereceptorofTNF␣.TNF␣isaclassical proinflammatorycytokinewithademonstratedroleinarangeof autoimmunediseasessuchasRA,SLE,multiplesclerosis(MS),and T1D,andalsoatargetforimmunotherapeuticinterventioninthose diseases (Aggarwal,2003).Furthermore, we foundhypomethy- latedDMRsinthegeneTNFSF14(encodinglymphotoxin-related inducibleligandthatcompetesforglycoproteinDbindingtoher- pesvirusentrymediatoronTcells,orLIGHT).LIGHTisapowerful stimulatoryfactorfor Tcellsthat maycontributetothebreach ofimmunologicaltoleranceinautoimmunediseases(Wangetal., 2001).BasedonthepivotalroleplayedbymembersoftheTNF superfamily in awide array ofautoimmune disorders, wesug- gestthatthefindingsreportedhereinindicatearolefortheTNF superfamilyalsointhepathogenesisofAAD.

It isalsoworthnoting thata considerableproportionofthe DMRsdemonstratingthequantitatively largestmethylationdif- ference betweenpatientsandcontrolsand thehighest number of probe hits, mapped promoters of genes with no previously known rolesin theimmune response or in autoimmunity; for example,GRIK5(encodingglutamatereceptor,ionotropic,kainite 5)and FBLN1(encoding fibulin1).The potentialrolesforthese factorsinAAD,ifany,arestilltobedescribed.Moreover,ofinter- estis alsothatthepromoter regionof thegeneencoding StAR (steroidogenicacuteregulatoryprotein)washypomethylatedin AADpatients. StARisessential insteroidhormone biosynthesis asthetransporterofcholesteroltotheinnermitochondrialmem- brane(PapadopoulosandMiller,2012).Anothergeneexpressedin adrenocorticalcellsfoundtobehypomethylatedinAADpatients isMC2R(melanocortinreceptor2),thereceptorforadrenocorti- cotropichormone (ACTH).Whetherthesefindings representan effectfromlong-termsteroidsupplementationintheAADpatients, orratheranintrinsicdysfunctionoftheadrenalcortexthatpredis- posesforautoimmuneadrenalitisremainanopenquestion.CYP21, againstwhichantibodiesistheprimeknownmarkerforAAD,was notfoundtobedifferentiallymethylatedinAADpatients.

0.0477 0.0452

0.045 0.0446

0.0439

0.0438 0.0434

0.0434 0.0428 0.0405 0.0394

0.0384 0.0383

0.0367 0.0367

0.034 0.034 0.0332

0.0332

0.0319 0.0319

0.0313 0.0275

0.0273 0.0273 0.0273

0.026 0.026 0.0254

0.0252

0.0242 0.0239

0.0236 0.0208

0.0168 0.0167

0.0143 0.0114

0.004

6.00 4.00 2.00

0.00 8.00 10.00 12.00 14.00

Establishment of localization [GO:0051234] (BP) Actin binding [GO:0003779] (MF) Retinoic acid metabolic process [GO:0042573] (BP) Glycerolipid metabolic process [GO:0046486] (BP) Contractile fiber part [GO:0044449] (CC) Potassium channel regulator activity [GO:0015459] (MF) Intermediate filament cytoskeleton [GO:0045111] (CC) Antiporter activity [GO:0015297] (MF) Regulation of lipid metabolic process [GO:0019216] (BP) Myofibril [GO:0030016] (CC) Transmembrane transport [GO:0055085] (BP) Intermediate filament [GO:0005882] (CC) Transport [GO:0006810] (BP) Response to ethanol [GO:0045471] (BP) Cytoskeletal part [GO:0044430] (CC) Terpenoid metabolic process [GO:0006721] (BP) Negative regulation of cell cycle process [GO:0010948] (BP) Spermatogenesis [GO:0007283] (BP) Male gamete generation [GO:0048232] (BP) Regulation of cGMP metabolic process [GO:0030823] (BP) Regulation of protein stability [GO:0031647] (BP) Serine-type endopeptidase activity [GO:0004252] (MF) Lipoprotein metabolic process [GO:0042157] (BP) Diterpenoid metabolic process [GO:0016101] (BP) Vitamin A metabolic process [GO:0006776] (BP) Retinoid metabolic process [GO:0001523] (BP) Regulation of cGMP biosynthetic process [GO:0030826] (BP) Regulation of cholesterol biosynthetic process [GO:0045540] (BP) Cytoskeleton [GO:0005856] (CC) Response to endogenous stimulus [GO:0009719] (BP) Intermediate filament-based process [GO:0045103] (BP) Response to chemical stimulus [GO:0042221] (BP) Transporter activity [GO:0005215] (MF) Vitamin metabolic process [GO:0006766] (BP) Exonuclease activity [GO:0004527] (MF) Response to organic substance [GO:0010033] (BP) Fat-soluble vitamin metabolic process [GO:0006775] (BP) Hormone metabolic process [GO:0042445] (BP) Receptor-mediated endocytosis [GO:0006898] (BP)

