integrated molecular phenotyping identifies genes and ......rachael coyle1, mercedes pardo1,...

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TITLE

Integratedmolecularphenotypingidentifiesgenesandpathwaysdisruptedinosteoarthritis

AUTHORS

GrahamR.S.Ritchie1,2,3,4,TheodorosI.Roumeliotis1,JuliaSteinberg1,AbbieL.A.Binch5,5

RachaelCoyle1,MercedesPardo1,ChristineL.LeMaitre5,JyotiS.Choudhary1,J.Mark

Wilkinson6#,EleftheriaZeggini1#

AFFILIATIONS

1WellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,Hinxton,Cambridge,10

CB101SA,UK

2EuropeanMolecularBiologyLaboratory,EuropeanBioinformaticsInstitute,WellcomeTrust

GenomeCampus,Hinxton,Cambridge,CB101SD,UK

3UsherInstituteofPopulationHealthSciences&Informatics,UniversityofEdinburgh,

Edinburgh,EH164UX,UK15

4MRCInstituteofGenetics&MolecularMedicine,UniversityofEdinburgh,Edinburgh,EH4

2XU,UK

5BiomolecularSciencesResearchCentre,SheffieldHallamUniversity,Sheffield,S11WB,UK

6DepartmentofOncologyandMetabolism,UniversityofSheffield,BeechHillRoad,Sheffield

S102RX,UK20

#[email protected]@sheffield.ac.uk

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not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was. http://dx.doi.org/10.1101/038067doi: bioRxiv preprint first posted online Jan. 28, 2016;

http://dx.doi.org/10.1101/038067

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ABSTRACT

Osteoarthritis(OA)isadegenerativejointdiseasewithsubstantialglobalhealtheconomic

burdenandnocurativetherapy.Hereweinvestigategenesandpathwaysunderpinning

diseaseprogression,combiningmethylationtyping,RNAsequencingandquantitative

proteomicsinchondrocytesfrommatcheddamagedandhealthyarticularcartilagesamples5

fromOApatientsundergoingkneereplacementsurgery.Ourdatahighlight49genes

differentiallyregulatedatmultiplelevels,identifying19novelgeneswithapotentialrolein

OAprogression.Anintegratedpathwayanalysisidentifiesestablishedandemerging

biologicalprocesses.Weperformaninsilicosearchfordrugspredictedtotargetthe

differentiallyregulatedfactorsandidentifyseveralestablishedtherapeuticcompounds10

whichnowwarrantfurtherinvestigationinOA.Overallthisworkprovidesafirstintegrated

viewofthemolecularlandscapeofhumanprimarychondrocytesandoffersinsightsintothe

mechanismsofcartilagedegeneration.Theresultspointtonewtherapeuticavenues,

highlightingthetranslationalpotentialofintegratedfunctionalgenomics.Alldatafrom

theseexperimentsarefreelyavailableforaccessintheappropriaterepositories.15


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INTRODUCTION

Osteoarthritis(OA)affectsover40%ofindividualsovertheageof70(1),andisaleading

causeofpainandlossofphysicalfunction(2).ThereisnotreatmentforOA;instead,disease

managementtargetssymptomcontrolandculminatesinjointreplacementsurgery.OAisa5

complexdisease,withbothheritableandenvironmentalfactorscontributingto

susceptibility(3).Despitetheincreasingprevalence,morbidity,andeconomicimpactofthe

disease(1,2,4),theunderlyingmolecularmechanismsofOApathogenesisandprogression

remainincompletelycharacterizedandthemainstayoftreatmentforadvanceddiseaseis

stilltotaljointreplacement.Thereasonsforthelimitedsuccessinunravelingthe10

pathogenesisofOAisdue,inpart,toreductionistapproachesappliedinmodelsthatdonot

accuratelyrecapitulatetheclinicaldiseasesufferedbypatients(5).

Emergenthighthroughputtechnologiesandbioinformaticsanalysesofclinicaltissuesoffer

thepromiseofnovelfunctionalapproachestodiseasecharacterizationandtherapeutic15

targetandbiomarkerdiscoveryincomplexdiseaseslikeOA.Inrecentyears,studies

individuallyexamininggeneexpression,DNACpGmethylationandproteomicshave

expandedourunderstandingofOApathogenesis,reviewedin(6,7).OAisprimarily

characterizedbycartilagedegeneration,thoughttobebroughtaboutbyanimbalance

betweenanabolicandcatabolicprocessesthroughacomplexnetworkofproteinsincluding20

proteasesandcytokines,reviewedin(8-10).Herewereportthefirstapplicationof

integratedomics,includingDNAmethylation,RNAsequencingandquantitativeproteomics

fromjointtissuetoobtainacomprehensivemolecularportraitofdiseasedversushealthy

kneecartilageinOApatients(Figure1a).Ourdatahighlightbothfamiliarandlesswell-

establisheddiseaseprocesseswithinvolvementacrossmultipleomicslevels,andreveal25

novelcandidatemolecularplayerswiththerapeuticordiagnosticpotential.


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RESULTS

Weextractedcartilageandsubsequentlyisolatedchondrocytesfromthekneejointsof12

OApatientsundergoingtotalkneereplacementsurgery.Weobtainedtwocartilagesamples

fromeachpatient,scoredusingtheOARSIcartilageclassificationsystem(11,12):one‘high-

gradedegenerate’samplewhichweclassifiedasdiseased,andone‘normal’or‘low-grade5

degenerate’samplewhichweclassifiedashealthy(SupplementaryFigure1).Wecompared

thehealthyanddiseasedtissueacrosspatient-matchedsamples.

Quantitativeproteomics

Weusedisobariclabelingliquidchromatography-massspectrometry(LC-MS)toquantifythe10

relativeabundanceof6540proteinsthatmappedtouniquegenes.Weidentified209

proteinswithevidenceofdifferentialabundance(SupplementaryTable1);ninetywere

foundathigherabundanceinthediseasedsamples,and119werefoundatlower

abundance.Fortworepresentativepatientswealsousedanorthogonallabel-freeapproach

toconfirmtheproteinquantificationdata(SupplementaryFigure2).Thisisthemost15

comprehensivedifferentialproteomicsstudyonOAsamplestodate.Oneofthemost

stronglydown-regulatedproteinsishyaluronanandproteoglycanlinkprotein1(HAPLN1),

whichbindshyaluronicacidintheextracellularmatrix.HAPLN1wasfoundtobemore

abundantlyreleasedtotheculturemediabyOAcartilageexplantscomparedtohealthy

controltissue(13).Intra-articularinjectionofhyaluronicacidisoneofthefewcurrent20

targetedtreatmentsforOApain(14).BasedonUniProtannotation(15)wefoundanumber

ofproteoglycans,cartilageandchondrocytefunction-relatedproteinsaswellasbasement

membraneproteinsconsistentlyatlowerabundancesinthediseasedsamples

(SupplementaryTable2).Thisispotentiallyareflectionoftheincreasedcartilagecatabolism

thatoccursinOA.AsisthecaseforHAPLN1,severalofthesearefoundincreasinglyinthe25

mediareleasedbyOAtissueorsynovialfluidfromOApatients(13,16).


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Wevalidatedthehigherlevelsindiseasedcartilageoffourofthedifferentiallyabundant

proteins(ANPEP,AQP1,TGFBIandWNT5B)byWesternblotting(SupplementaryFigure6).

ANPEP(aminopeptidaseE)isabroadspecificityaminopeptidasethathaspreviouslybeen

detectedinthesynovialfluidofOApatients(17),andthereforehaspotentialasanovelOA5

biomarker.TGFBI/BGH3isanadhesionproteininducedbyTGF-βthatbindstoECMproteins,

integrinsandtoperiostin,anotherup-regulatedproteininOAwhichpromotescartilage

degenerationthroughWNTsignaling(18,19).TGFBIproteinisreleasedbyOAtissueexplants

(13)andisalsofoundinthesynovialfluidofOApatients,butismoreabundantinthatof

rheumatoidarthritispatients(16).WNT5B,aligandforfrizzledreceptorsintheWNT10

signalingpathway,haspreviouslybeenreportedtobedifferentiallytranscribedin

osteoarthriticbone(20).

