HEDGEHOGSIGNALINGMODULATESCHOLESTEROLHOMEOSTASISINCHONDROCYTESANDINOSTEOARTHRITIS

by

ShabanaAmandaAli

AthesissubmittedinconformitywiththerequirementsforthedegreeofDoctorofPhilosophy

InstituteofMedicalScienceUniversityofToronto

©CopyrightbyShabanaAmandaAli2014

ii  

HEDGEHOGSIGNALINGMODULATESCHOLESTEROLHOMEOSTASISINCHONDROCYTESANDINOSTEOARTHRITIS

ShabanaAmandaAliDoctorofPhilosophy

InstituteofMedicalScienceUniversityofToronto

2014

Abstract

Osteoarthritis(OA)isacommondegenerativediseaseofthejointthatis

characterizedbydegradationandcalcificationofarticularcartilage,andsubchondral

bonechanges.Hedgehog(Hh)signalingisknowntobeactivatedinhumanandmurine

OA.SinceHhsignalingregulatesGli‐mediatedgeneexpression,weidentifiedHhtarget

genesthatareexpressedinchondrocytes.Microarrayanalyseswereperformedto

detectchangesingeneexpressionwhentheHhpathwaywasmodulatedinhumanOA

cartilagesamples.ResultsfromtheAffymetrixHumanGene1.0STmicroarraywere

analyzedfordifferentiallyexpressedgenesfromthreepatientsamples.Using

Ingenuity®Pathwayanalysis,severalgenesknowntobeinvolvedinsterolhomeostasis

werefoundtobemodulatedwithHhinhibition.WehypothesizedthatHhsignaling

regulatescholesterolbiosynthesisinchondrocytes,andthatmodulatingcholesterol

homeostasisimpactstheseverityofOA.

Toinvestigatethefunctionofcholesterolinthecartilage,micewith

chondrocyte‐specificcholesterolaccumulationweregenerated.Thiswasachievedby

excisingInsig1andInsig2,majornegativeregulatorsofcholesterolhomeostasis.Over

time,micewithchondrocyte‐specificcholesterolaccumulationexhibitedimpaired

growthofthelongbones.Withagingorsurgicallyinducedjointinstability,thesemice

iii  

developedmoresevereOAthancontrollittermates.TheyexpressedtypicalOA

markers,suchastypeXcollagen,indicatingchondrocytehypertrophyinthearticular

cartilage.GeneticmanipulationofHhsignalinginthesemicesuggeststhatHhsignaling

ismodulatingthephenotypebyregulatingsterolhomeostasis.Hhreductionandstatin

treatmentbothloweredcholesterolproductionandrescuedthephenotype,reducing

theseverityofOA.HereweidentifiednovelHhtargetgenesinchondrocyteswhich

regulateintracellularcholesterollevels,andfoundthatcholesteroldysregulationinthe

chondrocytespredisposestoOA.Thesedatasuggestthatpharmacologiccorrectionof

intra‐articularsterolimbalancecanbeusedasatreatmentforosteoarthritis.

iv  

Acknowledgements

Fortakingachanceonme,IthankmysupervisorDr.BenjaminAlman.Hisvision

sawbeyondthelimitedscientificexperienceIbroughttothelab,andhissupport

affordedmethetrainingenvironmenttoflourishasascientist.Tomypastandpresent

labmates,Iamforeverindebtedforthepatience,assistance,andinspirationprovided

tome.Fortheirscientificguidanceand/orexperimentalassistanceIthankDr.Louisa

Ho,Dr.ClaireHsu,Dr.ZhuJuan(Sue)Li,Dr.JasonRockel,Dr.SaeidAmini‐Nik,Dr.

FarasatZaman,Dr.AlvinLin,MushriqAl‐Jazrawe,HeatherWhetstone,RaymondPoon,

HenryMa,andPuviindranNadesan.Forhiscontributions,bothsignificant(helpwith

mathematicalcalculations)andtrivial(changingmyoutlookonlife),Ithankmydesk‐

mateDr.GurpreetBaht.

Iamthankfulforthescientificexpertiseofferedbymysupervisorycommittee,

Dr.ChristopherMcCulloch,Dr.KhosrowAdeli,andDr.JaneAubin.Theseindividuals

werecriticalinshapingthedirectionofthisproject.Theparticipationofmyesteemed

thesisexaminationcommittee,Dr.FrankBeier,Dr.MohitKapoor,andDr.DominicNg,is

greatlyappreciated.Forhisparticipationinbothmyreclassificationexamandmyfinal

committeemeeting,IthankDr.Chi‐ChungHui.Hisadvice,bothscientificandpersonal,

wasinstrumentaltomygraduateexperience.HeisadedicatedmentorandIam

gratefulforhisinvestmentinme.

Withoutcollaborationthisworkwouldnotbepossible.IamthankfultoDr.

KhosrowAdeli,Dr.PhilipConnelly,Dr.CarolynCumminsandtheirrespective

laboratoriesforprovidingexperimentalguidance,andtoDr.DavidBacksteinatMount

SinaiHospital(Toronto,Canada)forgrantingaccesstohumancartilagespecimens.

v  

FormakingmethepersonIamtoday,Ithankmygrandparents,myparents,my

brother,andallofmygoodfriendsalongtheway.Mygrandmotherwhoraisedme

duringtheearlyyearstaughtmemyfirst,andarguablymostimportant,lifelessons.Her

strugglewithosteoarthritismotivatedmetopursuethisfieldofresearch.Ifmysmall

contributionmakesadifference,itisforher.

Myparentsworkedtirelesslytoprovidemewithsupportofallkinds.Mymother

showedmethemeaningofstrength,ofhardwork,ofunconditionallove.Myfather

encouragedmycuriosity,gavemeasenseofworth,andinstilledinmetheimportance

ofeducation.Together,myparentscreatedalifeformethattheythemselvescouldnot

have.Despitetheroomforimprovementthatremains(ofwhichyou’realways

remindingme),IhopeIhavemadeyouproud.

MybiglittlebrotherJordanisconstantlypushingmetobebetter,leadingme

withhisownincredibleexample.Iknowthathewillalwaysbethereforme,becausehe

hasalwaysbeenthereforme.IdidthethingwhichIthoughtIcouldnotdo;timetoset

newgoals.MybestfriendNatalieisaperpetualsourceofinspiration,leadingmewith

herownincredibleexample.Sheseesthebestinme,andfirmlyremindsmewhenI

forget.Thesetwogivemetheperfectbalanceoftoughandunconditionallove,adjusted

accordinglyonadailybasis,attheirwiseandtrusteddiscretion.TothemIowemy

sanity(astatethatisconstantlychallengedingraduateschool).

Thesewordsonlybegintoexpressmygratitude.

Isharethisaccomplishmentwitheachofyou.

vi  

Contributions

UnderthesupervisionofDr.BenjaminAlman,Idesignedexperiments,

interpretedresults,andwrotethemanuscriptsresultingfromtheworkdescribedhere.

TheresultsdescribedinChapterTwo,aspartlypublishedinAnalyticalBiochemistry

(AliandAlman2012),wereacquiredindependently.TheresultsdescribedinChapters

ThreeandFour,themanuscriptforwhichisbeingpreparedforpublication,were

acquiredwiththeassistanceofthefollowingindividuals.MushriqAl‐Jazraweassisted

withhistologicalgrading,proteinblots,andstatisticalanalyses.HeatherWhetstone

assistedwithhistologicalsectioningandstaining.RaymondPoonandHenryMa

assistedwithprimaryhumanchondrocytecultureandreporterconstructexperiments.

SarahFarrandMarkNaplesfromthelaboratoryofDr.KhosrowAdeliperformed

radiotracerexperimentstoassesscholesterolbiosynthesis.Thecontributionsmadeby

theseindividualsarealsodescribedin‐text.

vii  

TableofContentsAbstract iiAcknowledgements ivContributions viListofFigures xListofTables xiiChapterOne:Introduction 1

Background

Osteoarthritis:Prevalence,Pathophysiology,Etiology,Burdenofdisease 2

Experimentalmodelsofosteoarthritis 7Articularjoints:Articularcartilage,Subchondralbone 9Articularchondrocytesandgrowthplatechondrocytes 12

Hedgehogsignalingandosteoarthritis 14Geneexpressionanalysesinosteoarthritis 17Systemicinfluencesinosteoarthritis 19

Cholesterolhomeostasis 21CholesterolhomeostasisandHedgehogsignaling 25Cholesterolhomeostasisandosteoarthritis 27

viii  

Currenttreatmentstrategiesinosteoarthritis:Pharmacologics,Lifestylechanges,Surgery,Cartilagerestoration 30

Rationale 35

Hypothesis 36

Objectives 36

ChapterTwo:Hhsignalingregulatesexpressionofcholesterolbiosynthetic genesinchondrocytes 37 Summary 38 Introduction 39 Results

OptimizedRNAextractionfromhumanosteoarthriticcartilage 41 Geneexpressionprofilinginosteoarthriticcartilage:microarrayanalyses 47IdentifyingHhtargets:cholesterolbiosyntheticgenes 52

Discussion 58 MaterialsandMethods 62ChapterThree:Hhsignalingmodulatescholesterolaccumulationinchondrocytes 67 Summary 68 Introduction 69 Results

Cholesterolaccumulationinchondrocytes:InsigDKOmice 72

ix  

Hedgehogsignalingregulatescholesterolbiosynthesis inchondrocytes 78Cholesterolaccumulationinchondrocytesimpairsgrowthofthelongbones 81 

Discussion 88 MaterialsandMethods 92ChapterFour:Cholesterolmodulationcanaltertheseverityofosteoarthritis 95 Summary 96 Introduction 97 Results

Cholesterolaccumulationinchondrocytespredisposestoosteoarthritis 99

Cholesterolinhibitionprotectsagainstosteoarthritisinmice 105

StatintreatmentreducesOAmarkersinhumancartilage 115

Discussion 118 MaterialsandMethods 122ChapterFive:Conclusionsandfutureresearch 127

FutureResearch 130Relevance 139

References 140Appendix:Microarraygenelist 155

x  

ListofFiguresFigure1.Representativetotalkneearthroplastysampleshowingfeaturesofosteoarthritis.Figure2.Schematicrepresentationofarticularchondrocytesandgrowthplatechondrocytes.Figure3.SchematicrepresentationoftheHedgehog(Hh)signalingpathway.Figure4.Schematicrepresentationofcriticalregulatorsofcholesterolhomeostasis.Figure5.RepresentativeelectropherogramscomparingRNAquality.Figure6.Hhinhibitioninhumanarticularcartilage.Figure7.Microarrayanalysis:genefiltering.Figure8.Heatmapofcholesterolhomeostaticgenes.Figure9.Real‐timePCRvalidationofcholesterolhomeostaticgenes.Figure10.WesternblotofINSIG1inhumanOA.Figure11.Insilicoanalysesofcholesterolhomeostaticgenes.Figure12.SREBF2chromatinimmunoprecipitation.Figure13.Schematicofthecholesterolbiosyntheticpathway.Figure14.WesternblotofINSIG1inInsigDKOmice.Figure15.Real‐timePCRofInsig1inmurinecartilage.Figure16.Visualizingtotallipidandsterolaccumulationinchondrocytes.Figure17.Quantifyingtotallipidandsterolaccumulationinchondrocytes.Figure18.Real‐timePCRforHhtargetgenesinmice.

xi  

Figure19.WesternblotofINSIG1inmicewithHhmodulation.Figure20.Quantifyingcholesterolbiosynthesisinchondrocytes.Figure21.GrosssizeofInsigDKOmice.Figure22.BonelengthinInsigDKOmice.Figure23.GrowthplateanalysesinInsigDKOmice.Figure24.SecondaryossificationcentredevelopmentinInsigDKOmice.Figure25.SkeletalanalysesinInsigDKOmice.Figure26.HistologicanalysesofInsigDKOcartilage.Figure27.TypeXcollagenstainingofInsigDKOcartilage.Figure28.RadiographsofInsigDKOknees.Figure29.Real‐timePCRofOAmarkersinInsigDKOcartilage.Figure30.Verificationofstatintreatmenteffectivenessinthecartilage.Figure31.Serumcholesterollevelfollowingstatintreatment.Figure32.StatintreatmentinControl,Col2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKOmice.Figure33.Statintreatmentinmicewithsurgically‐inducedOA.Figure34.TypeXcollagenstainingofCol2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKOcartilage.Figure35.TypeXcollagenstainingofcartilageaftersurgically‐inducedOA. Figure36.Real‐timePCRofOAmarkersafterstatintreatment.Figure37.ADAMTS5reporterconstructassay.Figure38.ADAMTS5promoteranalysis.Figure39.Workingmodel:cholesterolhomeostasismediatesHedgehogsignalinginosteoarthritis.

xii  

ListofTablesTable1.ComparingRNAobtainedfromdifferentextractionmethods.Table2.ComparinggeneexpressionresultsusingRNAobtainedfromdifferentextractionmethods.Table3.Microarrayanalysis:top20genechanges.Table4.IngenuityPathwayAnalysisofmicroarrayresults.Table5.ICRSScoringofGli2+/‐,InsigDKO,andGli2+/‐;InsigDKOcartilage.Table6.ICRSScoringofCol2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKOcartilage.Table7.ICRSScoringofcartilageaftersurgically‐inducedOA.

1  

ChapterOne:

Introduction

2  

BackgroundOsteoarthritis(OA)

Prevalence

Currently,10%ofmenand18%ofwomenover60yearsofagedevelop

osteoarthritis.Musculoskeletaldisorders,ofwhichOAisthethirdmostprevalent,are

thesecondlargestcontributorstoyearslivedwithdisability(Vosetal.2012).In

Canada,asmanyas1in8experiencesymptomsofthisdisease(Bombardieretal.

2011),andtheprevalencewillriseasthepopulationages(Felsonetal.1987).By2040,

itisexpectedthatalmost30%oftheCanadianlabourforcewillhavesymptomatic

osteoarthritis(Bombardieretal.2011).Thecostassociatedwithtreatment

interventionsandlostproductivitycreatesaneconomicburden(WoolfandPfleger

2003).