Fold Enrichment

0.0497

0.0494 0.0493

0.0472 0.0455

0.0439

0.0413 0.0368

0.034 0.0338 0.0335 0.033

0.0257 0.0239

0.0214 0.0185

0.0111 0.0103

0.00888 0.00703

0.00466

50 45 40 35 30 25 20 15 10 5 0 Microtubule organizing center [GO:0005815] (CC)

Melanocyte differentiation [GO:0030318] (BP) Amine binding [GO:0043176] (MF) Organelle organization [GO:0006996] (BP) Cellular macromolecule catabolic process [GO:0044265] (BP) Microtubule associated complex [GO:0005875] (CC) Cytoskeletal adaptor activity [GO:0008093] (MF) Centrosome [GO:0005813] (CC) Proteolysis [GO:0006508] (BP) Catabolic process [GO:0009056] (BP) Cytoskeleton [GO:0005856] (CC) Microtubule cytoskeleton [GO:0015630] (CC) Microtubule motor activity [GO:0003777] (MF) Protein catabolic process [GO:0030163] (BP) Macromolecule catabolic process [GO:0009057] (BP) Signal transduction [GO:0007165] (BP) Actin filament-based process [GO:0030029] (BP) Cell projection organization [GO:0030030] (BP) Actin cytoskeleton organization [GO:0030036] (BP) Cell motion [GO:0006928] (BP) Cytoskeleton organization [GO:0007010] (BP)

Fold Enrichment

A

B

Fig.3.(a)GOanalysesofgeneswithhypomethylatedDMRsintheproximalpromoterregion(−2000/+625).(b)GOanalysesofgeneswithhypermethylatedDMRsinthe proximalpromoterregion(−2000/+625).GOcategories:BP,biologicalprocesses;MF,molecularfunction;andCC,cellularcompartments.ThedifferentGOtermsaregiven totheleftfollowedbytheIDinbrackets.Thepvaluesaregivenbehindeachbar.

3.3. Geneontologyandpathwayanalyses

We nextperformeda GOanalysisonDMRslocalizedtothe proximalpromoterregion(2000/+625)asdescribedinSection2.9.

AnalyseswerecarriedoutforallDMRs(supplementaryfigureS1), andseparatelyforhypo-(Fig.3a,seeSupplementaryData,TableS3 forlistofgenesundereachGOterm)andhypermethylatedDMRs (Fig.3b,seeSupplementaryData,TableS4forlistofgenesunder

eachGOterm).NotethatfewhypermethylatedDMRswerefound inthe2000/+625region(12%;75/644).