RNAsequencing

WesequencedtotalRNAfromallsamplesandidentified349genesdifferentiallyexpressed15

ata5%falsediscoveryrate(FDR)(SupplementaryFigure3).Ofthese,296and53genes

demonstratedhighertranscriptionlevelsinthediseasedandhealthysamples,respectively

(SupplementaryTable3).Mostofthesegenesareannotatedasprotein-coding,butthe

shortlistalsoincludedseveralspeciesofnon-codinggenesincluding7longintergenicnon-

codingRNAgenes,3microRNAgenes,and7annotatedpseudogenes.Oneofthemost20

stronglydown-regulatedgenesinchondrocytesisolatedfromdamagedsiteswasCHRDL2,

thepresenceofwhichwasfurtherconfirmedbyimmunohistochemistry(IHC)

(SupplementaryFigure4).Thisgenewasalsofoundatlowerabundanceintheproteomics

data,andisabonemorphogeneticprotein(BMP)inhibitorthathaspreviouslybeen

reportedtobelostfromchondrocytesofthesuperficialzoneandshiftedtothemiddlezone25


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inOAcartilageinatargetedstudy(21).WealsoidentifyBMP1asup-regulatedattheRNA

level,suggestingdysregulationofbonemorphogeneticproteinpathwaysinOA.

Amongthe209proteinswithevidenceofdifferentialabundanceintheproteomicsdata,31

werealsoincludedinthesetofgenesidentifiedasdifferentiallyexpressedattheRNAlevel5

(hypergeometricp=5.3e-7,Figure1b).Ofthese,26genesshowedconcordantdirectionsof

effectbetweendiseasedandhealthysamples(binomialp=0.0002),whilethedirection

differedfor5genes(COL4A2,CXCL12,FGF10,HTRA3andWNT5B).Inallfivecasesthegene

wasfoundtobeover-expressedattheRNAlevelandlessabundantattheproteinlevelin

thediseasedtissue.UsingannotationsfromtheHumanProteinAtlas(22)wefoundthatall10

fiveproteinsencodedbythesegenesareannotatedaspredictedsecretedproteins,

suggestingtheseproteinsmightbeincreasinglyreleasedorsecretedinOA.Inagreement

withthis,severalcollagensaremoreabundantlyreleasedintotheculturemediafrom

diseasedtissuethanfromhealthytissue(13).Theseproteinshavepotentialvalueas

biomarkersofOA.15

DNAmethylation

WeusedtheIllumina450kmethylationarraytoassay~480kCpGsitesacrossthegenome.

Wefirstperformedaprobelevelanalysisandidentified9,896differentiallymethylated

probes(DMPs)at5%FDR(SupplementaryTable4).CpGmethylationisregionallycorrelated20

andwealsosoughttoidentifydifferentiallymethylatedregions(DMRs)thatmayhavemore

biologicalrelevance.Thisanalysisyielded271DMRs,associatedwith296uniqueoverlapping

genes(SupplementaryTable5).SixteenofthegeneswithanassociatedDMRwerealso

amongthelistofdifferentiallytranscribedgenes,including2membersoftheADAMTS

family:ADAMTS2andADAMTS4.Bothgenesareup-regulatedindiseasedsamplesbasedon25

theRNAsequencingdata,andwereconsistentlyassociatedwithhypo-methylatedDMRs.


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Wealsoidentifiedseveralothermembersofthisfamilytobeeithertranscriptionallyup-

regulated(ADAMTS12andADAMTS14)orassociatedwithahypo-methylatedDMR

(ADAMTS17).Wefoundnoevidencefordifferentialabundanceoftheassociatedproteins,

possiblybecausetheywouldhavebeensecretedintothematrix.Thesegenesencode

peptidasesthatcatabolisecomponentsoftheextracellularmatrix,includingaggrecan,which5

wasthemostdown-regulatedproteinindiseasedsamples.Previousstudieshaveimplicated

thisgenefamilyinOA(23)andhavesuggestedthatADAMTS4-mediatedaggrecan

degradationmaybeanimportantprocess(24).Accordingly,aggrecanismoreabundantin

OAsynovialfluid(16).

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Integrativeanalyses

Toinvestigatetheconcordancebetweenthetranscriptomeandproteomeassays,we

computedtheglobalcorrelationinallsamplesirrespectiveoftissuestatus,comparingthe

RNAfragmentsperkilobaseoftranscriptpermillionfragmentsmapped(FPKM)(25)to

normalisedpeptidespectralcounts(SupplementaryFigure5a).Wefoundasignificant15

positivecorrelation(Spearman’srho=0.29,p<2.2e-16)betweenRNAexpressionlevelsand

proteinabundance.ToestablishiftherewerealsoconcordantdifferencesinRNAand

proteinabundanceaccordingtotissuegrade,wecomputedthecorrelationsbetweenRNA

andproteinratiosbetweendiseasedandhealthysamples(Figure2a).Whenconsideringall

geneswithdataobtainedfrombothassays,weidentifiedasignificant,butsmall,positive20

correlation(Pearson’sr=0.17,p<2.2e-16).Themagnitudeofcorrelationbecame

substantiallystrongerwhenweonlyconsideredthe31genesthatwereexpressed

differentiallyinbothdatasets(Pearson’sr=0.43,p=0.01).

Toinvestigatetheconcordancebetweenthemethylomeandtranscriptomedata,we25

comparedtheaggregatemethylationstatusofpromoterregionCpGprobeswith


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transcriptionlevelsinallsamplesirrespectiveoftissuegrade(Methods,Supplementary

Table6)andfoundtheexpectednegativecorrelationbetweenpromoterregionmethylation

andgeneexpression(Spearman’srho=-0.43,p<2.2e-16,SupplementaryFigure5b).

ComparisonofthechangesinRNAexpressiontothedifferencesinpromoter-region

methylationvalueswhencomparingsamplesaccordingtotissuegradedemonstrateda5

smallbutsignificantcorrelation(Pearson’sr=-0.08,p<2.2.e-16,Figure2b).Takingonlythose

genescalledassignificantlychangingatboththemethylomeandtranscriptomelevel,the

correlationincreasedinmagnitude(Pearson’sr=-0.48,p=0.002).

Weidentified49geneswithevidenceofdifferentialregulationinchondrocytesextracted10

fromdiseasedvshealthycartilagefromatleasttwoofthethreeomicsanalyses

(SupplementaryTable7).Weidentifythreegenesconsistentlyacrossall3approaches:AQP1,

COL1A1andCLEC3B(Figure1b).All3geneswereidentifiedasup-regulatedindiseased

tissueinboththeRNA-seqandproteomicsanalyses(Figure2a),andashavingassociated

DMRs.AQP1andCOL1A1showedaconsistentdecreaseinmethylationofallCpGprobesin15

theirassociatedDMRs,commensuratewithanincreaseintranscription,whiletheDMR

associatedwithCLEC3Bshowedevidenceofincreasedmethylation(SupplementaryTable5).

UsingIHCweindependentlyconfirmedthepresenceofall3proteinswithinthe

chondrocytesincartilagesamples(SupplementaryFigure4).Thesethreegeneshave

previouslybeenimplicatedinOA.AQP1,encodingaquaporin-1,isamemberofafamilyof20

proteinsthatfacilitatewatertransportacrossbiologicalmembranes.Chondrocyteswelling

andincreasedcartilagehydrationhasbeensuggestedasanimportantmechanisminOA(26).