Riskfactorsforthisdiseasecanbeclassifiedintosystemicfactorsand

biomechanicalfactors(Felsonetal.2000).Systemicfactorsincludeage,sex,ethnicity,

bonedensity,genetics,weight,menstrualhistory,andnutrition.Localbiomechanical

factorsincludejointloading,injuryortrauma,jointdeformity,physicalactivity,and

muscleweakness.Despitebeingthemostcommondegenerativediseaseofthejoints,

thereiscurrentlynocureandonlylimitedtreatmentoptionsforosteoarthritis(Felson

etal.2000;NeogiandZhang2013)(seeCurrenttreatmentstrategiesin

osteoarthritis).

3  

Pathophysiology

Osteoarthritisprimarilyaffectsthearticularcartilageliningthejointsandthe

subchondralbone(Poole1999).ThemainfeatureofOAisdegradationanderosionof

thearticularcartilage.Chondrocytesarethecellsresponsibleforbuildingand

maintainingthecartilagematrix.DuringOA,thearticularchondrocytesundergo

changeswhichcontributetodiseasepathogenesis(SandellandAigner2001).The

structureofthematrixchangeswithalterationstochondrocytedistributionandmatrix

components(Mainil‐Varletetal.2003).Thecartilagebecomescalcifiedandfibrillated,

withdegradationcommonlybeginningatafocallesionandprogressingoutward

(Figure1).Inflammationofthesynovialjointcanalsooccur,butisusuallylocaltothe

joint(Poole1999).

AsOAseverityincreases,thecartilagebecomescompletelyeroded,exposing

subchondralbone.Changestothesubchondralboneincludeirregularityofthecontour,

increasesinboneremodeling,andsclerosis(increaseddensity/hardeningofthebone)

(Sanchezetal.2005).Osteophytes,orbonespurs,alsodevelopandarethoughttobean

attempttorestorestabilitytothejoint(KellgrenandLawrence1957)(Figure1).The

resultingdiscomfort,pain,stiffness,andswellingrestrictphysicalactivityforthose

afflicted(Felsonetal.2000).

Anyarticulatingjointcanbeaffectedbyosteoarthritis,especiallyfollowingan

injury.Themostcommonlyaffectedjointsincludetheknees,hips,hands,andspine,but

otherjointssuchastheshoulderscanalsobeaffected(Hassettetal.2006;Hunterand

Eckstein2009).Osteoarthritisofthekneeaccountsfor83%ofthetotalosteoarthritis

4  

burden,affecting250millionpeopleglobally,andalmosttwiceasmanywomenasmen

(Vosetal.2012).

Figure1.Representativetotalkneearthroplastysampleshowingfeaturesof

osteoarthritis.Typicalfeaturesofosteoarthritisincludecartilagefibrillation,cartilage

erosion,andosteophyte(bonespur)formation.Progressionofthisdiseasetypically

correlateswithdegenerationofthemenisci.Themedialsidetendstobemoreseverely

affected.

5  

Etiology Becauseweight‐bearingjointsaremostsusceptible,osteoarthritishasbeen

framedasamechanicaldiseasewhichresultsfromload,wear‐and‐tear(withaging),

and/orinstability(fromtrauma).Featuressuchasflatteningofthemeniscusinknee

joints(Figure1)supportthisnotion,butmechanicalfactorsalonecannotexplainthe

originsofthisdiseasesincenon‐weight‐bearingjointsarealsoaffected(Hassettetal.

2006).OAcanbeclassifiedasidiopathic,orprimaryOA,whenthecausesareunknown,

orsecondaryOA,whenthecausesareknown(Felsonetal.2000).

Inadditiontomechanicalfactors,otherknowncontributorstoOAinclude

mutationswhichcompromisejointarchitectureorcartilageintegrity(Y.Lietal.2007).

Regardingjointarchitecture,mutationswhichresultinchondrodysplasia

(compromisedjointandbonedevelopment)havebeenshowntocauseprematureOA

(PattersonandDealy2014).Regardingcartilageintegrity,mutationstotypeIIcollagen,

themajorstructuralcomponentoftheextracellularmatrixofcartilage,canleadto

osteoarthritis(Ala‐Kokkoetal.1990;Sahlmanetal.2004).Asidefromthesestructural

contributors,anemergingbodyofevidencesuggeststhatperturbationstosystemic

factorsmayalsocontributetoOApathogenesis.Regardlessofthecauseof

osteoarthritis,thebody’sinabilitytoregeneratecartilageleadstoprogressionin

severitywithage(Sudoetal.2008).Themulti‐factorialetiologyofOAnecessitates

carefulexperimentalmodelingforscientificstudyofthedisease.

6  

Burdenofdisease Thediseaseburdencreatedbyosteoarthritiscanbecategorizedintothree

groups:directcosts,indirectcosts,andintangiblecosts(Hunteretal.2014).Thedirect

costsaretheobviouscostsassociatedwithtreatmentinterventionssuchassurgery,

pharmacologictreatment,andnon‐pharmacologictreatment.BecauseOAisachronic

disease,therearedirectcostsassociatedwithhealthcareprovision,adverseeffectsof

treatment(suchasrevisionsrequiredfollowingtotaljointreplacement),andlong‐term

care(seeCurrenttreatmentstrategiesinosteoarthritis).

EasilyoverlookedaretheindirectcostsassociatedwithOA.Thesetypically

relatetolostorreducedproductivityintheworkforceorathome.Regardingthe

workforce,sufferersofOAcanexperiencereducedemploymentduetotheirlimited

physicalcapabilities,increasedabsenteeism,andearlyretirement(Hunteretal.2014).

Regardinghouseholdresponsibilities,sufferersofOAmayrequireassistancewithdaily

tasksfromacaregiver.Prematuredeathbecomesariskduetoavarietyofcontributing

factors,includingtheramificationsofthesedentarylifestylethatisfrequentlyadopted

(seeCurrenttreatmentstrategiesinosteoarthritis).

Reductioninsocialparticipationbeginstocrossoverfromtheindirectcoststo

theintangiblecostsassociatedwithOA.Thepainandfatiguethatiscommonly

experiencedlimitsactivity,includingsocialoutings,anddecreasesqualityoflife

(Hunteretal.2014).Thepsychosocialtollofchronicpainandlimitedphysicalabilityis

difficulttocapture.Mobilitycanbeequatedwithfreedomandindependence,so

sufferersofOA(aswellastheirsocialnetwork)arefacedwiththeaddedmentaland

emotionalburdenwhichresultsfromlimitedmobilityandinevitabledependence.

7  

Experimentalmodelsofosteoarthritis TostudyOA,manyexperimentalmodelsareavailable,eachofferingunique

advantagesincapturingthedisease.Mousemodelsareperhapsthemostwidelyused,

withgenetic,mechanical,age‐related,andsystemically‐inducedOAallbeingreported.

HarnessingtypeIIcollagenasatissue‐specificdriverforalterationstochondrocyte

geneexpressionhasproventobeapowerfultoolforinvestigatinggenefunctioninthe

cartilage(Grantetal.2000;GroverandRoughley2006).MicewithmutationsintypeII

collagenitself,suchastheCol2a1‐sedcmouse,developseveralcartilagedefects,

includingOA(Donahueetal.2003).

MorethanonesurgicalmodelofOAhasbeendescribed,withstudiescomparing

theresultingseverityfromdifferentsurgicalprocedures(Kamekuraetal.2005).

Whetherthemeniscusorvariousligamentsaretransected,theresultismechanical

instabilityinthejointwhichprovidesamodeloftrauma‐orinjury‐inducedOA(Janusz

etal.2002).Tocapturespontaneousage‐relatedOA,researchershaveallowedmiceto

reach12monthsofage,andeven18monthsinsomecases(Miyakietal.2010).A12‐

month‐oldmouseismiddle‐aged,estimatedtobeequivalenttoa42‐year‐oldhuman

(Flurkeyetal.2007).Thisfacilitatesquestionsrelatedtodiseasedevelopment;

capturingbothprogressivechanges(beginninginmiddleage,representativeofearly‐

stagedisease)andcumulativechanges(occurringinolderage,representativeoflate‐

stagedisease).Using6‐month‐oldmicecanproveadvantageousforinvestigating

preventativestrategiestoinhibitjointchangesandcartilagedegradationinearlier

stagesofOA.

8  

MousemodelsofOAcontinuetoevolve,withsystemicmodelsdescribing

cytokine‐anddiet‐inducedOA(Griffinetal.2010;Seeuwsetal.2010).Ingeneral,mice

areadvantageousbecausetheyarerelativelyinexpensive,theyareamenabletogenetic

manipulation(GroverandRoughley2006),andtheirkneeanatomyresemblesthatof

othermammals(Glassonetal.2010).However,giventhedifferences,suchasthefact

thatmiceareapproximately2500‐foldlighterinweightthanhumans(Glassonetal.

2010),alternativemodelshavebeendevelopedtostudyOA.Rats,guineapigs,rabbits,

horses,andotherlargeranimalshavebeenused(vandenBerg2008).

Themostclinicallyrelevantmodelisofferedbyhumanosteoarthriticcartilage

itself,whichisavailablefromtotaljointreplacementsurgeries(seeCurrenttreatment

strategiesinosteoarthritis).Primaryhumancartilagecanbetreatedasexplantsto

maintaintheextracellularenvironmentofthechondrocyte.Thisoffersamajor

advantageovercellculture,sincechondrocyteshavebeenshowntolosetheir

phenotypewhenculturedforextendedperiodsoftime(vonderMarketal.1977).

Explantsareasystemforinvestigatingwhetherexperimentalfindingsfromanimal

studiesaresustainedinhumansamples.Administeringpharmacologicstohuman

samplesinvitrofacilitatestranslationoffindings.Doseresponses,toxicity,andefficacy

ofpharmacologicscanallbequeriedsafelyandinexpensivelypriortoclinicaltrials.

Usedtogether,mousemodelsofOAandhumanOAsamplesarecomplimentaryfor

robusttestingofhypotheses.

9  

Articularjoints

Articularcartilage Articularchondrocytesareresponsibleforproducingandmaintainingthe

extracellularmatrixthatcomprisesthearticularcartilage.Thefunctionofthearticular

cartilageistolinetheendsoftheboneswhichmakeupthejoints,suchthatthejoints

canarticulateinafrictionlessmannerandabsorbimpact.Articularcartilageconsistsof

threedistinctzonesthatareidentifiablebyshapeoftheresidentchondrocytesandby

orientationofthetypeIIcollagenfibres(Figure2).Thesuperficialzone(ZoneI)

containsflattenedchondrocyteswithdense,horizontalcollagenfibrilsandminimal

proteoglycans.Themiddlezone(ZoneII)containsroundedchondrocyteswithcrossed,

intersectingcollagenfibrilsandmoreproteoglycans.Thedeepradialzone(ZoneIII)

containssphericalchondrocyteswithradialcollagenfibrilsandabundant

proteoglycans.Atidemarkcanbeobservedmicroscopicallyanddelineatesthe

transitionfromthelowerradialzonethatisun‐calcified(ZoneIV)totheunderlying

zoneofcalcifiedcartilage(ZoneV)(Lyonsetal.2006;Mainil‐Varletetal.2003).

10  

Figure2.Schematicrepresentationofarticularchondrocytesandgrowthplate

chondrocytes.Thearticularcartilageisdividedintoseveralzoneswhichcanbe

identifiedbyarticularchondrocyteshapeandtypeIIcollagenfibreorientation.The

growthplatechondrocytesundergoacontrolledprocessofdifferentiationfromresting,

toproliferating,topre‐hypertrophic,tohypertrophic.Thisenableselongationofthe

longbones.

11  

Subchondralbone BeneathZoneVliesthesubchondralbonewhichhasbeenshowntoundergoOA‐

associatedchanges.Theseincludeirregularityofthecontour,increasedremodeling,

sclerosis,necrosis,andfracture(Mainil‐Varletetal.2003).Hypotheseshavebeenput

forthtosuggestthatsubchondralbonealterationscanimpactarticularcartilage(R.

Zhangetal.2012).Forexample,changestomechanicalpropertiessuchasthestiffness

ofsubchondralbonemayreduceshockabsorptionandpromotecartilagedegeneration

(Muraokaetal.2007).Invitroexperimentsshowthatco‐cultureofsubchondral

osteoblastscandecreasegeneexpressionofchondrogenicmarkerssuchastypeII

collageninchondrocytes.Thesimultaneousshifttowardshypertrophy(asseeninOA)

suggeststhatthesubchondralbonemayplayaroleinthatshift(Sanchezetal.2005).A

recentstudyreportedthatkeyOA‐associatedgenesshowsimilarchangesinthe

subchondralboneastheydointhearticularcartilage(Chouetal.2013).Itremainsto

bedeterminedwhetherchangestothesubchondralboneprecedeorresultfrom

changestothearticularcartilage;nevertheless,thesereportssuggestthatOAshouldbe

consideredatotaljointdiseasewhichaffectsmultipletissues.

12  

Articularchondrocytesandgrowthplatechondrocytes Inosteoarthritis,thechondrocytesinthearticularcartilageundergo

characteristicchangeswhichrecapitulatethedifferentiationprocessofthe

chondrocytesinthegrowthplatecartilage(Tchetinaetal.2005).Normalbone

developmentbeginswithcondensationofmesenchymalcells.Endochondral

ossificationproceedsunderthecontrolofsignalingpathwayswhichdirectthecellsto

becomechondrocytes.ThechondrocytesproliferateandproduceamatrixrichintypeII

collagenandaggrecan(Grantetal.2000).Asubsetofchondrocytesthenbecomes

hypertrophic,producingamatrixrichintypeXcollagen.Thesehypertrophic

chondrocytes,whicharealsofoundinthearticularcartilageduringosteoarthritis,

regulatematrixmineralization.Bloodvesselsenterthedevelopingstructure,bringing

withthembone‐formingcells.Hypertrophicchondrocytesthenundergocelldeath,

leavingbehindamatrixonwhichbonecanbeformed(Kronenberg2003).