TheGOanalysisrevealedsignificantenrichmentofDMRswithin the categoriesbiological processes (BP),molecular function (MF), andcellularcompartments(CC).Theenrichmentofhypomethylated DMRsin theBP-category terms receptor-mediatedendocytosis, transportactivity,responsetostimulusbyorganic,chemicaland endogenoussubstancesandhormonemetabolicprocesses,aswell

asintheMF-categorytermtransporteractivity(Fig.3a),probably reflectthecombinationofsteroidhormonesupplementaltherapy andthelack ofendogenous steroidhormone production inthe AADpatients.CentralgenesinseveraloftheseBParepresentin ourdataset.TheseincludeAPOE(apolipoprotein E),SCAP(sterol regulatoryelement-bindingproteincleavage-activatingprotein), andABCG1(TP-bindingcassettesubfamilyGmember1)thatare crucialforregulationofcellularuptakeandtransmembranetraf- ficofcholesterol,theprecursorofallsteroidhormones(Mahley, 1988;Nakajimaetal.,1999;Schmitzetal.,2001).Moreover,genes encodingproteinsthataredirectlyinteractingwiththeglucocorti- coidreceptorandknowntomodulatetheeffectsofglucocorticoid hormones(e.g.,PPARA,encodingperoxisomeproliferator-activated receptoralphaandSTAT5B,encodingsignaltransducerandactiva- toroftranscription5B;Bougarneetal.,2009;Wyszomierskietal., 1999)werefoundtobehypomethylated.Itiswellestablishedthat excessiveamountsof endogenous orexogenous glucocorticoids mayleaveatransientandalsostableepigeneticimprints(Khulan andDrake,2012).AstheAAD patientsinthis studyhavetaken exogenousglucocorticoidsonadailybasisforperiodsof4–40years, animpactonDNAmethylation levelswassomewhatexpected.

Infact,severalhypomethylatedDMRsidentifiedinAADpatients inthepresentstudy,localizetogenespreviouslydescribedtobe hypomethylatedinchronicobstructivepulmonarydiseasepatients treatedwithsystemicsteroids(Wanetal.,2012).Thesehitsinclude genesencoding proteinswithdiverse biological functions(e.g., SLC22A18(solutecarrierfamily22member18),SCNN1A(amiloride- sensitivesodiumchannelsubunitalpha),DYRK4(dual-specificity tyrosine-(Y)-phosphorylationregulatedkinase4),IRF7(interferon regulatoryfactor7),andAIF1(Allograftinflammatoryfactor1,an interferon-␥inducibleCa²⁺bindingEFhandprotein).Fiverelated metabolicprocesstermswithintheBP-categoryweresignificantly enriched for hypomethylated DMRs (Fig.3a). These terms, the terpenoid-,diterpenoid-,retinoid-,vitamin A-,andretinoicacid metabolicprocessesallconsistedofthesamefourgenes,namely ALDH1A2(retinaldehydedehydrogenase 2),ALDH8A1 (Aldehyde dehydrogenase8family,memberA1),CYP26C1(cytochromeP450, family26,subfamilyc,polypeptide1),andRPE65(retinalpigment epithelium-specific65kDaprotein).Thisisaninterestingobserva- tioninlightofrecentdiscoveriesregardingtheproinflammatory andimmunoregulatoryrolesofretinoicacidandvitaminA(Hall etal.,2011).

WithintheMF-category,wefoundenrichmentofhypomethy- latedDMRsinthetermexonucleaseactivity,includinggeneslike APTX(aprataxin)andRAD9(RAD9),whicharebothinvolvedinDNA damagecontrol(NiidaandNakanishi,2006;Sperkaetal.,2012;

Fig.3a).Thiscouldberelatedtotheincreasedextentofgenomic DNAdamageandkillingoflymphocytesbyreactiveoxygeninter- mediates in many autoimmune diseases (Bashir et al., 1993).

Asecond MF-termthat showedenrichmentofhypomethylated DMRswasserine-typeendopeptidaseactivity,includinggeneslike FURIN(FURIN)andKLK6(Kallikrein-6;Fig.3a).Thesefactorsare involvedincleavageofviralproteinsandcytokineprecursors,and promotionoflymphocytesurvival,respectively(Scarisbricketal., 2011;Tayetal.,2012).TheCC-categoryrevealedsignificantenrich- mentofbothhypo-andhypermethylatedDMRsingenesconnected tothe cytoskeleton (Fig.3a and b),and related terms such as microtubulecytoskeletonandintermediatefilaments(Fig.3b).We

proposethatthismightreflecttheactivationandpolarizationofT cellsandlymphocytesthattakeplaceinaTcellmediatedautoim- munediseasesuchasAAD(Filbertetal.,2012).