Accordingly,AQP1hasbeenobservedasover-expressedinaratmodelofkneeOA(27),and

thereisonepreviousreportoftranscriptionalover-expressioninkneeOAinhumans(28).

CLEC3B(alsoknownasTNA)encodestheproteintetranectin,whichbindshumantissue25

plasminogenactivator(tPA)(29).PreviousstudieshaveidentifiedCLEC3Basup-regulatedin


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humanOA(30,31),mediatingextracellularmatrixdestructionincartilageandbone(32),and

acandidategeneassociationstudyfoundevidenceofassociationofacodingvariant

(rs13963,Gly106Ser)inCLEC3BwithOA(33)(althoughthisassociationhasnotbeen

replicatedinsubsequentstudies(34)).COL1A1isoneofseveralcollagenproteins(including

COL1A2,COL3A1,COL4A2,COL5A1,Figure2a),whichweidentifiedasdifferentially5

regulatedinthediseasedsamplesusingbothRNA-seqandproteomics.Collagensarethe

mainstructuralcomponentsofcartilageandseveralstudieshavehighlightedtheimportance

ofcollagendysregulationinOA(10,35).Arecentstudyalsoidentifiedup-regulationof

COL1A1andCOL5A1insynoviumfromhumanswithend-stageOA,inthesynoviumofmice

withinducedOA,andinhumanfibroblastsstimulatedwithTGF-β(36).10

Ofthe49geneswithevidenceofdifferentialregulationonatleasttwomolecularlevels,19

genes(39%)havenotpreviouslybeenimplicatedinOA(SupplementaryTable7).Novel

geneswithconvergentevidenceincludeMAP1AandMAP1B,encodingmicrotubule-

associatedproteins,bothofwhichwereup-regulatedsignificantlyatboththeRNAand15

proteinlevels(Figure2a).Theseproteinsareexpressedmostlyinthebrainandareinvolved

inregulationoftheneuralcytoskeleton(37).Cytoskeletalregulationisthoughttobean

importantprocessinOA(38)andaccordingly,recentstudieshaveimplicatedtheseproteins

inboneformation(39).ThePXDNgenewasup-regulatedattheRNAlevelandassociated

with2hypo-methylatedDMRs.PXDNencodesperoxidasin,whichissecretedintothe20

extracellularmatrixandcatalysescollagenIVcross-linking(40).Wealsoidentify2sub-units

ofcollagenIV,COL4A1andCOL4A2,asdifferentiallyregulatedatseverallevels

(SupplementaryTable7).Althoughmanycollagenshavebeenfoundtobedifferentially

regulatedinOA,COL4A2hasnotbeenreportedpreviouslyinassociationwithOA.Collagen

IVisthemajorstructuralconstituentofbasementmembranes,butithasalsobeen25

identifiedatthearticulatingsurfaceofnormalandosteoarthriticarticularcartilage(41).


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HTRA3,up-regulatedattheRNAlevel,butfoundatlowerabundanceattheproteinlevel

possiblyduetoincreasedsecretion,isamemberoftheHTRAfamilyofserineproteinases

involvedincartilagedegradationandtissueturnover,importantprocessesinOAprogression

(42).Anothermemberofthisfamily,HTRA1,istranscriptionallyup-regulatedinOAcartilage

(43)andhasalsobeenimplicatedinthealterationofchondrocytemetabolismbydisrupting5

thepericellularmatrix(44).GeneswithrelativelylittlecharacterizationincludeCRTAC1,

encodingcartilageacidicprotein1whichissecretedbychondrocytes(45),andPODN,

encodingpodocan,whichbindscollagenintheextracellularmatrix(46).Ourcombined

epigenetic,transcriptomicandproteomicanalysishasuncoveredasubstantialnumberof

genesassociatedwithOAprogression,someofwhichhaveknownconnectionstocartilage10

orbone-relatedprocesses,andotherswithnolinks,whosedetailedcharacterizationshould

bringnewinsightsintothemolecularmechanismsofOApathogenesis.

Genesetanalyses

Weperformedagenesetenrichmentanalysisontheshortlistedgenesfromeachseparate15

omicsanalysisandfoundthatseveralcommonbiologicalprocessesarehighlightedat

multiplelevels(SupplementaryTables8&9,Methods).Toidentifypathwaysjointlyaffected

bygenesidentifiedatmultiplemolecularlevels,weusedthegeometricmeanofthethree

enrichmentp-valuesfromeachanalysis,andcalculatedanempiricalp-valueforthisstatistic

withapermutationstrategyaccountingfortheoverlap(Methods).Weidentified1920

enrichedpathwaysfromKEGG&Reactomeatacombined5%FDR,and30GOannotations

(SupplementaryTables8&9).Oftheseenrichedgenesets,7fromKEGG&Reactomeand11

fromGOcontainatleastfivesignificantgenesfromeachofatleasttwoomicsapproaches

(Figure3,SupplementaryFigure7).

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Acommonthemeinthehighlightedpathwaysiscartilagematrixregulationand

degeneration,inagreementwiththenotionthatincreasedECMturnoverisacrucial

componentinOApathogenesis.Pathwaysincluding“extracellularmatrixorganisation”and

“collagenformation”wereaffectedbygenesidentifiedbyallthreeomicsanalyses,although

thecomponentgenesdonotalloverlap(Figure3).Resultsfromthe3analysesconvergeon5

sharedmechanisms,supportingtheimportanceofutilisingevidencefromanintegrated

perspective.TheGOtermanalysisuncoveredconsistentevidencefromallthreeomics

assaysforgenesannotatedwiththeterms“extracellularmatrixdisassembly”and“collagen

catabolicprocess”.Inthesepathwayswealsofindsuggestiveevidenceofalinktogenetic

OAriskloci.ThesesignalswouldnothavebeenidentifieddirectlyfromGWASdata10

(SupplementaryResults),highlightingtheimportanceofsynthesizingdataatmultiple

molecularlevelstoobtainamorepowerfulintegratedview.

Furtherinterestingpathwaysandbiologicalprocessesenrichedatmultiplelevelswere

“positiveregulationofERK1/2cascade”,“heparin-binding”,“plateletactivation”,allofwhich15

areinterconnectedthroughcommongenes.Severalstudieshavelinkedtheextracellular

signal-regulatedkinase(ERK)cascadetoOA(8,47-50).Heparin-bindinggrowthfactorshave

alsobeenshowntobeinvolvedinOA(51-54),someinparticularthroughactivationofthe

ERKsignalingpathway.Injectionofplatelet-richplasmainOAkneesleadstosignificant

clinicalimprovement(55,56)andthereisevidencetosuggestthatthiseffectismediatedvia20

theERKcascade(57).Ourfindingsprovidestrongevidencesupportingaroleforthis

pathwayinOApathogenesis.

Wealsofoundenrichmentofgenesinvolvedintheregulationofangiogenesisatmultiple

levels.Thegrowthofbloodvesselsandnervesarecloselylinkedprocessesthatshare25

regulatorymechanisms,includingtheERKcascadeandheparin-bindingproteinsmentioned


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above(58).Accordingly,pathwayslikeNCAMsignalingforneuriteoutgrowthandPDGF

signalingthatplayasignificantroleinbloodvesselandnervoussystemformationwere

highlightedbythepathwayanalysis.ToinvestigatethisfurtherweusedtheHumanProtein

Atlastoannotatetheprotein-codinggenesidentifiedintheRNA-seqandproteomics

experiments,andfoundasignificantenrichmentinplasmaproteins(RNA-seq5

hypergeometricp=6.9e-11,proteomicsp=1.8e-5).Thissupportsaroleforangiogenesisand

nervegrowthinOAprogression(58,59).Indeed,histologicalexaminationwithinthesamples

weinvestigatedshowedgreaterbloodvesselingrowthintissueswithmoreadvancedOA

(SupplementaryFigure1).Resultsfromthethreemolecularanalysesconvergeonshared

biologicalmechanismsthatarerelevanttothepathogenesisofOA,supportingthe10

importanceofutilizingevidencefromanintegratedperspective.Thesedatashouldbeuseful

inpinpointingcandidatetargetstohelpimprovetherapeuticintervention.