Throughthecontrolledprocessofendochondralossification,thegrowthplate

architectureisestablishedfortheprimaryandsecondaryossificationcentres(Figure

2).Restingchondrocytestransitionintoproliferatingchondrocytes,whichchangefrom

aroundshapetoaflatshape,andorganizeintocolumns.Pre‐hypertrophic

chondrocytesmodifytheirexpressiontobecomehypertrophicandarereplacedby

boneasdescribedabove(Figure2).Developmentalsignalingpathways,suchasthe

Hedgehogpathway,tightlyregulatethisprocessanddeterminetherateofgrowthof

thelongbones(Goldringetal.2006;ProvotandSchipani2005).

Severalofthephenotypicchangeswhichoccurinthegrowthplatechondrocytes

areobservedinarticularchondrocytesduringosteoarthritisdiseaseprogression.

13  

Recapitulatingthebehaviorofgrowthplatechondrocytes,articularchondrocytesinOA

proliferate(becomeclonal),becomehypertrophic,andformbone(osteophytes).The

proliferationthatreplenishesthechondrocytepoolinthegrowthplateishypothesized

tobemimickedbythechondrocyteclonalitythatisseeninOAarticularchondrocytes

(SandellandAdler1999).Articularchondrocyteshavebeenshowntoadaptananabolic

profileduringOA,secretingmatrixinanattempttorebuildandcompensateforthe

degradationthatoccurs.Thisisashiftawayfromthenormalrestingphenotypeof

articularchondrocytes(Kruegeletal.2008).Chondrocytehypertrophyisahallmarkof

osteoarthritis,wherechondrocytesbecomeenlargedandbeginexpressingtypeX

collagen,exactlyastheydointhegrowthplate(Girkontaiteetal.1996).Finally,the

entireprocessofendochondralossificationhasbeenhypothesizedtoberecapitulated

inosteophyteformation(SandellandAigner2001).

ThesimilaritiesthatcanbedrawnbetweengrowthplatechondrocytesandOA

articularchondrocytessuggestthattheremaybeacommonbiologybehindthe

controlledchangesthatoccurinbothpopulationsofcells.Insupportofthis,markers

thataretypicallyfoundindifferentiatinggrowthplatechondrocytesbecomeexpressed

inthearticularcartilageduringOAprogression(Pfanderetal.2001).Thissuggeststhat

thegeneticprofileofarticularchondrocytesshiftstowardsthatofgrowthplate

chondrocytes(Tchetinaetal.2005).Signalingpathways,suchastheHedgehog

pathway,whichgoverngrowthplatedevelopment,alsobecomeactivatedinOA.As

such,thesetwopopulationsofchondrocytesareinextricablylinked,andinformationon

thebiologyofthegrowthplatecanbeusedtoinformhypothesesonthebiologyofOA.

14  

Hedgehogsignalingandosteoarthritis

PreviousdatahaveshownthatmodulationoftheHedgehog(Hh)signaling

pathwayimpactstheseverityofosteoarthritis(Linetal.2009;J.Zhouetal.2014).Of

thethreeextracellularHhligands,Indianhedgehog(Ihh)hasbeenshowntoregulate

chondrocyteproliferationandhypertrophyinthegrowthplateofthelongbones(Lai

andMitchell2005;Vortkampetal.1996).TheHhsignalingcascadebeginswith

extracellularHhligandbindingtothetransmembranereceptorPtch1(Figure3).This

relievesinhibitionofSmo,asecondtransmembraneprotein.Throughamechanismthat

isincompletelyelucidated,SmogovernsprocessingoftheGlitranscriptionfactors

(RockelandAlman2011).Invertebrates,HhsignalingismediatedbythreeGli

transcriptionfactors:whileGli1isnotessentialfordevelopment(H.L.Parketal.2000),

Gli2isthoughttobethemaintranscriptionalactivatorandGli3themain

transcriptionalrepressor(JiangandHui2008;RuiziAltaba1999).Thesetranscription

factorsregulatetheexpressionofHhtargetgenes,includingGli1,Ptch1,andHhip

(McMahon2000;Wangetal.2007)(Figure3).

AberrantactivationofHedgehogsignalinginarticularchondrocytescausesthem

toundergophenotypicchangesthatresemblegrowthplatechondrocytedifferentiation

(Aigneretal.2007;Maketal.2008).Forexample,changessuchaschondrocyte

hypertrophyareobservedinOA(seeArticularchondrocytesandgrowthplate

chondrocytes).Basedonthesimilaritiesinchondrocytephenotypeinthegrowthplate

andarticularcartilageduringOA,Linetal.hypothesizedandultimatelydemonstrated

thatHhsignalingwasinvolvedinmediatingOApathogenesis(Linetal.2009).

15  

Figure3.SchematicrepresentationoftheHedgehog(Hh)signalingpathway.In

theabsenceofHhligand,thetransmembranereceptorPtchrepressesasecond

transmembranereceptorSmo,resultinginpathwayinhibition.InthepresenceofHh

ligand,repressionofSmobyPtchisrelieved,andtheGlitranscriptionfactorsbecome

active,resultinginpathwayactivationandupregulationoftargetgenes.PM=plasma

membrane.

16  

WhenOAissurgicallyinducedinmicebymedialmeniscectomy,theHhpathway

becomesactivatedinthearticularcartilage.AnincreaseintheexpressionofOA‐

associatedgenesAdamts5,Col10a1,andMmp13,isaccompaniedbyHhactivation.In

thismodel,HhpathwayblockadereducestheseverityofOA,suggestingthatthis

pathwayismediatingthephenotype(J.Zhouetal.2014).Ingeneticallymodifiedmice

withchondrocyte‐specificHhactivation,inwhichGli2isexpressedundertypeII

collagen(Col2a1)regulatoryelements(Col2a1‐Gli2),anOAphenotypeisobservedby6

months(Hopyanetal.2002).Thisphenotypeincludestypicalradiographicand

histologicfeaturesofOAinthekneesofmutantscomparedtowildtypelittermates.In

humanarticularcartilage,OAseveritycorrelateswithHhpathwayactivation.Severity

isevidencedbyupregulatedexpressionoftypicalOAmarkers,ADAMTS5,COL10A1,and

MMP13,andaccompanyingHhactivationisdemonstratedbyanincreaseinexpression

oftheHhtargetsPTCH1,GLI1,andHHIP(Linetal.2009).

Takentogether,theevidencefrommouseandhumanstudiesindicatethat

activatedHhsignalingpredisposestoOA.Theapplicationsofthisareevidencedin

existingpatentswhichdescribetheuseofIhhasamarkertopredictOAandtheuseof

HhinhibitorstopreventOA(Bumcrot2005;L.Wei2012).Despitethis,themechanisms

bywhichHhsignalingresultsinOAremainunknown.

17  

Geneexpressionanalysesinosteoarthritis Geneexpressionanalysescanbeusedtoimproveourunderstandingofspecific

pathwaysandsystemswithinmulti‐factorialpathologiessuchasOA.Withtechnologies

suchasreal‐timepolymerasechainreaction(PCR),microarray,andsequencing,our

abilitytoassessandvalidategeneexpressionchangesinahigh‐throughputmanneris

improving(Lovenetal.2012).Theutilityofgeneexpressiondataisfar‐reaching,asit

canbeusedtoidentifymutations,dysregulatedbiologicalnetworksorsignaling

pathways,andothergeneticsignaturesinnormalanddiseasedtissues.

Identifyingperturbationstogeneexpressionpatternsinthecontextofdisease

canelucidateunderlyingcausesandilluminatetargetsfortreatment.Microarray

analyseshavebeenconductedtocomparenormalandosteoarthriticsamplessuchas

thecartilage(Aigneretal.2006;Karlssonetal.2010),subchondralbone(R.Zhangetal.

2012),synovium(Katoetal.2007),andblood(Ramosetal.2013),butnotwithout

limitations(Lovenetal.2012).Theinputandoutputofgeneexpressionexperiments

mustbecarefullyconsideredtomaximizetheutilityoftheresults,theinputbeinghigh

qualityRNA,andtheoutputbeingfinaloutcomesofdownstreamanalysis.

Geneexpressionprofilesofthearticularcartilagearecommonlyprobed,but

articularcartilagepresentsspecificchallengestotheisolationofhigh‐qualityRNAthat

issufficientforexperimentation.ArticularcartilageconsistsprimarilyoftypeII

collagenandtheproteoglycanaggrecan(CarneyandMuir1988;RoughleyandLee

1994).Itisalargelyacellulartissue,containingonly1%to2%articularchondrocytes.

Becausetheproteoglycanscarryanegativecharge,theytendtoco‐purifywithRNA,

andbecausethenumberofcellsislimited,thereisasmallamountofRNApergramof

18  

tissue.WithoutproperRNAisolationtechniques,thereisariskoflow‐qualityRNA

beingusedtoconductgeneexpressionexperiments,theresultsofwhichmaybe

inaccurateandmisleading(Perez‐Novoetal.2005).

Theresultsthatareobtainedfromhigh‐throughputgeneexpressionscreening

canbeoverwhelminganddifficulttointerpret.Thevalueofglobalgeneassessmentcan

belostifanalysesaretoobroadortoofocused,ifdataarenotnormalizedcorrectly,if

resultsarenotverifiableinindependentsamples,andsoon.Resultsfromexpression

experimentsaretypicallyfiltereddowntoalistofgeneswhichshowchangesbasedon

criteriasetbytheinvestigator.Becausethislistcancontainhundredsofgenes,itis

commontochooseonegene(orasmallsubsetofgenes)asthefocusofsubsequent

analyses(Ijirietal.2008;H.Zhangetal.2002).Unfortunatelythisstrategyfailsto

capturetheglobaloverviewthatistheadvantageofhigh‐throughputexperiments.

Withevolvingtechnologiesforassessinggeneexpressioncomenewplatforms

foranalyzingandinterpretingresults(Downey2006).Ifgenelistsarecategorized

functionallyintobiologicalnetworksorsignalingpathways,thenthehigh‐throughput

valueismaintained(Lovenetal.2012),investigatorshaveafunctionalnetworkor

pathwaywithwhichtoconductsubsequentanalyses(Olexetal.2014),andtargeted

treatmentforpathologiescanbepursued.

19  

Systemicinfluencesinosteoarthritis OAhasbeendescribedasametabolicdiseasewherebysystemicfactors(suchas

lipids,inflammatorymolecules,neuroendocrinefactors)impactskeletaltissue

developmentandintegrity.Disruptiontocartilageintegrityleavesitsusceptibleto

secondaryfactorswhichcaninducecartilagedegradationandothertypicalfeaturesof

OA(Aspdenetal.2001).FramingOAasatotaljointdiseasewithsystemiccontributors

offersanexplanationastowhyOAaffectsnon‐weight‐bearingjointssuchasthehands.

Jungmannetal.examinefourmetabolicriskfactors,includinghighabdominal

circumference,hypertension,highfatconsumption,andself‐reporteddiabetesmellitus,

inthecontextofOA.Atbaseline,theauthorsreporteachoftheseriskfactorstobe

independentlyassociatedwithcartilagedegeneration,butthisassociationwaslost

afterthetwo‐yearfollow‐upperiod(Jungmannetal.2013).Othergroupshave

describedanassociationbetweenobesityandOA,notonlyfortheincreasein

mechanicalloadtothejoints,butalsofortheincreaseinfactorsthatinduce

inflammation(Karvonen‐Gutierrezetal.2013).Itisthoughtthatexcessiveadipose

tissueservesasasourceofinflammatoryfactorswhichcaninducecartilage

degradation(Griffinetal.2010;IssaandGriffin2012).Theseresultspointtoa

relationshipbetweensystemicmetabolismandOA,butthemechanismandcausality

remainunclear.Thisproblemiscommonwithepidemiologicalstudies(Al‐Arfaj2003;

Sturmeretal.1998).

Studieswhichexaminemetabolicfactorstendtoassayserumlevelsofmarkers

suchascytokinesorsterols,whileneglectingtoexamineintra‐articularlevels(Al‐Arfaj

2003;Griffinetal.2010).Giermanetal.reportthatahighcholesteroldietinmice

20  

predisposestoOAdevelopment,buttheyreportonlyonserumcholesterolandofferno

mechanisticexplanationastowhysomedrugswhichlowerserumcholesterolare

effectiveatpreventingOAwhileothersarenot(Giermanetal.2014).ConsideringOAa

totaljointdisease,disruptionstocellandtissuehomeostasis(inthecartilage,bone,

muscle,ligaments,jointcapsule,andsurroundingfat)mustbeconsidered.

Becausechondrocytesaretheonlycelltypeinthecartilageandareresponsible

formaintainingcartilageintegrity,theirhomeostaticbalanceisofcriticalimportanceto

OApathogenesis.OAcartilagedoesshowalteredlipidmetabolism,withcholesteroland

lipidlocalizationinthesuperficialzone(Cillero‐Pastoretal.2012;Villalvillaetal.

2013).OAchondrocytesshowintracellularlipiddepositsandalteredexpressionof

regulatorsoflipidandsterolhomeostasis(Kostopoulouetal.2012;Tsezouetal.2010).

Furthermore,articularchondrocyteshavebeenshowntodifferfromgrowthplate

chondrocytesintermsoflipidcontentandfreecholesterol(LeLousetal.1981).

Takentogether,existingliteraturesupportaroleforbothlocalandsystemic

factorsinOApathogenesisduetotheirpotentialimpactonchondrocytemetabolism

andhomeostasis.Becauseserumlevelsmaynotalwaysberepresentativeofintra‐

articularlevelsofvariousfactors(Preteetal.1993),futurestudiesshouldfocusmore

closelyontheintra‐articularenvironment(seeCholesterolhomeostasisand

osteoarthritis).InordertoelucidatethepathophysiologyofOA(thebiologybehind

cartilagedegradation)andunderstandtheetiologyofOA(whynon‐weight‐bearing

jointsareaffected),amorecarefulexaminationofcontributorstochondrocyte

homeostasisisrequired.

21  

Cholesterolhomeostasis Cholesterolisessentialtocellandtissuehomeostasis.Itisamajorcomponentof

mammaliancellmembranes,aconstituentofmyelininthenervoussystem,anda

precursortohormonesandothersignalingmolecules(Jira2013).Inthelipidbilayerof

thecellmembrane,cholesterolcontributestomembranefluidity,andexistsin

unesterified(“free”)form.Withinthecellortissue,cholesteroltypicallyexistsin

esterifiedform,boundtophospholipids(Tabas2002).