To determine the potential enrichment of pathways in our dataset,we usedtheKEGGfunctionalannotationtool inDAVID (asdescribed inSection 2.9).Althougha substantialnumber of thegenescontaininghypomethylatedDMRsintheirproximalpro- moterregionsareinvolvedinimmunologicalpathways,noneof these pathwayswere enriched. The only significantly enriched pathwayswerefound forhypermethylated DMRsand included melanogenesis and axon guidance(Table 1).Since the number ofgeneswithhypermethylatedDMRsineachpathwaywasonly three,andsincethepvaluesapproachedtheborderlineofsignif- icance,thisfindingshouldbeinterpretedwithcaution.However, theenrichmentofhitswithinthemelanogenesispathwayisnote- worthysinceoneofthecardinalsignsofAADishyperpigmentation of theskindue toexcessive production ofACTH (Bratland and Husebye,2011).

In thepresentstudy, weutilized DNA isolatedfromCD4+ T cellsasthestartingmaterialtoidentifyDMRs.Thispoolconsists ofdifferentsubpopulationsofcells,andinthebody,therelative abundanceofeachsubtypewilldependonvarioussignalspresent intheirmilieu.Infact,thedistributionofCD4+Tcellsubtypesisfre- quentlyalteredinautoimmunediseases(Buckner,2010).InAAD, anincreasedpercentageofactivatedTcellsisobservedinpatients withrecentonsetofdisease,andmoreover,adecreasedpercentage ofsuppressiveT_REGSisevident(Colesetal.,2005;Rabinoweetal., 1984).Thischaracteristicofautoimmunediseases,togetherwith thefactthattheDNAmethylomediffersbetweenCD4+Tsubtypes (Leeetal.,2009)canpotentiallyconfoundourresults.Forexample, inourdataset,DMRswerelocalizedtothepromoterregionsofLIF (leukemiainhibitoryfactor),PIAS3(E3SUMO-proteinligasePIAS3), and RORC(retinoicacidreceptor (RAR)-relatedorphan receptor C).Ithaspreviouslybeendemonstratedthatthesegenescanbe regulatedbymethylation,andmoreoverthesegenesaredifferen- tiallyexpressedinCD4+Tcellpopulations(Bixleretal.,2013;Cao etal.,2011;Klugeetal.,2011;Myckoetal.,2012;Santarlascietal., 2012;Shinetal.,2011;Thomasetal.,2012).Althoughthelimited numberofcellshamperedexperimentsonindividualCD4+Tcell subpopulationsatthistime,suchexperimentsshouldbepossible inthecomingyearsastheDNAmethylationtechniquesadvance andallowsatisfactoryanalysesonfewcells.Itisalreadypossible to separate most known subpopulations based on cell-specific markers (Bendall et al., 2011). Another limitation is that the assaydoesnotprovideinformationaboutwhethertheidentified DMRsare causesor consequences of the disease.Moreover,as indicated by the GO-analyses (discussed in Section 3.3), it is possiblethattheabnormalmethylationpatterninAADpatients hasdeveloped partly asa result of medication. However, such changesarealsovaluabletomonitorastheymayhinttogenomic regions and cellular pathways that are important to take into accountforupcomingproposalsforimprovedpatienttreatment andcare.Futurestudiesshouldbedesignedtodeterminewhen and where differences in methylation status arise. Important toolsfor suchanalyseswillbecohorts ofindividuals that have notyetdevelopedAAD,buthaveCYP21-Ab,andanimalmodels that allow cell tracking in combination with DNA methylation analyses.

Table1

Pathwayanalyses(bytheKEGGfunctionalannotationtoolinDAVID)ongeneswithhypermethylatedDMRsintheproximalpromoterregion(−2000/+625).

PathwayID(KEGG) Pathwayterm Genes Foldenrichment pvalue

hsa04916 Melanogenesis EDNRB,MITF,KRAS 7.337662338 0.05679425

hsa04360 Axonguidance NCK2,KRAS,PLXNC1 5.631229236 0.09016057