InsilicoscreenfornewOAmodifyingdrugs

Themolecularsignatureshighlightedaboveprovidenovelinvestigativecandidatesas15

prognosticbiomarkersforOAandpointtonoveltherapeuticopportunities.Toidentify

existingdrugsthatcouldbeappliedtoOA,wesearchedDrugbank(60)usingthe49

differentiallyregulatedgenesidentifiedbyatleasttwoofthefunctionalgenomics

approaches.Weuncovered29compoundswithinvestigationalorestablishedactionsonthe

correspondingproteins.AfterfilteringtoincludeonlyagentswithcurrentFoodandDrug20

AdministrationMarketingAuthorizationforuseinhumans,weidentifiedtenagentswith

actionsonnineofthedysregulatedproteins(Table1).Theseagentscoverabroadrangeof

mechanismsofactionandrepresentnovelinvestigationaltargetsfor‘firstindisease’studies

ofOAprogression.Thesedrugshaveestablishedsafetyprofilesandpharmacokineticdata

foruseinman,whichwouldshortentheinvestigativepipelinetoclinicaluseinOA.Oneof25

theidentifiedgroupoftenagents,thoseactiveagainstprostacyclinsynthase(NSAIDs),


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alreadyhavemarketingauthorizationforthesymptomatictreatmentofOA.Anotherdrug

identifiedinthissearchwasphylloquinone(vitaminK1),anagonistofosteocalcin(BGLAP).

Periostin,aproteinwithelevatedexpressioninOA,inthisstudyandothers(13,18,61),isa

vitaminK-dependentproteinthatinducescartilagedegeneration(62).Interestingly,arecent

studyhasassociatedsub-clinicalvitaminKdeficiencywithkneeOAincidence(63),5

warrantingfurtherinvestigationofthiscompoundasadisease-modifyingagentinOA.Thus,

ourworkcouldhelpprioritizetherepurposingofexistingdrugsforthetreatmentofOA.


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DISCUSSION

Althoughpreviousstudieshaveindividuallyinvestigatedmethylation(64-66),transcription

(31,67),andproteinexpression(7,68)inOAtissue,theseresultsprovidethefirstintegrated,

systematicandhypothesis-freeanalysisofthebiologicalchangesinvolvedinhumanOA

progressionatallthreemolecularlevels.Usingthismulti-levelfunctionalgenomicsapproach,5

wehaveprovidedafirstintegratedviewofthemolecularalterationsthataccompany

cartilagechangesresultingindebilitatingjointdisease.Wealsohighlighttheclinical

translationimplicationsfordrugrepurposingtoslowOAprogression.Herewehavefocused

onOAanddemonstratethepotentialofmulti-omicsapproachesusingarelativelysmall

sampleset.Largersamplesizeswillberequiredforamorepowerfulcharacterisationofthe10

diseaseprogression-relatedmolecularlandscapechanges.Theintegrativefunctional

genomicsapproachillustratedhereoffersanopportunitytoidentifymolecularsignaturesin

disease-relevanttissues,therebygaininginsightsintodiseasemechanism,identifying

potentialbiomarkers,anddiscoveringdruggabletargetsforintervention.Akeyfuture

challengewillbethedevelopmentofpowerfulstatisticalapproachesfortheintegrationof15

high-dimensionalmoleculartraitsinthecontextofcomplexdiseases.Alldataarisingfrom

theexperimentsdescribedherearefreelyavailabletoresearchersintheappropriateonline

repositories.

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METHODS

Patientconsent&studyapproval

Allsubjectsprovidedwritten,informedconsentpriortoparticipationinthestudy.Tissue

sampleswerecollectedbetweenOctober2013andFebruary2014underHumanTissue

Authoritylicense12182,SheffieldMusculoskeletalBiobank,UniversityofSheffield,UK.All5

sampleswerecollectedfrompatientsundergoingtotalkneereplacementforprimary

osteoarthritis.Patientswithdiagnosisotherthanosteoarthritiswereexcludedfromthe

study.ThestudywasapprovedbyOxfordNHSRECC(10/H0606/20).

Sampleprocessing10

Extractionofchondrocytesfromosteochondraltissuetakenatkneereplacement

OsteochondralsamplesweretransportedinDulbecco'smodifiedEagle'smedium(DMEM)/F-

12(1:1)(LifeTechnologies)supplementedwith2mMglutamine(LifeTechnologies),100

U/mlpenicillin,100μg/mlstreptomycin(LifeTechnologies),2.5µg/mlamphotericinB

(Sigma-Aldrich)and50μg/mlascorbicacid(Sigma-Aldrich)(serumfreemedia).Halfofeach15

samplewastakenforchondrocyteextractionandtheremainingtissuewasfixedin10%

neutralbufferedformalin,decalcifiedinsurgipathdecalcifier(Leica)andembeddedto

paraffinwaxforhistologicalandimmunohistochemicalanalysis.Chondrocytesweredirectly

extractedfromeachpairedmacroscopiccontrolandOAgradecartilageinordertoremove

theextracellularmatrixallowingahigheryieldofcellstobeloadedontotheQiagencolumn.20

Cartilagewasremovedfromthebone,dissectedandwashedtwicein1xPBS.Tissuewas

digestedin3mg/mlcollagenasetypeI(Sigma-Aldrich)inserumfreemediaovernightat37°C

onaflatbedshaker.Theresultingcellsuspensionwaspassedthrougha70µmcellstrainer

(FisherScientific)andcentrifugedat400gfor10minutes;thecellpelletwasthenwashed25

twiceinserumfreemedia,followedbycentrifugationat400gfor10minutes.Theresulting


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cellpelletwasresuspendedinserumfreemedia.Cellswerecountedandtheviability

checkedusingtrypanblueexclusionandtheCountesscellcounter(Invitrogen).Theoptimal

cellnumberforQiagencolumnextractionfromcellsisbetween4x106and1x107.Cellswere

pelletedat400gfor10minutesandhomogenizedinQiagenRLTbuffercontainingβ-

MercaptoethanolandusingtheQIAshreddercolumnandDNA,RNAandproteinextractions5

wereperformedasoutlinedfortissueextraction.RNA,DNAandproteinwerequantified

usingaNanodrop.

Histologicalexamination

Fourmicronsectionsofparaffin-embeddedcartilagetissueweremountedontopositively10

chargedslides.SectionsweredewaxedinSub-X,rehydratedinIMS,washedindistilledwater,

stainedin1%w/vAlcianblue/glacialaceticacid(pH2.4)for15minutes,counterstainedin

1%w/vaqueousneutralredfor1minuteorstainedwithMassonTrichrome(Leica)

accordingtothemanufacturer’sinstructions.Sectionsweredehydratedandmounted.

CartilagetissuewasgradedusingtheMankinScore(0-14)withadditionalscoresfor15

abnormalfeatures(0-4)andcartilagethickness(0-4)basedontheOARSIscoringsystem

(11,12).Thetotalscoreswereusedtodeterminetheoverallgradeofthecartilageaslow-

grade(median:4.5;IOR:3-5.5;n=12)whichwedefineas‘healthy’,orhighgradedegenerate

(median:14;IOR:11.75-18;n=12),whichwedefineas‘diseased’.