Becausedietaryintakeofcholesterolcanvary,andinsufficientorexcessive

cholesterolcanhavedeleteriouseffects,cholesterolbiosynthesisandtraffickingis

tightlycontrolledtomaintainhomeostasis.TheLDLreceptor(LDLR)isresponsiblefor

cellularcholesteroluptakewhiletheATP‐bindingcassettetransporterA1(ABCA1)is

responsibleforcholesterolremoval(GoedekeandFernandez‐Hernando2012)(Figure

4).Thesetransporters,andothergenesinvolvedinthecholesterolbiosynthetic

pathway,areregulatedbymechanismswhichdetectintracellularcholesterollevelsand

maintainthemwithinaphysiologicrange.

22  

Figure4.Schematicrepresentationofcriticalregulatorsofcholesterol

homeostasis.Intracellularcholesterollevelsaregovernedviacholesterolefflux(ATP‐

bindingcassettetransporterA1;ABCA1),cholesterolimport(low‐densitylipoprotein

receptor;LDLR),andcholesterolbiosynthesiswhichisregulatedbyINSIGs(insulin‐

inducedgenes),SCAP(SREBPcleavage‐activatingprotein),SREBFs(sterolregulatory

element‐bindingfactors),andHMGCR(3‐hydroxy‐3methylglutarylcoenzymeA

reductase).Onceprocessed,theSREBFstranslocatetothenucleustoregulate

expressionofcholesterolhomeostaticgenesviatheSREbindingsite.PM=plasma

membrane.ER=endoplasmicreticulum.

23  

Lowintracellularcholesterollevelsaredetectedbyaproteincomplexconsisting

ofInsig,Srebf,Scap,andHmgcr,allofwhicharetetheredtotheendoplasmicreticulum

(ER)membrane(Figure4).CholesteroldepletioncausesScaptoundockfromthe

complexandtakeSrebftotheGolgiforprocessingtoactivatorforms(Brownand

Goldstein1997;Engelkingetal.2005).Srebfproteinsaretranscriptionfactorswhich

regulateexpressionofalllipidandsterolregulatorygenes.Srebf1aandSrebf1care

thoughttoregulatefattyacidsynthesisgenesandSrebf2isthoughttoregulate

cholesterolbiosynthesisgenes(Hortonetal.2003).WhenSrebf2targetgenesare

upregulated,cholesterolproductioninthecellultimatelyincreases.

Whenintracellularcholesterollevelsrise,cholesterolbindstothesterol‐sensing

domainofScapandpreventsitsdissociationfromtheproteincomplexintheER

membrane.ThiscausesnuclearSrebf2todecreasealongwithtranscriptionofitstarget

genes,andcholesterolbiosynthesisdecreases(BrownandGoldstein1997;Engelkinget

al.2005).ThetwomammalianInsiggenes,Insig1andInsig2,functionredundantlyto

regulatecholesterolbiosynthesisandintheirabsence,miceaccumulatecholesteroland

triglycerides(Engelkingetal.2005;Engelkingetal.2006).Inadditiontotetheringthe

proteincomplextotheERmembrane,Insighasbeenshowntoregulatetheproteolytic

degradationofHMG‐CoAreductase(Hmgcr)(Severetal.2003).Thisestablishesthe

Insigproteinsasmajornegativeregulatorsofcholesterolbiosynthesis.

Insig1isregulatedatbothtranscriptandproteinlevels.Whenintracellular

levelsofsterolsarelowandtheScap/SrebfcomplexdissociatesfromInsig,residue‐

specificubiquitinationofInsig1targetsitforproteasomaldegradation(Gongetal.

2006).ProcessedSrebf2translocatestothenucleustoactivatetranscriptionof

24  

cholesterol‐relatedgenes,includingInsig1.NewlysynthesizedInsig1bindsthe

Scap/Srebfcomplexandpreventsfurtheractivationofthecholesterolpathway(Gonget

al.2006).Thisisanimportantfeatureofthefeedback‐inhibitionwhichmaintains

homeostaticlevelsofsterolsinthecell.

ThenuclearreceptorsLXRs(liverXreceptors)actascholesterolsensorswhich

alsoregulatelevelsofintracellularcholesterol.TheLXRsareactivatedbyendogenous

oxysterolswhichaccumulatewithincreasingintracellularcholesterol.Topreventover‐

accumulationofcholesterol,LXRactivationinducestranscriptionofgeneswhichare

involvedincholesteroltransportandregulation,amongothergenes(Zhaoand

Dahlman‐Wright2010).Thisrepresentsanothermechanismthroughwhichcholesterol

homeostasisismaintainedinthecell.

25  

CholesterolhomeostasisandHedgehogsignaling

Multiplelinesofevidencepointtoarelationshipbetweencholesterol

homeostasisandHedgehogsignaling.Hhligandshavecholesterolmoieties,Hhpathway

proteinshavesterol‐sensingdomains,anddefectsineitherHhsignalingorcholesterol

homeostasisproducesimilarcentralnervoussystemabnormalities,facial

dysmorphisms,andskeletaldefects(Eaton2008).Smith–Lemli–Opitzsyndrome(SLOS)

includesmultiplemalformationandmentalretardationwhichisknowntoresultfrom

defectsincholesterolsynthesis(Jira2013).Becausesomeofthedevelopmental

abnormalitiesobservedinSLOSarecomparabletothosewhichresultfrom

compromisedHhsignaling,ithasbeenproposedthattheabnormalcholesterol

metabolismofSLOSimpairsHhsignaling(Koideetal.2006).Insupportofthis,Cooper

etal.showthatinsufficientlevelsofintracellularsterolcandiminishresponsetoHh

signalinembryonicchicktissues(Cooperetal.2003).

WhereasdepletionofintracellularsterolhasbeenhypothesizedtodiminishHh

signaling,increasedintracellularsterolhasbeenhypothesizedtoaugmentHhsignaling.

OxysterolshavebeenshowntoregulateHhsignaling,with25‐hydroxycholesterol

activatingHhsignalinginmedulloblastomacells(CorcoranandScott2006).Lipidrafts

intheplasmamembranehavebeenshowntoconcentrateSmoothened(Figure3)and

potentiatetransductionofHhsignal(Shietal.2013).Theseandotherstudiesindicate

thatHhsignaltransductiondependsoncholesterolmetabolism(Stottmannetal.2011).

CholesterolhasbeenhypothesizedtoplayaroleinHhligandtrafficking,

includingsecretionfromthecellanddistributionacrossthetissue(Eaton2008).Inthe

growthplate,Hhligandisreleasedbypre‐hypertrophicchondrocytesbutsignals

26  

distallytoregulatetherateofchondrocytedifferentiation(Vortkampetal.1996).

Modulationofsterollevelhasbeenshowntocausealteredgrowthplatephenotypes,

whichcouldpotentiallyarisefromimpairedHhligandtrafficking.Impairedgrowthof

thelongbones,asdescribedtoresultfromcholesteroldepletion(Gofflotetal.2003;S.

WuandDeLuca2004),hasalsobeenshowntoresultfromdecreasingHhsignaling

(Kobayashietal.2005).WhiletheseoverlappingphenotypesinSLOSandthegrowth

platehighlighttheinterdependencybetweenHhsignalingandsterolhomeostasis,the

exactregulatoryrelationshipremainsunclear.

27  

Cholesterolhomeostasisandosteoarthritis

Theeffectsofdisruptedcholesterolhomeostasisarecommonlyconsideredin

thecontextofcardiovasculardiseases(GoedekeandFernandez‐Hernando2012;Ng

andHegele1993),buttherelativeimportanceofcholesterolanditsfunctionstocell

homeostasismakesperturbationstothismoleculedeleteriousinothersystemsaswell.

Deficienciesincholesterolmetabolismduringdevelopmenthavebeenreportedto

resultinavarietyofhumandisorders,includingskeletaldefects,behaviouraldeficits,

andSmith‐Lemli‐Opitzsyndrome(SLOS;describedinHedgehogsignalingand

cholesterolhomeostasis),amongothers(Jira2013;Porter2003).

Regardingskeletaldevelopment,disruptiontocholesterolequilibriuminthe

cartilagemayhavedirectorindirecteffectsonjointarchitecture,chondrocyte

metabolism,matrixsynthesisordegradation,boneremodeling,andsignalingpathways

(Girkontaiteetal.1996;Ijirietal.2008;Kanbeetal.2006;Woodsetal.2009;H.Zhang

etal.2002).Inratstreatedwithpharmacologiccholesterolinhibitors,impaired

development(Gofflotetal.2003)andgrowthofthelongbones(S.WuandDeLuca

2004)havebeenreported.Aberrantlimbpatterning(Gofflotetal.2003)andreductions

ingrowthplatechondrocyteproliferationandhypertrophy(S.WuandDeLuca2004)

arehypothesizedtobecausedbyimpairedHedgehogsignalingresultingfrom

cholesteroldeficiency.Suchperturbationstonormalchondrocytebehaviourmay

contributetoOAdevelopment.

Todate,studiesexaminingtherelationshipbetweensterolprofilesandthe

occurrenceofOAinhumanshavebeenlargelyepidemiological,withconfounding

variablesmakingresultsdifficulttointerpret(Al‐Arfaj2003;Sturmeretal.1998).Al‐

28  

Arfajshowsanassociationbetweenincreasedserumcholesterollevelsand

osteoarthritiswhichpersistswhenadjustedforpotentialconfoundingvariables

includingage,sex,bodymassindex,serumuricacid,andserumtriglycerides(Al‐Arfaj

2003).Thisstudyislimitedbytherelativelysmallsamplesize(246patients),theshort

duration(7months)forachronicdisease,andtheoversightofotherpotential

confoundingvariables(activitylevel,diet,ethnicity,andsoon).Otherepidemiological

studiesshowasimilarpositiverelationshipbetweensystemicsterollevelsandOAbut

aresubjecttothesameconfounds(Sturmeretal.1998).

Arecentstudyprofiledphospholipidsinhumansynovialfluid(aviscousfluid

fillingthearticularjoint,thoughtprimarilytoprovidelubrication).Kosinskaetal.used

massspectrometrytoidentifylipidspeciesinthesynovialfluidandshowedsignificant

differencesbetweencontrols,earlyOA,andlateOA.Theauthorsspeculatethat

alterationstophospholipidprofilesmayaffectjointlubrication,scavengingofreactive

oxygenspecies,orinflammatoryprocesses,allofwhichcancontributetoOA

pathogenesis(Kosinskaetal.2013).Withrespecttothearticularcartilage,reportsof

cholesterollocalizationtothesuperficialzonehavebeenmade(Cillero‐Pastoretal.

2012),andcomparisonsofcandidatelipidsbetweengrowthplateandarticular

chondrocyteshavebeenmade(LeLousetal.1981),butnocompleteprofilesofsterol

orlipidspeciesinosteoarthriticchondrocyteshavebeenmade.

StatinsarewidelyuseddrugswhichinhibitHMG‐CoAreductaseandlower

cholesterolproduction.Basedonepidemiologicalstudiesassociatingelevated

cholesterolandosteoarthritis,statinshavebeensuggestedasapotentialtherapeutic

interventionforOA(Bakeretal.2011).Themajorlimitationofthesestudiesisthe

29  

assumptionthatsystemicsterollevelsarerepresentativeofintra‐articularsterollevels.

Preteetal.compileddatashowingthattotalcholesterollevelsofcontrolpatients

rangedfrom148‐269mg/dLintheplasmato7‐8mg/dLinthesynovialfluid,whilethat

ofOApatientsrangedfrom127‐252mg/dLintheplasmato4‐169mg/dLinthe

synovialfluid(Preteetal.1993).Thissuggeststhatanypharmacologiccholesterol

inhibitorshouldtargetthesynovialjointspecifically,toimproveOAprognosis.

Mousestudiesofferfurthersupportfortargetingcholesterolintracellularlyin

OA.Giermanetal.reportarescueoftheOAphenotypewhenmiceweretreatedwitha

statin,whichreducesintracellularproductionofcholesterol,butnotwithezetimibe,

whichreducesabsorptionofdietarycholesterol.Whileserumcholesterolwaslowered

bybothdrugs,onlystatin(presumablybyintracellularreductionofcholesterol)was

effectiveinattenuatingOA.Unfortunatelytheauthorsdonotexplicitlyreportonthe

efficacyofstatintreatmentinthechondrocytes(Giermanetal.2014).Inotherreports,

statinshavebeenshowntopreventcartilagedegradationbyreducingenzymessuchas

MMP13(Barteretal.2010),andpromoteanabolismbyinducingexpressionofcollagen

typeIIandaggrecan(Simopoulouetal.2010).Thesefindingspointtoarolefor

cholesterolhomeostasisinOApathogenesis.

30  

Currenttreatmentstrategiesinosteoarthritis

Pharmacologics Thereisnocureforosteoarthritisandtreatmentoptionsarelimited.Thethree

mostcommoninterventionsarepharmacologic,lifestylechanges,andsurgical

(Bombardieretal.2011).Emerginginterventionsincludecelltherapiesandcartilage

replacementstrategies,butthesearestilllargelyexperimental.Analgesicsandanti‐

inflammatorydrugsarecommonlyprescribedtopatientssufferingfromthechronic

painassociatedwithOA,buttheside‐effectscausedbylong‐termuseofthesedrugscan

provetobedetrimental.

Formulationsofglucosaminehavebeeninvestigatedfortheirputativebenefits

incartilagemaintenance,butinconsistentresultsfromdifferentstudiesmake

conclusionsdifficulttodraw(Altmanetal.2006;Kapooretal.2012;Uitterlindenetal.

2007).Illustratingthispoint,Vladetal.foundtheheterogeneityamongglucosamine

trialstobelargerthancouldbeexpectedbychancealone.Theauthorsspeculatethat

thedifferencesineffectsizeacrosstrialscouldbeduetodifferencesinglucosamine

preparationsorindustrybias/involvement,amongotherfactors(Vladetal.2007).

Anotherchallengeiswhetheroralpharmacologicscanreachthesynovialjointandbe

effective,orwhetherlocaladministrationviaintra‐articularinjectiondirectlyintothe

synovialjointisrequired.Theseareimportantconsiderationsforanypharmacological

treatmentofOA.

31  

Lifestylechanges ObesityisamongthetopmodifiableriskfactorsforOAdevelopment.Although

commonlythoughttoincreaseloadandexacerbatewear‐and‐tearonthejoints,obesity

hasalsobeenshowntocorrelatewithOAsymptomsinnon‐weight‐bearingjoints

(Griffinetal.2010)(seeArticularjoints).Weightmanagementcanhelpreduce

symptomsofOAbyreducinginflammationandalleviatingloadonthejoints

(Bombardieretal.2011;Messieretal.2013b).Weightreductionhasbeenshownto

haveadose‐responseeffectinalleviatingsymptomsofkneeOA.Messieretal.followed

patientsoveran18‐monthperiodwithcombinationdietandexerciseinterventions

(Messieretal.2013b).“High”weightlossconsistedof32.5%to10.1%,“medium”9.9%

to5.0%,and“low”4.9%to9.9%bodyweight.Foroutcomesincludingknee

compressiveforce,inflammation,pain,andfunction,participantsinthe“high”weight

losscategoryhadsignificantlylessjointload,reducedsystemicinflammationandpain,

andimprovedfunction(Messieretal.2013b).

Lifestylechangeswhichpromoteweightreductionsuchasdietaryrestriction

andregularexercisehavebeenshowntoimproveOAoutcomes(HunterandEckstein

2009;Messieretal.2013b).Recommendingexercisetoapopulationofpatientswith

restrictedphysicalmobilitymayseemcounterintuitiveandevendetrimental,but

studiesindicatethatmoderateexercisecanimproveOAoutcomes,whileexcessive

exercisecanexacerbateoutcomes(Galoisetal.2004).Amajorconsiderationregarding

exerciseisthefrequency,duration,andtype(aerobicvs.strength)recommended.

Differentregulatorybodiesmakedifferentrecommendations.Forexample,the

WorldHealthOrganization(WHO)recommends30minutesofmoderatephysical

32  

activity5daysperweek,whiletheOsteoarthritisResearchSocietyInternational

(OARSI)guidelinesrecommendreferraltoaphysicaltherapistandsubsequent“regular

exercise”includingaerobic,strengthening,andrangeofmotionexercises(Iversen

2010).Theseguidelinesareambiguousatbest,since“strengthening”exercisescan

refertoavarietyofexercises.Currentclinicaltrialsseektoassesstheeffectsof

localizedstrengthtrainingtothethighmusclespecifically,inanattempttoreducepain

andimprovemobility(Messieretal.2013a).Studiesofthisnaturearerequiredto

elucidateaspecificdoseofexercisethatphysicianscanprescribetoOApatients.

Despitetheknownbenefitsofexercise,patientssufferingfromlate‐stageOA

mayfindphysicalactivityprohibitiveduetopain,stiffness,andswellinginthejoints.As

such,weightgainmaybebothacauseandresultofOA,sincesufferersbecome

sedentary.AlthoughpeopledonotdiefromOAdirectly,theycanexperiencepremature

mortalityfromOAindirectly,duetothesedentarylifestyletheyadopt.Rahmanetal.

showthatpatientswithOAhadahigherriskofcardiovasculardisease(Rahmanetal.

2013).Whilemetabolicperturbationsmaybeatplay,physicalinactivitymayalso

contribute.Forthesereasons,thosewhoareableshouldpursueweightlossthrough

dietandexercisetoreduceOAsymptoms.

33  

Surgery Despitesomearthroscopicinterventionsshowingminimalresultsinalleviating

pain(Moseleyetal.2002),surgeryisstillcommonlyprescribedtopatientswhosuffer

fromdisablingOA(Bombardieretal.2011).Jointreplacementsurgeriesaremost

frequentlyperformedonthekneeandhip,butcanalsobedonefortheshoulderand

ankle(Proffenetal.2013).IntheUnitedStatesbetween1991and2010,thevolumeof

annualtotalkneereplacementsurgeriesincreased162%.Patientsaretypicallywhite

(~90%)andfemale(~65%),withameanageof74(Crametal.2012).Thisoptionis

drasticandinvasive,comingwithallthecomplicationsofsurgeryinadditiontosuch

risksasjointinfection,thrombosisandembolism,andmechanicalfailureofimplants

resultingindislocationorevenbonefracture(Proffenetal.2013).

Althoughimplantsurvivalratescanreach85‐95%evenafter10‐15years

(Proffenetal.2013),asignificantproportionofpatientsareunsatisfiedwithpost‐

operativeresultsandmanyrequirehospitalreadmissionorsurgicalrevision(Cramet

al.2012;Liddleetal.2013).Totaljointreplacementsarerestrictedtopatientswith

advanceddisease(Liddleetal.2013),makingthisexpensiveprocedureonethatcan

onlyofferreliefafteryearsofsuffering(Bombardieretal.2011).Withouraging

population,thedemandforjointreplacementsurgerieswillcontinuetorise,especially

ifimprovedpreventativemeasuresarenotsought(Carretal.2012).

34  

Cartilagerestoration StillinexperimentationaremorecontroversialtreatmentstrategiesforOA

whichincludemechanicaltherapies,cartilagerestoration,andvariouspharmacologics.

Brightonetal.describedeliveryofaspecificelectricalsignaltothearticularcartilageto

stimulateanabolicactivityandreduceproteaseexpression(Brightonetal.2008).

Proteaseinhibitorshavebeenmetwithchallenges,butarestillindevelopment,

especiallyagainstMMP13andADAMTS5,thetwomajorproteasesknowntomediate

cartilagedegradationinOA.Forafullreviewofpotentialdisease‐modifying

osteoarthritisdrugs,seeAbramsonetal.,2006(Abramsonetal.2006).

Cartilagegraftsandcell‐basedtherapiesarebecomingmorefeasibleasour

understandingofcartilagebiologyimproves(Craftetal.2013).Thisimproved

understandingalsofacilitatesdevelopmentofpharmacologicswhichtargetspecific

biologicalpathways.ThediscoverythatHhmodulationcanaltertheseverityofOA(Lin

etal.2009;J.Zhouetal.2014)hasledtopatentsdescribingHhinhibitorsforthe

treatmentofOA(Bumcrot2005;L.Wei2012)(seeHedgehogsignalingand

osteoarthritis).TheobservationthatlipidandsterolimbalancecorrelateswithOA

severity(Al‐Arfaj2003;Aspdenetal.2001)hasledtopatentsdescribingstatinsforthe

treatmentofOA(M.L.Hoetal.2013)(seeCholesterolhomeostasis).Current

treatmentoptionsareclearlyinadequate,butadvancesarebeingmadeasour

understandingofthepathophysiologyofOAimproves.

35  

Rationale

HedgehogsignalinghasbeenshownimpactOAseveritybutthemechanismsby

whichthisoccursremainunknown(Linetal.2009;J.Zhouetal.2014).Giventhe

technologiesavailableforstudyinggeneexpressioninahigh‐throughputmanner

(Lovenetal.2012),identifyingHhtargetgenesinOAcartilageisaviablestrategyfor

elucidatingthoseunknownmechanisms.Usinghumancartilagesamplesthatare

availablefromtotalkneereplacementsurgeries,IwillidentifyHhsignalingtargetgenes

inosteoarthriticchondrocytes.Bygroupingthosetargetgenesfunctionally,Iwill

elucidatebiologicalnetworksthatareregulatedbyHhsignaling.Usinggenetically

modifiedmice,Iwillinvestigatetheimportanceofachosenbiologicalnetworkin

chondrocytehomeostasis,andassesstheeffectsinthegrowthplatecartilageandthe

articularcartilage.Finally,Iwilltargetthebiologicalnetworkpharmacologicallyinboth

mouseandhumansamples,todeterminewhethermodulationcanaltertheseverityof

osteoarthritis.

36  

HypothesisHhsignalingregulatescholesterolbiosynthesisinchondrocytes,andmodulating

cholesterolhomeostasiscanimpacttheseverityofOA.

Objectives1. IdentifyHhsignalingtargetgenesinchondrocytes.

2. DeterminewhethermodulationofHhsignalingtargetgenescanimpactchondrocyte

homeostasis.

3. AssesstheimpactofHhsignalingtargetgenemodulationontheseverityof

osteoarthritis.

37  

ChapterTwo:

Hhsignalingregulatesexpressionofcholesterolbiosyntheticgenesinchondrocytes

RNAextractionfromhumanarticularcartilagebychondrocyteisolation

(AliandAlman2012)

PublishedinAnalyticalBiochemistry

38  

Summary

Geneexpressionanalysescanbeusedtoelucidatetheroleofsignaling

pathways,suchastheHhsignalingpathway,inosteoarthritis.Thesestudiesrequire

sufficientquantitiesofhigh‐qualityRNA,andfunctionalanalysisofresultstouncover

thebiologicalrelevance.HerewedescribeanoptimizedmethodforRNAextraction

fromhumanarticularcartilage.Chondrocytesareisolatedfromtheextracellularmatrix

andmodificationsaremadetothetraditionalTRIzol®protocol,includingtwoRNA–

DNA–proteinphaseseparations.Withtheoptimizedmethoddescribed,RNArecovery

increasedbyapproximately1µgper100mgofcartilage,andRNAintegritynumber

(RIN)improvedfrom2.0to7.5.Usingthismethod,RNAwasisolatedfromthreehuman

OAcartilagesampleswhichweretreatedwithapharmacologicalHhinhibitorto

modulatetheHhsignalingpathway.AfterbeingtestedforeffectiveHhinhibition,these

samplesweresubjectedtotheAffymetrixHumanGene1.0STmicroarray.Using

Ingenuity®Pathwayanalysistocaptureglobalchangesingenenetworks,severalgenes

knowntobeinvolvedinsterolhomeostasiswerefoundtobedysregulatedwithHh

inhibition.IndependenthumancartilagesampleswereusedtoverifyHh‐mediated

changesinexpressiontocholesterolbiosyntheticgenes.Apotentialmechanismbehind

thisrelationshipisidentified,wherebyHhsignalingregulatesexpressionofSREBF2,

themajortranscriptionalactivatorofallcholesterolbiosyntheticgenes.Takentogether,

weshowthatgenesinvolvedincholesterolhomeostasisaresubjecttoregulationbyHh

signaling.

39  

Introduction

Geneexpressionstudiesofhumanarticularcartilagecanimproveour

understandingofOA,buttranscriptomicsrequiresufficientquantitiesofhigh‐quality

RNA(Geyeretal.2009).Samplesfrompatientsundergoingtotaljointreplacement

surgeriesserveasphysiologicallyrelevantmodelsforexperimentation,butthelow

cellularityandhighproteoglycancontentofcartilagemakeefficientandeffective

isolationofhigh‐qualityRNAdifficult(CarneyandMuir1988;RoughleyandLee1994).

Becausetotalcartilageconsistsofonly1%to2%chondrocytes,RNAcontentpergram

oftissueislimited;andbecausethemajorproteoglycan,aggrecan,isnegatively

charged,RNApurityiscompromisedbyco‐purificationofprotein.Existingmethodsfor

RNAextractionfromhumancartilagedonotsucceedinaddressingthesespecific

challenges.Somemethodsrequirespecializedequipmentsuchasmicro‐

dismembranatorsandfreezermills,whichmaynotbereadilyavailableinall

laboratories.ThesemethodsdescribetheuseofcolumnsforRNAextractionor

purification(e.g.,QiagenRNeasycolumns)whichimprovequalitybutcompromiseyield

(Gehrsitzetal.2001;McKennaetal.2000).Giventhevalueofstudyinggeneexpression

inchondrocytes,theinherentchallengesinisolatingRNAfromcartilage,andthe

limitationsofcurrentmethodologies,weoptimizedamethodforRNAextractionfrom

humanarticularcartilage(AliandAlman2012).

OuroptimizedmethodofRNAextractionwasusedtoconductaseriesof

microarrayexperimentsinordertoidentifyHhsignalingtargetgenesinhuman

osteoarthriticcartilage.BecauseHhsignalinghasbeenshowntobedysregulatedin

humanandmouseOA(Linetal.2009;J.Zhouetal.2014),wehypothesizethataberrant

40  

expressionofHhtargetgenesinchondrocytespromotesOApathogenesisbydisrupting

normalarticularchondrocytebehaviour.Toelucidatethebiologicalnetworksthat

couldberesponsibleforthisdisruption,functionalpathwayanalysisofmicroarraydata

wasconducted(Downey2006).RegulationoftargetgenesbyHhsignalingwasverified

usingindependenthumansamplesthatweretreatedwithaHhantagonistoraHh

agonist.Additionally,insilicoanalyseswereconductedtodeterminewhethertarget

genescontainedGliconsensusbindingsitesandwhethertargetgeneswere

dysregulatedinosteoarthriticcartilagewhencomparedtonormalcartilage.Theresults

oftheseexperimentsidentifyHhtargetgenesthatareexpressedinosteoarthritic

cartilage.

41  

ResultsOptimizedRNAextractionfromhumanosteoarthriticcartilage

TomaximizeRNAyieldwhileminimizingdegradationandcontamination,an

optimizedmethodforRNAextractionfromhumanosteoarthriticcartilagewas

developed(AliandAlman2012).Sampleswerecollectedfrom20patientsundergoing

totalkneereplacementsurgeryforthetreatmentofclinicallydiagnosedOA.Informed

consentwasobtainedfromeachpatient.Articularcartilageexplantswereharvested

anddividedintogroups,thenincubatedovernightat37°Cand5%CO2inculturemedia.

Forafulldescriptionofthisprotocol,seeMethods.

Foracomparisongroup,somecartilagesamplesweresubjectedtothe

traditionalTRIzol®protocol(Invitrogen,CA).Thesesampleswerefrozenwithliquid

nitrogen,pulverizedusingmortarandpestle,suspendedin1mLofTRIzol®reagent

per50mgofcartilage,andhomogenizedbyPolytronsonication.RNAextractionwas

conductedaspertheTRIzol®manufacturer’sprotocol.Asubsetofthesesampleswas

subjectedtoQiagenRNeasycolumnpurification(Qiagen,MD),accordingtothe

manufacturer’sprotocol.