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Proteomics

ProteinDigestionandTMTLabeling

Theproteincontentofeachsamplewasprecipitatedbytheadditionof30μLTCA8Mat

4°Cfor30min.Theproteinpelletswerewashedtwicewithicecoldacetoneandfinallyre-

suspendedin40μL0.1Mtriethylammoniumbicarbonate,0.05%SDSwithpulsedprobe25

sonication.ProteinconcentrationwasmeasuredwithQuickStartBradfordProteinAssay


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(Bio-Rad)accordingtomanufacturer’sinstructions.Aliquotscontaining30μgoftotalprotein

werepreparedfortrypsindigestion.Cysteinedisulfidebondswerereducedbytheaddition

of2μL50mMtris-2-carboxymethylphosphine(TCEP)followedby1hincubationinheating

blockat60°C.Cysteineresidueswereblockedbytheadditionof1μL200mMfreshly

preparedIodoacetamide(IAA)solutionand30minincubationatroomtemperatureindark.5

Trypsin(Pierce,MSgrade)solutionwasaddedatafinalconcentration70ng/μLtoeach

sampleforovernightdigestion.Afterproteolysisthepeptidesamplesweredilutedupto100

μLwith0.1MTEABbuffer.A41μLvolumeofanhydrousacetonitrilewasaddedtoeachTMT

6-plexreagent(ThermoScientific)vialandaftervortexmixingthecontentofeachTMTvial

wastransferredtoeachsampletube.Labelingreactionwasquenchedwith8μL5%10

hydroxylaminefor15minafter1hincubationatroomtemperature.Sampleswerepooled

andthemixturewasdriedwithspeedvacconcentratorandstoredat-20°Cuntilthehigh-pH

ReversePhase(RP)fractionation.

Peptidefractionation15

OfflinepeptidefractionationbasedonhighpHReversePhase(RP)chromatographywas

performedusingtheWaters,XBridgeC18column(2.1x150mm,3.5μm,120Å)onaDionex

Ultimate3000HPLCsystemequippedwithautosampler.Mobilephase(A)wascomposedof

0.1%ammoniumhydroxideandmobilephase(B)wascomposedof100%acetonitrile,0.1%

ammoniumhydroxide.TheTMTlabelledpeptidemixturewasreconstitutedin100μLmobile20

phase(A),centrifugedandinjectedforfractionation.Themulti-stepgradientelutionmethod

at0.2mL/minwasasfollows:for5minutesisocraticat5%(B),for35mingradientto35%

(B),gradientto80%(B)in5min,isocraticfor5minutesandre-equilibrationto5%(B).

Signalwasrecordedat280nmandfractionswerecollectedinatimedependentmanner

everyoneminute.ThecollectedfractionsweredriedwithSpeedVacconcentratorand25

storedat-20°CuntiltheLC-MSanalysis.


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LC-MSAnalysis

LC-MSanalysiswasperformedontheDionexUltimate3000UHPLCsystemcoupledwiththe

high-resolutionLTQOrbitrapVelosmassspectrometer(ThermoScientific).Eachpeptide

fractionwasreconstitutedin40μL0.1%formicacidandavolumeof10μLwasloadedto5

theAcclaimPepMap100,100μm×2cmC18,5μm,100Ȧtrappingcolumnwithauser

modifiedinjectionmethodat10μL/minflowrate.Thesamplewasthensubjectedtoa

multi-stepgradientelutionontheAcclaimPepMapRSLC(75μm×50cm,2μm,100Å)C18

capillarycolumn(Dionex)retrofittedtoanelectrosprayemitter(NewObjective,FS360-20-

10-N-20-C12)at45°C.Mobilephase(A)wascomposedof96%H2O,4%DMSO,0.1%formic10

acidandmobilephase(B)wascomposedof80%acetonitrile,16%H2O,4%DMSO,0.1%

formicacid.Thegradientseparationmethodatflowrate300nL/minwasasfollows:for95

mingradientto45%B,for5minupto95%B,for8minisocraticat95%B,re-equilibrationto

5%Bin2min,for10minisocraticat5%B.

15

Thetenmostabundantmultiplychargedprecursorswithin380-1500m/zwereselected

withFTmassresolutionof30,000andisolatedforHCDfragmentationwithisolationwidth

1.2Th.Normalizedcollisionenergywassetat40andtheactivationtimewas0.1msforone

microscan.TandemmassspectrawereacquiredwithFTresolutionof7,500andtargeted

precursorsweredynamicallyexcludedforfurtherisolationandactivationfor40seconds20

with10ppmmasstolerance.FTmaxiontimeforfullMSexperimentswassetat200msand

FTMSnmaxiontimewassetat100ms.TheAGCtargetvaleswere3×10e6forfullFTMSand

1×10e5forMSnFTMS.TheDMSOsignalatm/z401.922718wasusedasalockmass.

DatabaseSearchandProteinQuantification25


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TheacquiredmassspectraweresubmittedtoSequestHTsearchengineimplementedonthe

ProteomeDiscoverer1.4softwareforproteinidentificationandquantification.The

precursormasstolerancewassetat30ppmandthefragmentionmasstolerancewassetat

0.02Da.TMT6plexatN-termimus,KandCarbamidomethylatCweredefinedasstatic

modifications.DynamicmodificationsincludedoxidationofMandDeamidationofN,Q.5

Maximumtwodifferentdynamicmodificationswereallowedforeachpeptidewith

maximumtworepetitionseach.PeptideconfidencewasestimatedwiththePercolatornode.

PeptideFDRwassetat0.01andvalidationwasbasedonq-valueanddecoydatabasesearch.

AllspectraweresearchedagainstaUniProtfastafilecontaining20,190Humanreviewed

entries.TheReporterIonQuantifiernodeincludedacustomTMT6plexQuantification10

Methodwithintegrationwindowtolerance20ppmandintegrationmethodtheMost

ConfidentCentroid.Foreachidentifiedproteinanormalizedspectralcountvaluewas

calculatedforeachoneofthe6-plexexperimentsbydividingthenumberofpeptide

spectrummatches(PSMs)ofeachproteinwiththetotalnumberofPSMs.Median

normalizedspectralcountsperproteinwerecomputedacrossthedifferentmultiplex15

experiments.

Differentialabundance

Toidentifythoseproteinswithevidenceofdifferentialexpression,weshortlistedproteins

withabsolutemedianabundanceratiosbetweendiseasedandhealthysamples>=0.75,20

wherethemedianabundanceratiowasgreaterthanthestandarddeviationinallsamples

withdata,andwithevidencefromatleast5patients.Thisanalysisidentified209proteins

(SupplementaryTable1).

Westernblotting25


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Samplepairswereadjustedtothesameproteinconcentration.Twentymicrogramsof

proteinpersamplewereelectrophoresedon4-12%Bis-TrisNuPAGEgels(LifeTechnologies)

andtransferredtonitrocellulosemembranes.Primaryantibodiesusedwereasfollows:

ANPEP,ab108382;AQP1,ab168387;COL1A,ab14918;TGFB1,ab89062;WNT5B,ab124818

(Abcam);GAPDH,sc-25778(SantaCruzBiotechnologies).Chemiluminescencedetectionwas5

carriedoutusingECLPrime(GEHealthcare)orECLUltra(Lumigen)andImageQuant

LAS1400(GEHealthcare).DensitometrywasperformedwithImageQuantToolBox(GE

Healthcare).IntensityvalueswerenormalisedtoGAPDHloadingcontrolbeforeratio

calculation.

10

Labelfreequantificationofrepresentativesamples

Foraselectionoffourrepresentativecontrolanddiseasesamples,peptidealiquotsof500ng

withoutTMTlabellingwereanalysedontheDionexUltimate3000UHPLCsystemcoupled

withtheOrbitrapFusion(ThermoScientific)massspectrometerforlabelfreequantification

andvalidation.Tandemmassspectrawereacquiredoverduplicaterunsof120minwitha15

topspeediontrapdetectionmethodanddynamicexclusionat10secandMSR=120,000.