InPart1ofthemodifiedmethod(seeMethods:IsolationofPrimary

Chondrocytes),chondrocyteswereisolatedfromthesurroundingproteoglycan‐rich

matrix(Goldring2005).Briefly,cartilageexplantswereincubatedintrypsinwhilein

rotationwithglassbeadstoprovidephysicalhomogenization.Afterbeingwashed,

collagenaseA1wasappliedtotheexplantsfora6‐hourorovernightdigestion.

Followingchondrocyteisolation,anaverageof1.1x106(±0.3x106,n=5)viablecells

wereobtainedpergramofcartilage,asdeterminedbythetrypanblueexclusionassay.

42  

IsolatedchondrocyteswerelysedwithTRIzol®,transferredtocryotubescontaining

smallerglassbeads,andstoreduntilfurtheruse.

InPart2(seeMethods:RNAExtractionbyRepeatedPhaseSeparations),

RNAwasextractedfromthechondrocytesbymodifyingtheTRIzol®manufacturer’s

protocol(Dell'accioetal.2008).ThekeymodificationwastotheRNA–DNA–protein

phaseseparationstep.TheaqueousphasecontainingRNAwastransferredtoafresh

tube,butbeforeprecipitatingtheRNA,anequalvolumeofTRIzol®wasadded.

IncubationandphaseseparationwererepeatedtoremoveresidualproteinandDNA

withoutcompromisingRNAyield.RNAintheaqueousphasewasthenprecipitatedand

resuspendedinnuclease‐freewater.

RNAextractedbytraditionalandoptimizedmethodswascomparedby

spectrophotometry,Bioanalyzer,andreal‐timePCR.RNAqualitywasdefinedbyRNA

integrity(lackofdegradation)andRNApurity(absenceofcontaminants).TheAgilent

2100Bioanalyzer(RNA6000NanoLabChip,AgilentTechnologies,CA)wasusedto

determinetheRNAintegritynumber(RIN),ameasureofRNAdegradation(Schroeder

etal.2006)where0representsthepoorestqualityordegradedRNAand10represents

thebestqualityorintactRNA.Integrityisvisualizedbyelectropherogrambasedonthe

identificationof18Sand28SribosomalRNA(rRNA)peaks/bands.TheNanoDrop1000

spectrophotometer(NanoDropTechnologies,DE)wasusedtodetermineRNApurity

andconcentration.LowA260:A280andA260:A230ratios(<1.5)reflectRNAthatis

contaminatedwithprotein,phenol,andotherorganiccompounds,whileratiosof

approximately2.0reflectrelativelypureRNA.Inparticular,lowA260:A280ratiosreveal

43  

proteincontaminationgiventhatnucleicacidsabsorbmaximallyat260nmand

proteinsat280nm.

UsingtheoptimizedRNAextractionprotocol,1.1µgofRNAper100mgof

cartilagewasrecovered,whereasonly0.2µgofRNAwasrecoveredfromthesame

amountofcartilageusingthetraditionalTRIzol®protocolwithcolumnpurification

(Table1).RNAfrombothmethodsshowedreasonableA260:A280ratiosandlow

A260:A230ratios,buttheoptimizedprotocolsignificantlyimprovedA260:A230from0.42to

1.33(Table1).Additionally,RNAintegritywasimprovedwhenthemodifiedprotocol

wasused,withanincreaseinRINfrom2.0to7.5(Table1).RNAfromthetraditional

TRIzol®protocolwasdegraded,withlow‐molecular‐weightcontaminationand

backgroundnoise(Figure5a).RNAfromtheoptimizedprotocolexhibitedclearly

definedpeaksandbandsfor18Sand28SrRNAontheelectropherogram,

demonstratingitshighquality(Figure5b).

Table1.ComparingRNAobtainedfromdifferentextractionmethods.In

comparisonwiththetraditionalTRIzol®protocol,themodifiedprotocolimprovesthe

qualityandquantityofRNAextractedfromhumanarticularcartilage.RIN(RNA

integritynumber)andRNAparametersarepresentedwithmeanvaluesinboldandthe

rangeofvaluesinparentheses(*P<0.05).

44  

Figure5.RepresentativeelectropherogramscomparingRNAquality.RNA

extractedusingthetraditionalTRIzol®methodcoupledwithQiagencolumn

purification(a,RIN2.3)andRNAextractedusingthemodifiedprotocol(b,RIN8.2).

Peaksandbandslabeledas18Sand28SrepresentrRNA.RIN=RNAintegritynumber.

TodeterminetheeffectsofRNAqualityongeneexpressionassays,cartilage

fromarepresentativesamplewasdividedandRNAwasextractedusingthetraditional

45  

TRIzol®methodandtheoptimizedmethod.RNAwasreversetranscribedto

complementaryDNA(cDNA)usingSuperscriptII(Invitrogen)asperthe

manufacturer’sprotocol.ToshowtheimpactofRNAqualityongeneexpressionassays,

real‐timePCRwasperformedforCOL2A1,COL10A1,MMP13,andβ‐ACTINusing

TaqManassaysfromAppliedBiosystems(Carlsbad,CA).Resultsarepresented

accordingtothecomparativeCTmethod(∆∆CT)ofanalysis(LivakandSchmittgen

2001).AlthoughtheRNAyieldsfromthetwomethodsappeartobecomparable,the

valueforthetraditionalTRIzol®methodlikelyreflectsproteincontamination,as

shownbythelowA260:A280ratio(Table2a).Resultsfromabicinchoninicacid(BCA)

assayreveal0.36ngofproteinpernanogramofRNAextractedfromthetraditional

method,whereasnoproteinwasdetectedinRNAextractedfromtheoptimizedmethod.

Real‐timePCRresultsshowthattheRNAisolatedusingthetraditionalmethod

consistentlyrequiresmoreamplificationcycles(higherCTvalues)fordetectionof

COL2A1,COL10A1,MMP13,andβ‐ACTINtranscripts,genesthataretypicallyassayedin

cartilageexperiments(Table2b).Theresultingdifferencesin∆CTvalues(Table2b)

ultimatelyalter∆∆CTcalculationsinthedeterminationofgeneexpressiongiventhata

∆CTof1equalsa2‐foldchangeinexpression(LivakandSchmittgen2001).

Furthermore,sincestandarddeviationshowsanincreaseinerrorwithhigherCT

values,geneexpressionresultsthatarebasedonhigherCTvaluesareconsideredless

accurate(Karlenetal.2007).ThisindicatesthatpoorRNAqualitylimitsaccurate

detectionofgeneexpressionchanges.Takentogether,theseresultsdemonstratethat

ourreportedoptimizedmethodforRNAextractionfromhumanarticularcartilage

improvestheyieldofhigh‐qualityRNAthatissuitableforgeneexpressionanalyses.

46  

Table2.ComparinggeneexpressionresultsusingRNAobtainedfromdifferent

extractionmethods.(a)ComparingqualityandquantityofRNAisolatedfromthe

samecartilagesampleusingtwodifferentextractionmethods.(b)CTvaluesforgenes

typicallyassayedincartilageexperiments,COL2A1,COL10A1,andMMP13.Inthis

representativesample,thequalityofRNA[shownin(a)]impactsgeneexpression

resultsbyaltering∆CTvalues.

47  

Geneexpressionprofilinginosteoarthriticcartilage:microarrayanalysesToelucidatethemechanismsbywhichHhactivationpredisposestoOA(Linet

al.2009;J.Zhouetal.2014),wesoughttoidentifyHhtargetgenesinhumanOA

cartilageusingmicroarrayanalysis.Withtheconsentofeachpatient,humanOA

sampleswereobtainedfromtotalkneearthroplastysurgeries.Articularcartilage

explantswereincubatedovernightwiththeHhantagonistN‐[(3S,5S)‐1‐(2H‐benzo[3,4‐

d]1,3‐dioxolan‐5‐ylmethyl)‐5‐(piperazinylcarbonyl)pyrrolidin‐3‐yl]‐N‐[(3‐

methoxyphenyl)methyl]‐3,3‐dimethylbutanamide(C31H42N4O5)orcarrier(Chenetal.

2002;Williamsetal.2003).OuroptimizedprotocolforRNAextractionwasusedto

maximizethequalityandquantityofRNAisolatedfromthecartilage,whichisknownto

havelowcellandhighproteoglycancontent(AliandAlman2012;CarneyandMuir

1988;Mankinetal.1971)(seeOptimizedRNAextractionfromhuman

osteoarthriticcartilage).Toverifymodulation,real‐timePCRwasusedtoconfirm

downregulationoftheknownHhtargetgeneGLI1inthetreatedgroup(Figure6).

Figure6.Hhinhibitioninhumanarticularcartilage.InhibitionofHhsignaling

followingtreatmentwithaHhantagonistisverifiedbydownregulationoftheknown

HhtargetgeneGLI1.Errorbaris95%confidenceinterval(n=3;*P<0.05).

48  

Microarrayanalyseswereperformedtoassessdifferentialgeneexpressionusing

theAffymetrixHumanGene1.0STplatform(Affymetrix,CA).Unlikearrayswitha3’

bias,thisarrayofferstheadvantageofassayingthewholegenetranscriptwithprobes

designedagainstwell‐annotatedexons.Inaddition,thetargetlabelingprotocolthatis

usedgeneratesbiotinylatedsenseDNAversuscRNA.Thisallowstarget‐probe

hybridizationtooccurasDNA‐DNAduplexeswhichhavebeenshowntobemore

specificthancRNA‐DNAduplexes(Pradervandetal.2008).Followinghybridization,

microarraychipswerescannedusingtheAffymetrixGeneChipScanner3000

(generatingCELfiles),byTheCentreforAppliedGenomicsatTheHospitalforSick

Children.Probesetdataweregenerated(CHPfiles)foreachofthechipsusingthe

AffymetrixExpressionConsole.Thesedataandadescriptionofthemicroarray

experimenthavebeenenteredintotheGEOdatabaseunderGSE54749.

Foreachofthreebiologicalreplicates,datafromtreatedsampleswere

normalizedtodatafromcontrolsamplesusingPartek®GenomicsSuite.Thisgenerated

threelistsofgenes(oneforeachofthethreebiologicalreplicates)whichwere

differentiallyexpressedinthetreatmentgroupoverthecontrolgroup.Duetothe

geneticvariabilitythatisinherenttohumansamples,genechangeswerenotaveraged

acrossdatasetsbutratherdifferentiallyexpressedgeneswerefilteredforthosewhich

changedinthesamedirectioninthreeofthreedatasets(Figure7).Noadditional

criteria,suchasarbitraryfold‐changecutoff,wereusedforgenefiltering.Acomplete

listofthe511downregulatedand1189upregulatedtranscriptscanbefoundin

Appendix:Microarraygenelist.

49  

Figure7.Microarrayanalysis:genefiltering.Eachcolourrepresentsadifferent

microarraydatasetafteridentifyinggenechangesinthetreated(Media+HhAntagonist)

groupoverthecontrolgroup(Media).Eachspikerepresentsadifferentgene,where

inwardspikesrepresentdecreasesingeneexpressionandoutwardspikesrepresent

increasesingeneexpression.Differentiallyexpressedgeneswerefilteredforthose

whichchangedinthesamedirectionacrossdatasets.

50  

Alistofthetop10upregulatedgenesandthetop10downregulatedgeneswas

generated(Table3).ThetopthreegenestoshowanincreaseinexpressionincludeSCD

[stearoyl‐CoAdesaturase(delta‐9‐desaturase)],LDLR(lowdensitylipoprotein

receptor),andINSIG1(insulininducedgene1).Thetopthreegenestoshowadecrease

inexpressionincludeTGFBI(transforminggrowthfactor,beta‐induced),MXRA5

(matrix‐remodellingassociated5),andFAM29A(familywithsequencesimilarity29,

memberA)(Table3).

TocapturetheglobalchangestogeneexpressionthatoccurredasaresultofHh

modulation,pathwayanalysiswasusedtoidentifysignalingpathways,molecular

networks,andbiologicalprocessesthatwererepresentedinthemicroarraydata

(Table4).ResultsfromIngenuity®Pathwayanalysisidentifiedthetopdysregulated

networkstobelipidmetabolism,andendocrinesystemdevelopmentandfunction.The

topdysregulatedbiologicalfunctionsincludedtheinflammatoryresponse,lipid

metabolism,andtissuemorphology.Amongthetopdysregulatedcanonicalpathways

werethebiosynthesisofsteroids,andLXR/RXRactivation.Thetoptoxicitylistwas

cholesterolbiosynthesis(Table4).Fromtheseanalysesemergedacommontheme,

genesandnetworksrelatedtolipidmetabolismandcholesterolbiosynthesiswere

dysregulatedwithHhmodulation.

51  

Table3.Microarrayanalysis:top20genechanges.Thetop10upregulatedandtop

10downregulatedgenesinhumanOAchondrocyteswithHhinhibition,afterfiltering

forgeneswhichchangedinthesamedirectionacrossthreedatasets.

Table4.IngenuityPathwayAnalysisofmicroarrayresults.Microarrayresultswere

analyzedinanunbiasedmannerforbiologicalnetworks,signalingpathways,andother

functionalgenegroupsusingIngenuityPathwayAnalysis.Thetopresultsforeach

categoryareshown.

52  

IdentifyingHhtargets:cholesterolbiosyntheticgenes

Microarrayanalysesrevealedgenesinvolvedinthecholesterolhomeostatic

pathwaytobeamongthetopdysregulatedwithHhpathwayinhibition(Figure8).

Figure8.Heatmapofcholesterolhomeostaticgenes.ResultsfromAffymetrix

HumanGene1.0STmicroarrayofhumanosteoarthriticcartilagetreatedwithHh

antagonist.Eachofthreepairsofsamples(Controlvs.Treated)wereanalyzed

independentlyandfilteredforgeneswhichwereeitherupregulatedordownregulated

acrossallthreesamples.Greaterintensityofredrepresentsincreasedgeneexpression

withHhinhibition(Treated).