DatabasesearchwasperformedonProteomeDiscoverer1.4withtheSequestHTengineand

normalizedspectralcountswerecomputedbasedonthetotalnumberofpeptide-spectrum

matchesattributedtoeachproteinpersampledividedbythemaximumvaluealongthe

differentsamples.Withaminimumrequirementofatleasttotal14spectraperproteinwe20

foundexcellentagreementinthedirectionofchangebetweenisobariclabellingandlabel

freequantificationforatleast32proteinswhichisapproximately90%ofthecommon

proteinsbetweentheTMTchanginglistandthelabelfreeidentifiedlist(Supplementary

Figure2).

25

RNA-seq


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RNAsequencing

UsingIllumina'sTruSeqRNASamplePrepv2kits,poly-AtailedRNA(mRNA)waspurified

fromtotalRNAusinganoligodTmagneticbeadpull-down.ThemRNAwasthenfragmented

usingmetalion-catalyzedhydrolysis.Arandom-primedcDNAlibrarywasthensynthesised

andthisresultingdouble-strandcDNAwasusedastheinputtoastandardIlluminalibrary5

prep:endswererepairedwithacombinationoffill-inreactionsandexonucleaseactivitiyto

producebluntends.A-tailingwasperformed,wherebyan"A"basewasaddedtotheblunt

endsfollowedbyligationtoIlluminaPaired-endSequencingadapterscontainingunique

indexsequences,allowingsamplestobepooled.Thelibrariesthenwentthrough10cycles

ofPCRamplificationusingKAPAHifiPolymeraseratherthanthekit-suppliedIlluminaPCR10

Polymeraseduetobetterperformance.

Sampleswerequantifiedandpooledbasedonapost-PCRAgilentBioanalyzer,thenthepool

wassize-selectedusingtheLabChipXTCaliper.Themultiplexedlibrarywasthensequenced

ontheIlluminaHiSeq2000,75bppaired-endreadlength.Sequenceddatawasthen15

analysedandqualitycontrolled(QCandindividualindexedlibraryBAMfileswereproduced.

Readalignment

TheresultingreadsthatpassedQCwererealignedtotheGRCh37assemblyofthehuman

genomeusingasplice-awarealigner,bowtieversion2.2.3(69),andusingareference20

transcriptomefromEnsemblrelease75(70),usingthe–library-typeoptionfr-firststrandto

bowtie.Welimitedthealignmentstouniquelymappingreads.Wethencountedthenumber

ofreadsaligningtoeachgeneinthereferencetranscriptomeusinghtseq-countfromthe

HTSeqpackage(71)separatelyforeachsampletoproduceareadcountmatrixcountingthe

numberofreadsmappingtoeachgeneinthetranscriptomeforeachsample.Toquantify25

absolutetranscriptabundancewecomputedthefragmentsperkilobaseoftranscriptper


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millionfragmentsmapped(FPKM)(25)foreachgeneusingthetotalreadcountsfromthis

matrix,andtheexoniclengthofeachgenecalculatedfromgenemodelsfromEnsembl

release75.Weobtainedameanof49.3millionuniquelymappingreadsfromeachsample

(range:39.2-71.4million)withameanof84%ofreadsmappingtogenes(range:67.9%-

90.6%)whichwereusedforthedifferentialexpressionanalysis.5

Differentialexpressionanalysis

WeusededgeRversion3.0(72)toidentifydifferentiallyexpressedgenesfromtheread

countmatrix.Werestrictedtheanalysisto15,418geneswith>1countspermillioninat

least3samples(similartotheprotocoldescribedbyAndersetal.(73)).Wefollowedthe10

processingstepslistedinthemanual,usingageneralisedlinearmodelwithtissuestatus

(diseasedorhealthy)andindividualIDascovariates.349genesweredifferentiallyexpressed

betweenthediseasedandhealthysamplesat5%FDR(296up-,54down-regulatedin

diseasedtissue).Thegenesdifferentiallyexpressedat5%FDRhadsomewhathigherexonic

lengththantheremaininggenes(Wilcox-testp=0.00013;4804vs4153bases),hencewe15

adjustedforgenelengthintherandomisationsforgenesetanalyses.

Methylation

Illumina450kBeadChipassay

Samplesubmission:samplesweretestedforqualityandthenquantifiedto50ng/ulbythe20

onsitesamplemanagementteampriortosubmissiontotheIlluminaGenotypingpipeline.

Beforeprocessingbegins,manifestsforsubmittedsamplesareuploadedtoIlluminaLIMS

whereeachsampleplateisassignedanidentificationbatchsothatitcanbetracked

throughoutthewholeprocessthatfollows.

25


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BisulfiteConversion:BeforePre-AmplificationsampleDNArequiresbisulfiteconversion

usingtheZymoEZ-96DNAMethylationassay.ThisiscompletedmanuallyasperZymoSOP

guidelines.

Pre-Amplification:DuetothedifferencesinsampleplatesbetweenthecompletedZymo5

assayandtheIlluminaassay,pre-AmplificationisperformedmanuallyfollowingtheIllumina

MSA4SOP.Oncecomplete,sampleandreagentbarcodesarescannedthroughtheIllumina

LIMStrackingsoftware.Fourmicro-litres(200ng)ofsampleisrequired(Illuminaguidelines)

forthepre-Amplificationreaction–thereisnoquantificationstepafterthecompletionof

theZymoassay.10

Post-Amplification:Overthreedays,Post-Amplification(Fragmentation,Precipitation,

Resupension,HybrisationtobeadchipandxStaining)processesarecompletedasper

IlluminaprotocolusingfourTecanFreedomEvos.Followingthestainingprocess,BeadChips

arecoatedforprotectionanddriedcompletelyundervacuumbeforescanningcommences15

onfiveIlluminaiScans,fourofwhicharepairedwithtwoIlluminaAutloader2.Xs.

ImageBeadchip:TheiScanControlsoftwaredeterminesintensityvaluesforeachbeadtype

ontheBeadChipandcreatesdatafilesforeachchannel(.idat).Genomestudiousesthisdata

fileinconjunctionwiththebeadpoolmanifest(.bpm)toanalysisthedatafromtheassay.20

QC:Priortodownstreamanalysis,allsamplesundergoaninitialQCtoestablishhow

successfultheassayhasperformed.IntensitygraphsinGenomestudio’sControlDashboard

identifysampleperformancebymeasuringdependentandnon-dependentcontrolsthatare

manufacturedontoeachBeadChipduringproduction.25


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Probe-levelanalysis

TheintensityfilesforeachsamplewereprocessedusingtheChAMPpackage(74).Probes

mappingtochromosomesX&Y,andthosewithadetectionpvalue>0.01(n=3,064)were

excluded.Thebetavaluesforeachprobewerequantile-normalised,accountingforthe

designofthearray,usingthe‘dasen’methodfromthewateRmelonpackage(75).Wealso5

excludedanyprobeswithacommonSNP(minorallelefrequency>5%)within2basepairs

oftheCpGsite,andthosepredictedtomaptomultiplelocationsinthegenome(76)

(n=45,218),leavingatotalof425,694probesfortheprobe-leveldifferentialmethylation

analysis.Weannotatedallprobeswithgenomicposition,geneandgeniclocation

informationfromtheChAMPpackage.10

ToidentifyprobeswithevidenceofdifferentialmethylationweusedtheCpGassocpackage

(77)tofitalinearmodelateachprobe,withtissuestatusandindividualIDascovariates.

Thisanalysisyielded9,867differentiallymethylatedprobes(DMP)betweendiseasedand

healthysamplesat5%FDR.15

Toidentifydifferentiallymethylatedregions,weusedcustomsoftware(availableupon

request)toidentifyregionscontainingatleast3DMPsandnomorethan3non-significant

probeswithnomorethan1kbbetweeneachconstituentprobe,followingpreviousanalyses

(66).Weusedbedtools(78)toidentifygenesoverlappingeachDMR,usinggeneannotations20

fromEnsemblrelease75,andextendingeachgene’sboundtoinclude1500basepairs

upstreamofthetranscriptionstartsitetoincludelikelypromoterregions.Thisanalysis

yielded271DMRswithameanof4.04DMPsperregion,andameanlengthof673basepairs.