Becausecholesterolisvitalforcellularprocessesandbecauseitsdysregulationhas

beenimplicatedinOA,wesoughttoassesstheroleofHhsignalinginregulatingthe

53  

expressionofcholesterolbiosyntheticgenes(Kostopoulouetal.2012;Sturmeretal.

1998).HumancartilagesamplesweresubjectedtoHhmodulationandassayedfor

expressionofHMGCR,HMGCS1,DHCR7,LDLR,ABCA1,andINSIG1.Thesegenes,which

areknowntobeinvolvedinsterolhomeostasis(Gilletal.2008;Hortonetal.2003),

showincreasedexpressionwithHhinhibition(evidencedbydownregulationofGLI1,

Figure9a)anddecreasedexpressionwithHhactivation(evidencedbyupregulationof

GLI1,Figure9b).Inadditiontovalidatingmicroarrayresultsbyconfirmingchangesto

topdysregulatedgenessuchasLDLR,INSIG1,andHMGCS1(Table3),thesedata

confirmHh‐mediatedregulationofcholesterolhomeostaticgenes.

Figure9.Real‐timePCRvalidationofcholesterolhomeostaticgenes.Validationof

Hhregulationofcholesterolhomeostaticgenesidentifiedbymicroarray.Independent

humanosteoarthriticcartilagesamplesweretreatedidenticallytothoseusedin

microarrayanalyses.Expressioninthecontrolgroupwasarbitrarilydefinedas‘1’

(dashedline)anddatafromthegroupstreatedwith(a)Hhantagonistand(b)Hh

agonistgivenasthemean.Errorbarsare95%confidenceintervals(n=3;*P<0.05).

54  

Todeterminewhethertheobservedchangesingeneexpressiontranslateto

changesinproteinexpression,WesternblotanalysiswasperformedusinghumanOA

cartilagewithHhmodulation.ResultsdemonstratethatchangestoINSIG1translateto

theproteinlevel,sincetreatmentwithHhantagonistincreasedINSIG1proteinand

treatmentwithHhagonistdecreasedINSIG1protein(Figure10).Insilicoanalyses

wereperformedtodeterminewhethercholesterolhomeostaticgenescontainGli

bindingsites.Mulananalyses(http://mulan.dcode.org)(Ovcharenkoetal.2005)

revealedGliconsensusbindingsitesthatareconservedbetweenhumansandmicein

10ofthe19genesidentifiedbymicroarray(Figure11).Analysisofpubliclyavailable

microarraydatacomparingnormalcartilagetoosteoarthriticcartilageidentified8of

the19genestobedysregulatedinosteoarthritis(Karlssonetal.2010;Kostopoulouet

al.2012)(Figure11).

Figure10.WesternblotofINSIG1inhumanOA.RepresentativeWesternblot

showingINSIG1proteinfromhumanOAcartilageexplantstreatedwithHhantagonist

orHhagonist.ACTINisshownasaloadingcontrol.

55  

Figure11.Insilicoanalysesofcholesterolhomeostaticgenes.Mulaninquiries

(http://mulan.dcode.org)wereconductedtoexaminethe5kbpromoterregionof

cholesterolhomeostaticgenesidentifiedbymicroarrayanalyses(3of3arrays)forGli

consensussequence‐bindingsitesthatareconservedbetweenhumansandmice(Gli

site).Publiclyavailableexpressiondatacomparingosteoarthriticcartilagetonormal

cartilagewerefilteredforchangestocholesterolbiosyntheticgenes(OAvs.normal).

GeneswithaGlisiteorwithdysregulatedexpressioninOAaredesignatedbyblack

boxesintherespectivecolumns.

56  

Frominsilicoanalyses,SREBF2wasfoundtohaveconservedGliconsensus

bindingsitesandtobedysregulatedinOA(Kostopoulouetal.2012).SREBF2isthe

majortranscriptionalregulatorofcholesterolhomeostaticgenessuchasINSIG1,which

negativelyregulatescholesterolbiosynthesis(Hortonetal.2003).Whenintracellular

cholesterolishigh,theInsigproteinstetherthetranscriptionalactivatorsofcholesterol

biosyntheticgenes(Srebfs)totheERmembrane,effectivelypreventingexpressionof

targetgenesandsubsequentcholesterolsynthesis(Figure4).Thisend‐product

feedbackinhibitionstipulatesthatwhenintracellularcholesterolislow,theSrebfs

translocatetothenucleustoactivatetranscriptionandrestorehomeostasis(Engelking

etal.2005)(seeChapterOne:Cholesterolhomeostasis).

ToconfirmwhetherSREBF2istargetedbyHhsignaling,weperformeda

chromatinimmunoprecipitationassaywithantibodiesdirectedagainstGli1and

relevantcontrols.PCRresultsusingprimersdesignedspecificallyforhumanSREBF2

confirmedthatGlitranscriptionfactorsbindtothisgene(Figure12).Thissuggeststhat

HhsignalingmayberegulatingexpressionofcholesterolhomeostaticgenesviaGli‐

mediatedregulationofSREBF2.AsaknowntargetofSREBF2andacriticalregulatorof

cholesterolbiosynthesis,thegeneandproteinchangesobservedtoINSIG1provides

supportfortheroleofHhsignalinginregulatingcholesterolhomeostaticgenes.

57  

Figure12.SREBF2chromatinimmunoprecipitation.Chromatinfromprimary

humanOAchondrocyteswasprobedwithanti‐Gli1,IgG(Neg),andanti‐H3(Pos)

antibodies.PCRamplificationoftheSREBF2geneshowsenrichmentintheGli1fraction,

withprimersdesignedtoflanktheputativeGlibindingsite.Additionalcontrols

includedprimersdesignedadjacenttotheGli‐bindingsiteandprimersdesignedto

amplifyrandomlyselectedDNAonthesamechromosome.

58  

Discussion

Toobtainsufficientquantitiesofhigh‐qualityRNAforgeneexpressionanalyses,

wehaveoptimizedamethodforRNAextractionfromhumanarticularcartilage.

Becauseofthelowcelldensityandhighproteoglycancontentofcartilage(McKennaet

al.2000;RoughleyandLee1994),therearemanyadvantagestoisolatingchondrocytes

fromtheextracellularmatrixpriortoRNAextraction:proteincontaminationis

minimized,homogenizationissimplified,exposuretonucleasesislimited,andRNA

yieldismaximized.Removingtheproteinaceousmatrixpreventscopurificationof

proteinwithRNA,effectivelyimprovingRNApurity.Homogenizationismadeeasywith

celllysisinTRIzol®,whichimmediatelyprotectsRNAfromnucleasesandprevents

degradation,effectivelyimprovingRNAintegrity.Finally,chondrocyteisolationallows

concentrationofRNAsuchthattheyieldpersamplepreparationismaximized.These

improvementsmakechondrocyteisolationapracticalstrategyforamelioratingRNA

qualityandyieldfromhumancartilage.

Whenchondrocytesareisolatedfromthecartilaginousmatrix,geneexpression

patternsmaybealtered(Haymanetal.2006).Whetherchondrocyteisolationchanges

geneexpressioncouldnotbeobjectivelydeterminedherebecausetwovariablesare

altereddependently:chondrocytemicroenvironmentandRNAquality(Ruettgeretal.

2010).Haymanetal.reportedthatchondrocyteswhichunderwenta6‐hourdigestion

hadthefewestgeneexpressionchangesrelativetonativecartilage,sothissupportsthe

useofa6‐hourdigestion(Haymanetal.2006).Whetherthechangesingeneexpression

theyobservedarearesultofchondrocyteisolationorRNAqualitycannotbe

ascertainedbecausetheauthorsdidnotreporttheRNAqualityofthesamples

59  

compared.ThisisrelevantsinceRNAqualityhasbeendemonstratedtohavean

independenteffectongeneexpressionassayssuchasreal‐timePCR(Perez‐Novoetal.

2005).

Theexplantmodeldescribedherecanbeconsideredmorephysiologically

relevanttoOApathologythaninvitroexperimentswithextendedcellcultureperiods;

theextracellularmatrixispreservedduringexperimentalmanipulationsandisdigested

justpriortoRNAextraction(vonderMarketal.1977).Topreservetheeffectsongene

expressionduringchondrocyteisolation,pharmacologicalmodulatorsorother

effectorsofgeneexpressioncanbespikedintothedigestionmedia.Changestogene

expressionthatareduetothechondrocyteisolationprocessareequalizedinallgroups,

removingitasanindependenteffectorongeneexpression(Haymanetal.2006).When

comparingexperimentaltocontrolgroups,suchasHhantagonist‐treatedversus

untreatedgroupsasdonehere,changesingeneexpressionthatareduetothe

experimentalmanipulation(suchasHhpathwayinhibition)areidentified.

ForgroupsthatpreferextractingRNAfromtotalcartilage,traditional

homogenizationmethodscanbeused,followedbymultipleroundsofRNA–DNA–

proteinphaseseparationtotrapproteinandDNAintheorganicphasewithout

diminishingRNAintheaqueousphase.Dell’Accioetal.describedamethodthat

involvesthreephaseseparationsusingasequentialphenol–chlorophorm–isoamyl

alcoholprocedurewithincubationandcentrifugationtimesextendingover4hours

(Dell'accioetal.2008).Theoptimizedprotocoldescribedhereprescribesonlytwo

roundsofphaseseparationwithTRIzol®reagentthatcanbecompletedin

approximately2hours.Furthermore,Dell’Accioetal.reportedarecoveryrateof5ng

60  

RNApermilligramofadulthumanarticularcartilage(Dell'accioetal.2008),butusing

thesametissuewithouroptimizedmethod,wewereabletorecoveranaverageof10

ngRNApermilligramofcartilage.

TheoptimizedmethoddescribedhereforchondrocyteisolationandRNA

extractionconferstheabilitytoefficientlyextracthigh‐qualityRNAfromhuman

articularcartilage.ThismethodwasappliedtothestudyofHhsignalingtargetgenesin

osteoarthriticcartilage.Followingmicroarrayanalysis,globalchangestogene

expressionwereassessed,andseveralbiologicalnetworkswereidentified.Fromthese

data,changestothecholesterolbiosyntheticpathwaywereevident.Genechangesto

masterregulatorssuchasINSIG1andSREBF2,aswellastheirtargets(seeChapter

One:Cholesterolhomeostasis),suggestthatHhsignalingisinvolvedinregulatingthis

network.

ThisisthefirststudytoidentifyHhsignalingasaregulatorofcholesterol

homeostaticgenes.SeverallinesofevidencepointtoarelationshipbetweenHedgehog

signalingandcholesterolhomeostasis.Hhligandscarrycholesterolmoieties,Hh

pathwayproteinscontainsterol‐sensingdomains,anddeficitsineitherHhsignalingor

cholesterolhomeostasisproducesimilarcentralnervoussystemabnormalities,facial

dysmorphisms,andskeletaldefects(Eaton2008).Sterollevelshavebeenshownto

regulateHhsignaling(CorcoranandScott2006),andHhsignaltransductionhasbeen

showntorequirecholesterolmetabolism(Stottmannetal.2011).Cholesterolhasbeen

hypothesizedtoplayaroleinHhligandtrafficking,includingsecretionfromthecell

anddistributionacrossthetissue(Eaton2008).Thepresentstudyidentifiescholesterol

homeostaticgenesasHhsignalingtargets,supportingthepossibilityofamutual

61  

regulatoryrelationshipbetweenHhsignalingandcholesterolbiosynthesis.While

othershaveinvestigatedtheroleofsterolsinmodulatingHhsignaling(Corcoranand

Scott2006;Stottmannetal.2011),oursisthefirstevidencetodemonstratetheroleof

Hhsignalinginregulatingsterolhomeostasis.

AsdescribedinChapterOne,cholesterolisvitalforcellularfunctionand

perturbationsinitsregulationcandisrupttissuehomeostasis;cholesterolandlipid

dysregulationhasbeenimplicatedinOApathogenesis;andfinally,aninterplayexists

betweencholesterolandHhsignaling,suggestingamutualregulatoryrelationship.For

thesereasons,wepursuedthehypothesisthatcholesterolhomeostaticgenesaretrue

targetsofHhsignaling.Resultsfromreal‐timePCRvalidationexperiments(Figure9),

Westernblotexperiments(Figure10),insilicoanalyses(Figure11),andchromatin

immunoprecipitation(Figure12)allconfirmedaroleforHhsignalinginregulating

expressionofcholesterolhomeostaticgenes.

WeputforthregulationofSREBF2asthemechanismthroughwhichHh

signalingregulatescholesterolhomeostasis,butotherpossibilitiesexist.Forexample,

LXR/RXRactivationwasidentifiedbymicroarrayanalyses.ModulationoftheLXR

pathwayisanothermechanismthroughwhichHhsignalingmayimpactcholesterol

homeostasis.ThesedatasuggestthatHhsignalingregulatescholesterolhomeostasisin

thecartilage,andthatdysregulationmaycontributetochondrocyte‐related

pathologies.

62  

MaterialsandMethodsIsolationofPrimaryChondrocytes

Humancartilagesampleswereobtainedfrompatientsundergoingtotalknee

replacementsurgeryforclinicallydiagnosedosteoarthritis(meanage64.7years).All

sampleswereobtainedwithinformedconsentundertheapprovaloftheMountSinai

HospitalResearchEthnicsBoard(Toronto,Canada).Articularcartilagewasdissected

awayfromsubchondralboneandconnectivetissue.Cartilagewasweighedthen

washedwithPBS2‐3timesina10‐cmdish.ANo.10scalpelwasusedtofinelymince

thecartilageintopiecesoflessthan1mm3.ForHhmodulation,HhantagonistorHh

agonistwasaddedtotheculturemedia,andcartilagewasincubatedat37°Cand5%

CO2overnight.CulturemediaconsistedofmodifiedDMEM(Wisent,CatNo.319‐005‐

CL)supplementedwith50ug/mLascorbicacid,0.1%BSA,10mMglycerol‐2‐

phosphate,and1Xpenicillin‐streptomycinsolution(Wisent,CatNo.450‐201‐EL),

passedthrougha0.20‐micronfilter.