Gene-levelanalysis25


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Weassignedprobesinthepromoterregionofeachgeneusingtheprobeannotationsfrom

theChAMPpackage,andassignedtoeachgeneanyprobewiththeannotation“TSS1500”,

“TSS200”,“5’UTR”and“1stExon”inordertocaptureprobesinlikelypromoterregions.We

thencomputedthemeannormalisedbetavalueofassignedprobesforallgeneswithat

least5associatedprobesforeachsampleseparately,toproduceasinglemethylationvalue5

foreachgeneineachsample.Weusedapairedt-testtoidentifygeneswithdifferential

promoter-regionmethylationbetweendiseasedandhealthysamples,anda5%FDRcutoff

tocallagene’spromoterregionasdifferentiallymethylated.Notethatthepairedt-test

assumesanequivalentmodeltothelinearmodelusedfortheprobe-levelanalysis.

10

Immunohistochemistry

Toidentifywhethernativechondrocytesdemonstratedexpressionofthekeyfactors

immunohistochemistrywasdeployed.Fourmicronsectionsweredewaxed,rehydrated,and

endogenousperoxidaseblockedusing3%hydrogenperoxidefor30minutes.Afterwashing

sectionswithdH2O,antigenswereretrievedin0.01%w/vchymotrypsin/CaCl2(Sigma,UK),15

for30minutesat37˚C.FollowingTBSwashing,nonspecificbindingsiteswereblockedat

roomtemperaturefor2hourswitheither25%w/vgoatserumorrabbitserum(Abcam,UK)

in1%w/vbovineserumalbumin(Sigma,UK)inTBS.Sectionswereincubatedovernightat

4˚Cwitheithermousemonoclonalprimaryantibodiesorrabbitpolyclonalantibodies.

NegativecontrolsinwhichrabbitandmouseIgGs(Abcam,UK)replacedtheprimary20

antibodyatanequalproteinconcentrationwereused.SlideswerewashedinTBSanda

biotinylatedsecondaryantibodywasapplied;eithergoatanti-rabbitorrabbitanti-mouse,

bothantibodieswereappliedat1:400dilutionin1%w/vBSA/TBSfor30minutesatroom

temperature.Bindingofthesecondaryantibodywasdisclosedwithstreptavidin-biotin

complex(VectorLaboratories,UK)techniquewith0.08%v/vhydrogenperoxidein0.6525

mg/mL3,3'-diaminobenzidinetetrahydrochloride(Sigma,UK)inTBS.Sectionswere


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26

counterstainedwithMayer'shaematoxylin(Leica,UK),dehydrated,clearedandmounted

withPertex(Leica,UK).AllslideswerevisualisedusinganOlympusBX60microscopeand

imagescapturedusingadigitalcameraandsoftwareprogramQCaptureProv8.0

(MediaCybernetics,UK).

5

Proteinatlasannotation

WedownloadedannotationsfromHumanProteinAtlasversion13,andannotatedeach

protein-codinggenefromthe3experimentswiththefollowingtermstakenfromthe

annotationfile:“Predictedsecretedprotein”,“Predictedmembraneprotein”,“Plasma

protein”.Thesecretedandmembraneproteinpredictionsarebasedonaconsensuscall10

frommultiplecomputationalpredictionalgorithms,andtheplasmaproteinannotationsare

takenfromthePlasmaProteinDatabase,asdetailedinUhlenetal.(22).

IdentificationofpreviouslyreportedOAgenes

Inordertoidentifywhethersomeofthegeneswehighlighthavepreviouslybeenreported15

asassociatedwithOAwesearchedPubMedinJune2015.Weusedan“advanced”searchof

theform“(osteoarthritis)AND(<gene_name>)”where<gene_name>wassettoeachHGNC

genesymbolandwereportthenumberofcitationsreturnedforeachsearch.

Genesetanalyses20

Individualdatasets

Weaimedtotestwhetherparticularbiologicalgenesetswereenrichedamongthe

significantgenesfromeachoftheRNA-seq,methylation,andproteomicsdatasets.Tothis

end,wedownloadedKEGG(79)andReactome(80)geneannotationsfromMSigDB(version

4)(81).WealsodownloadedGeneOntology(GO)biologicalprocessandmolecularfunction25

geneannotationsfromQuickGO(82)on4February2015.ForGO,weonlyconsidered


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annotationswithevidencecodesIMP,IPI,IDA,IEP,andTAS.Genesannotatedtothesame

termweretreatedasa“pathway”.KEGG/ReactomeandGOannotationswereanalysed

separatelyandonlypathwayswith20to200geneswereconsidered(555for

KEGG/Reactome,811forGO).Enrichmentwasassessedusinga1-sidedhypergeometrictest

andonlyconsideringgeneswithannotationsfromaparticularresource.Forexample,5

amongthe15418geneswithRNAsequencingdata,4787geneshadKEGG/Reactome

annotations,and65geneswereannotatedto“extracellularmatrixannotation”inKEGG.Of

the350significantlydifferentiallyexpressedgenes,134hadKEGG/Reactomeannotations,

and12geneswereannotatedto“extracellularmatrixannotation”.Consequently,the

enrichmentof“extracellularmatrixannotation”genesamongthedifferentiallyexpressed10

geneswasassessedbycomparing12of134to65of4787genes.Multiple-testingwas

accountedforbyusinga5%FDR(separatelyforKEGG/ReactomeandGO,andforRNAseq,

methylation,andproteinexpressiondata).

Empiricalp-valuesfortheenrichmentswereobtainedfromrandomisationsaccountingfor15

overlapofsignificantgenesamongtheRNAseq,methylation,andproteinexpression

datasets(seebelow).

Integrativegenesetanalyses

WeaimedtointegratethegenesetsanalysesfortheRNAseq,methylation,andprotein20

expressiondatasets.Foreachgeneset,weaskedwhethertheassociationacrossthethree

datasets(calculatedasgeometricmeanofthep-values)washigherthanexpectedbychance.

Tothisend,weobtained1-sidedempiricalp-valuesfrom100,000setsof“randomRNAseq

genes,randommethylationgenes,andrandomproteinexpressiongenes”.The“random”

setswerechosentoconservativelymatchtheoverlapobservedamongthesignificantgenes25


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asfollows.WeperformedtherandomisationseparatelyforKEGG/ReactomeandforGO,as

weonlyconsideredgeneswithatleastoneannotationintheresource.

TojointlyconstructoneseteachofrandomRNAseqgenes,randommethylationgenes,and

randomproteinexpressiongenes,wepicked:5

1) randomgenesfortheoverlapofRNAseq,methylation,andproteinexpression

(KEGG/Reactome:2;GO:3);

2) additionalrandomgenesfortheoverlapofRNAseqandmethylation


3) additionalrandomgenesfortheoverlapofRNAseqandproteinexpression10


4) additionalrandomgenesfortheoverlapofmethylationandproteinexpression


5) additionalRNAseqrandomgenes(KEGG/Reactome:115;GO:182);

6) additionalmethylationrandomgenes(KEGG/Reactome:73;GO:102);15

7) additionalproteinexpressionrandomgenes(KEGG/Reactome:62;GO:113);

Randomgeneswerepickedtoaccountforgenelengthasfollows.Instep1,wesubdivided

allgenespresentintheRNAseq,methylation,andproteinexpressiondatainto100binsby

increasingexoniclength.Iftheoriginalsignificantgenesintheoverlaphadggenesina

particularbinb,wepickedgrandomgenesfromthatsamebin;thiswasdoneforall10020

bins.Steps2to7weredoneanalogously.

Wetestedthat100binswereenough:choosing50or200binsgaveverysimilarresults

(Pearsoncorrelation>0.99forempiricalp-valuesinallenrichmentanalyses).Wealso

confirmedthat10,000randomgenesetswereenough:repeatingtheanalysisgavevery25


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29

similarresults(Spearmancorrelation>0.99forempiricalp-valuesinallenrichmentanalyses).

Toconfirmthelowerempiricalp-values,wecarriedout100,000randomisations.

arcOGENgene-setassociationanalysis

Weaskedwhetherthe18genesetswithstrongevidenceforassociationfromthefunctional5

genomicsdata(Figure4,SupplementaryFigure7)arealsoassociatedwithOAbasedon

GWAS.Tothisend,weusedthearcOGENGWAS,primarilythe3498caseswithkneeOAand

all11009controls.WeassignedaSNPtoageneifitwaslocatedwithinthegeneboundaries

(GenomeAssemblyGRCh37).ASNPwasassignedtoagenesetifitwasassignedtooneof

thegenesinthegivenset.10

First,weaskedwhethertheaverageSNPp-valueinagenesetwaslowerthanexpectedby

chance.Weusedthegenesettestinplink,filteringindependentSNPsatr2=0.2,and10000

case-controlphenotypepermutationstoobtainempiricalone-sidedp-values.Fiveofthe18

genesetshadempiricalp-valuessignificantat5%FDR(SupplementaryTable10).Allofthese15

fivegenesetswerealsosignificantlyassociatedat5%FDRwhenconsideringall7410knee

or/andhipOAcasesand11009controlsfromarcOGEN,withsimilarresultswhenfiltering

independentSNPsatr2=0.5(datanotshown).

Second,weaskedwhethertheresultswereconfoundedbypopulationstructure.Totestthis,20

werepeatedtheanalysisaccountingforpopulationstructurebyusinglogisticregression

withthe10firstprincipalcomponentsobtainedfromEIGENSTRATwhenconsideringall7410

casesand11009controlstogetherwithHapMaprelease23afounderindividuals.Theresults

wereasabove(SupplementaryTable10).

25


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30

Third,weaskedwhetherthefivegenesetswithassociationinthefirststepwerealso

associatedcomparedtoothergenesets,inparticular,accountingforgenesetsize.We

consideredthe250genesetsfromGO,KEGG,andReactomewiththehighestnumbersof

SNPsassigned.Foreachofthefivehighlightedgenesets,wechose100genesetswiththe

closestnumbersofassignedSNPs.Atleastoneintenoftheothergenesetshadempiricalp-5

valuesaslowasthehighlightedgeneset(SupplementaryTable10).

arcOGENhypothesis-freegene-setanalysis

Wealsoaskedwhetherwewouldhaveidentifiedthe18genesetsifwehadonlyconsidered

thearcOGENkneeOAGWASdatainahypothesis-freeapproach.Weusedtwocommon10

methods–agene-basedoverrepresentationtestasanaloguetothefunctionalgenomics

work,andadirectset-basedtestasabove.

First,weaskedwhetherthegenesetshighlightedfromthefunctionalgenomicsworkare

amongthegenesetsover-representedamongthe25%geneswiththelowestp-values.We15

calculatedp-valuesforeachgeneusingplinkgenesetanalysisasabove.Nogeneset

enrichmentwassignificantat5%FDRwhenconsideringallGOgenesets,and,separately,all

KEGGandReactomegenesets.(WhenconsideringallarcOGENcasesandcontrols,oneGO

andfiveKEGG/Reactomegenesetsweresignificantat5%FDR;theydonotoverlapwithany

ofthe18genesetshighlightedfromthefunctionalgenomicsanalyses.)20

Second,weaskedwhetherthegenesetshighlightedfromthefunctionalgenomicswork

wouldhavebeenamongthesignificantresultswhenallgenesetsareanalysedforlow

averageSNPp-values.Here,weusedtheplinkgenesettestandchi-squaredSNPp-valuesas

above.OftheGOgenesets,26weresignificantat5%FDR,including“plateletactivation”25

and“ECMdisassembly”,twoofthelargestgenesetshighlightedfromthefunctional


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31

genomicswork.OftheKEGG/Reactomegenesets,52weresignificantat5%FDR,including

“signallingbyPGDF”.Allofthesethreegenesetshadq-values>0.03andwerethusnot

amongthemostsignificantgenesetsidentified.


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32

AUTHORCONTRIBUTIONS

Studydesign:EZ,JMW.Omicsdataanalysis:GRSR,TIR,JS.Samplecollection:JMW.

RNA/DNA/Proteinextraction:CLLM,ALAB.Proteomics:JSC,TIR.Histologyand

immunohistochemistry:CLLM,ALAB.Westernblotting:MP,RC.Manuscriptwriting:GRSR,

EZ,JS,TIR,JSC,MP,JMW,CLLM.5


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33

ACKNOWLEDGEMENTS

ThisworkwasfundedbytheWellcomeTrust(WT098051).TheauthorswishtothankSara

DunnandCliveBuckleforcontributiontoextractionofRNA/DNA/proteinfromchondrocyte

samples,DanielleWalkerforresearchadministration,andPei-ChienTsaiandJordanaBell

foradviceonthemethylationanalyses.Theauthorswouldliketoacknowledgethe5

contributionoftheWellcomeTrustSangerInstituteSampleManagement,IlluminaBespoke,

andGenotypingteamstothiswork.GRSRwassupportedbytheEuropeanMolecularBiology

LaboratoryandtheWellcomeTrustSangerInstitutethroughanEBI-SangerPostdoctoral

Fellowship.ThisstudyutilizedgenotypedatafromarcOGEN(http://www.arcogen.org.uk/)

fundedbyaspecialpurposegrantfromArthritisResearchUK(grant18030).Thisstudy10

makesuseofdatageneratedbytheWellcomeTrustCase-ControlConsortium(the1958

BritishBirthCohortcollectionandtheUKBloodServicesCollection).Afulllistofthe

investigatorswhocontributedtothegenerationofthedataisavailablefrom

www.wtccc.org.uk.

15


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34

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FIGURESFigure1:(a)Aschematicviewofthe3functionalgenomicsexperimentsidentifyingthenumberofgenesshortlistedforeach.(b)Venndiagramidentifyingthenumberofoverlappingshortlistedgenesfromeachindividualexperiment.5


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Figure2:(a)AcomparisonofthefoldchangesbetweenallgenesidentifiedinboththeproteomicsandRNA-seqexperiments.Eachgeneisrepresentedasasinglepoint,andthecolourcorrespondstowhetherthegeneisidentifiedasdifferentiallyexpressedusingedgeRintheRNA-seqorproteomicsexperiments,orboth.Thetrendlinesarederivedfromalinearregressionineachsubset.Positivefoldchangesindicateincreasedexpressionindiseased5

samples.(b)ComparisonofRNA-seqfoldchange,andpromoterregionmethylation.Thetrendlinesarederivedfromalinearregressionineachsubset.

10


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Figure3:SignificantgenesetenrichmentsfromKEGG/Reactome(a)andGeneOntology(b).Thecircosplotsshowenrichedgenesets,withgenesdifferentiallyregulatedinatleastoneofthemethylation,RNA-seq,orproteomicsexperiments.Linesconnectgenesthatoccurinseveralgenesets.Thethreeoutsidecirclesshowboxesforgeneswithsignificantlyhigher(black)orlower(red)methylation,gene,orproteinexpressiondata.Aredboxwithblack5

borderindicatedagenethatoverlapshyper-aswellashypo-methylatedDMRs.


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TABLESTable1:ResultsofDrugbank15(www.drugbank.ca)searchfortherapeuticcompoundswithcurrentFDAmarketingauthorizationforaclinicalindicationandapotentialroleinOAtreatment.ThemechanismsofactionandreferencesaretakenfromDrugbank.5


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integrated molecular phenotyping identifies genes and ......rachael coyle1, mercedes pardo1,...

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