Cartilagepiecesweretransferredto50‐mLpolypropylenetubescontaining5mL

of5.0‐mmglassbeadsand10mLof0.25%trypsinpergramofcartilage.Withend‐to‐

endrotation,tubeswereincubatedfor45minat37°C.Afterwashingawaytrypsinwith

PBS,10mLof1mg/mLcollagenaseA1pergramofcartilagewasaddedtothetubes.

DigestionmediaconsistedofmodifiedDMEM(Wisent,CatNo.319‐005‐CL)

supplementedwith1Xantibiotic‐antimycoticsolution(Wisent,CatNo.450‐115‐EL)

andcollagenaseA1,passedthrougha0.20‐micronfilter.HhantagonistorHhagonist

wasalsoaddedtothemedia,tomaintainmodulationofHhsignalingduringdigestion.

63  

Oncecartilagewas95%digested,thecellsuspensionwasfilteredthrougha70‐

microncellstrainerintoafresh50‐mLtubeandcentrifugedat1000gfor5minutesat

roomtemperature.CellswerewashedtwicewithPBS,andlysedwithTRIzol®reagent

(approximately1mLpergramofdigestedcartilage).TRIzol®(withcells)was

transferredto2‐mLpolypropylenecryotubescontaining0.25mLof1.0‐mmglassbeads

andtubeswerestoredat‐80°CuntilRNAextraction.

RNAExtractionbyRepeatedPhaseSeparations

IsolatedchondrocytesstoredinTRIzol®werethawedfrom‐80°C.Sampleswere

homogenizedthriceusingaMini‐BeadbeaterTM(BioSpecProducts,OK)for30seconds

at4200rpm,alternatingbetweenice.Followinga20‐minuteincubationonice,tubes

werecentrifugedat10000gfor10minutesat4°Ctoremovecellulardebris.The

supernatantwastransferredtofresh1.5‐mLpolypropylenemicrocentrifugetubes,and

0.2mLchloroformper1mLTRIzol®wasadded.Aftershakingtubesbyhandfor30

secondsanda2‐minuteincubationonice,tubeswerecentrifugedat10000gfor12‐15

minutesat4°C.Theaqueousphasewastransferred(~50%oftotalvolume)tofresh

tubesandmixedwithanequalvolumeofTRIzol®.Theorganicphaseswerestoredat

‐80°Cforfutureproteinextraction.Followinga30‐minuteincubationonice,0.2mL

chloroformper1mLTRIzol®wasaddedagain.Aftershakingtubesbyhandfor30

secondsanda2‐minuteincubationonice,tubeswerecentrifugedat10000gfor12‐15

minutesat4°C.Again,theaqueousphasewastransferred(~50%oftotalvolume)to

freshtubes,butthistimemixedwithanequalvolumeofisopropanol(0.5mLper1mL

TRIzol®).Sampleswereincubatedonicefor5minutesandcentrifugedat12000gfor

64  

25‐30minutesat4°C.TheresultingRNApelletwaswashedwith1mLof70%or75%

ethanolper1mLTRIzol®andcentrifugedat7000gfor5minutesat4°C.The

supernatantwasdiscardedandtheRNApelletwasdriedfor5minutesatroom

temperature.TodissolvetheRNApellet,nuclease‐freewater(approximately25‐40uL

pergramofdigestedcartilage)wasaddedandsampleswereincubatedfor5minutesat

roomtemperature(vs.55‐60°CasrecommendedbytheTRIzol®protocol).

Microarray

Theexperimentaldesignincludedthreehumanosteoarthriticarticularcartilage

samplestreatedinvitrowithaHhantagonistorcontrol,asdescribedaboveinIsolation

ofPrimaryChondrocytes.TheAffymetrixHumanGene1.0STplatformwasused,

whichisspottedwithsynthesizedoligonucleotidesdesignedagainstexons.RNAwas

extractedfromhumanchondrocytestreatedwith10μMC31H42N4O5(Hhantagonist)or

control,asdescribedaboveinRNAExtractionbyRepeatedPhaseSeparations(Ali

andAlman2012).LabelingandhybridizationswereconductedbyTheCentrefor

AppliedGenomicsattheHospitalforSickChildren(Toronto,Canada).Resultswere

analyzedindependentlyforpairedsamplesfromeachofthethreepatients(Control1vs.

Hhantag1,Control2vs.Hhantag2,Control3vs.Hhantag3)usingAffymetrixExpression

ConsoleandPartek®GenomicsSuite.Differentiallyexpressedgeneswerefilteredfor

thosewhichwereeitherupregulatedordownregulatedacrossallthreesamples.

Ingenuity®Pathwayanalysiswasusedtoidentifyfunctionalgenenetworks

representedinthemicroarraydata.Multiple‐sequencealignmentanalysis

(http://mulan.dcode.org/)wasusedtodetectconservedtranscriptionfactorbinding

65  

sitesingenesofinterest(Ovcharenkoetal.2005).Datahavebeenreportedin

compliancewithMIAME(minimuminformationaboutamicroarrayexperiment)

(Brazmaetal.2001),andcanbeaccessedthroughtheGEOdatabase(GSE54749).

Real‐timePCR

Real‐timePCRexperimentswereconductedusingTaqManassaysfromApplied

Biosystems.Resultswerenormalizedtoanendogenouscontrol(ASNS

Hs00155888_m1,ACTBHs99999903_m1,orGAPDHHs99999905_m1),andanalyzed

accordingtothecomparativeCTmethod(∆∆CT).RNAqualitywasassessedusing

COL2A1(Hs00264051_m1),COL10A1(Hs00166657_m1),andMMP13

(Hs00233992_m1).TodeterminetheeffectivenessofHhantagonist[10μMN‐[(3S,5S)‐

1‐(2H‐benzo[3,4‐d]1,3‐dioxolan‐5‐ylmethyl)‐5‐(piperazinylcarbonyl)pyrrolidin‐3‐yl]‐

N‐[(3‐methoxyphenyl)methyl]‐3,3‐dimethylbutanamide(C31H42N4O5)]andHhagonist

[(10μMpurmorphamine,CaymanChemicalCompanyorHhligand(5μg/mlShh‐N,

R&DSystems)]inmodulatingpathwayactivity,levelsoftheHhtargetgeneGLI1

(Hs00171790_m1)wereassayed.MicroarrayvalidationwasperformedusingHMGCR

Hs00168352_m1,HMGCS1Hs00940429_m1,DHCR7Hs01023087_m1,LDLR

Hs00181192_m1,ABCA1Hs01059118_m1,andINSIG1Hs00175767_m1.

Statisticalanalyses

Real‐timePCRdataanalysiswasconductedsuchthatexpressioninthecontrol

groupwasarbitrarilysetto1.Dataarereportedasthemeanwith95%confidence

66  

intervals.StatisticalanalyseswereconductedusingtheStudent’sttestfortwosamples

assumingunequalvarianceswithalphasetto0.05.

Westernblotanalysis

HumancartilageexplantsweretreatedidenticallyasdescribedinIsolationof

PrimaryChondrocytes.Whole‐cellproteinlysatesfromisolatedchondrocyteswere

harvestedusingReporterLysisBuffer(Promega),accordingtothemanufacturer’s

instructions.AntibodiesagainstINSIG1(1:100,sc‐25124‐R,SantaCruz)wereused,

withACTIN(1:5000,A5441,Sigma)asaloadingcontrol.Thesignalsweredetectedand

quantifiedusingtheChemiDocMPImagingSystem(Bio‐Rad,Hercules,CA).These

experimentswereconductedwiththeassistanceofMushriqAl‐Jazrawe.

ChromatinImmunoprecipitation(ChIP)

ChIPwasperformedusingtheChIP‐ITkit(ActiveMotif)accordingtothe

manufacturer’sprotocol.Briefly,primaryhumanchondrocytes(freshlyisolatedfrom

cartilageexplantsasdescribedaboveinIsolationofPrimaryChondrocytes)were

fixedwith1%formaldehydetomaintainprotein‐DNAbinding.DNAwasshearedwith

ninepulsesatpowerleveltwousingaSonicDismembrator(FisherScientific),with15

secondsofsonicationfollowedby30secondsincubationonice.AGli1antibody

(AF3324,R&DSystems),negativecontrolIgG(ActiveMotif)andpositivecontrol

histoneH3(ActiveMotif)wereusedtoimmunoprecipitateDNA‐proteincomplexes

whichwerepurifiedandanalyzedbyPCR.

67  

ChapterThree:

Hhsignalingmodulatescholesterolaccumulationinchondrocytes

CholesterolhomeostasismediatesHedgehogsignalinginosteoarthritis

ShabanaAmandaAli,MushriqAl‐Jazrawe,HeatherWhetstone,RaymondPoon,HenryMa,SarahFarr,MarkNaples,KhosrowAdeli,BenjaminAlman

Manuscriptunderpreparation

68  

Summary

ResultsfromgeneexpressionanalysesinChapterTwosuggestthatHh

signalingregulatesexpressionofcholesterolhomeostaticgenesinchondrocytes.

Becauseseveralimportantregulatorsofcholesterolhomeostasiswerefoundtobe

regulatedbyHhsignaling,theneteffectontotalcholesterollevelswasunclear.Herewe

generatemicewithrobustchondrocyte‐specificcholesterolaccumulation.Using

geneticallymodifiedmice,weexciseInsig1,themajornegativeregulatorofcholesterol

biosynthesis,fromCol2a1‐expressingcells,inabackgroundInsig2knockout.To

interrogatetheroleofHhsignalinginregulatingcholesterolaccumulation,wecrossthe

resultingprogenytomicewithHhactivation(Col2a1‐Gli2)andmicewithHhreduction

(Gli2+/‐).Usingassaystomeasurelipidaccumulationandcholesterolbiosynthesis,we

showthatHhactivationincreasescholesterolproduction,andHhreductiondecreases

cholesterolproduction.Cholesterolhomeostasisisshowntobeimportantfornormal

growthofthelongbones,ascholesterolaccumulationresultsindelayeddifferentiation

ofthegrowthplatechondrocytesanddelayeddevelopmentofthesecondary

ossificationcentre.ThisisthefirstevidencetodemonstratearoleforHhsignalingin

regulatingcholesterolbiosynthesisinchondrocytes.

69  

Introduction WithevidencefromgeneexpressionanalysestosupportaroleforHhsignaling

inregulatingcholesterolhomeostaticgenes,wesoughttodeterminetheeffectofHh

signalingontotalcholesterollevels.Becausethereweregeneexpressionchangesto

manyregulatorsofcholesterolbiosynthesisandhomeostasis(Figure13),theneteffect

ofHhmodulationontotalcholesterollevelswasunclear.Asamajornegativeregulator

ofcholesterolbiosynthesis,theinverserelationshipweobservedbetweenINSIG1

expressionandHhsignalingsuggestedthatHhreductiondecreasedcholesterollevels

andconversely,thatHhactivationincreasedcholesterollevels.Thisisconsistentwith

theincreaseintranscriptionofcholesterolbiosyntheticgenesweobservedwithHh

inhibition(Figure8),anincreasewhichtypicallyoccurswhenintracellularcholesterol

islowinanattempttorestorehomeostasis(seeCholesterolhomeostasis)(Engelking

etal.2005).

ToassesstheroleofHhsignalinginmodulatingcholesterollevelsandthe

subsequentroleofcholesterolinchondrocytebiology,arobustmodelwithcholesterol

perturbationinchondrocyteswasneeded.Cholesterolhomeostasishasbeenthe

subjectofmanystudiesandasaresult,thereareseveraltransgenicmouselineswhich

targetcholesterolhomeostasis(Hortonetal.2002).Becausecholesterolissovitalto

cellhomeostasis,geneticmanipulationofsomegeneshasminimaleffect,likelydueto

functionalredundancybyothergenesand/orregulatorymechanismswhichmaintain

cholesterollevelswithinhomeostaticrange.Forsomecholesterol‐relatedgenes,

ubiquitoustransgenicmiceexhibitlethality(Engelkingetal.2005),whichagainreflects

theimportanceofcholesterolhomeostasis.BrownandGoldstein,whowonthe

70  

Figure13.Schematicofthecholesterolbiosyntheticpathway.Colouredboxes

signifyalteredgeneexpressionwithHhinhibition,asdeterminedbymicroarray

experiments.Redindicatesupregulationandgreenrepresentsdownregulation.Figure

adaptedfromGenMAPP2.0.

NobelPrizeinMedicinein1985fortheirworkoncholesterolhomeostasis,describea

mousewithafloxedInsig1geneandgermlinedisruptionoftheInsig2gene(Brownand

71  

Goldstein1996;Engelkingetal.2005;Engelkingetal.2006).WhensubjectedtoCre‐

mediatedexcisionofInsig1(Nagy2000),theresultingInsigdouble‐knock‐out

(InsigDKO)miceshowrobustaccumulationofcholesterolandtriglyceridesinthetarget

tissue(Engelkingetal.2005;Engelkingetal.2006).

Todeterminewhethersteroldysregulationplaysaroleincartilagehomeostasis,

wegeneratedachondrocyte‐specificknockoutoftheInsig1gene,usingthemouseline

describedbyBrownandGoldstein(Engelkingetal.2005;Engelkingetal.2006).To

determinetheroleofHhsignalinginmodulatingcholesterol,weusedtransgenicmice

whichhavehigher(Hopyanetal.2002)orlower(Moetal.1997)levelsofGli2,the

majortranscriptionalactivatorofHhtargetgenes,inordertoactivateorinhibitHh

signaling,respectively.Bycrossingthesemice,transgenicsweregeneratedinwhichHh

signalingandcholesterolbiosynthesisweremodulatedseparatelyandtogether.This

allowedustoinvestigatetheroleofHhsignalingincholesterolhomeostasis,andto

characterizetheroleofcholesterolhomeostasisinchondrocytes.

72  

ResultsCholesterolaccumulationinchondrocytes:InsigDKOmice

Tomodulatecholesterolbiosynthesisinchondrocytes,weremovedthemajor

negativeregulator,Insig1.Double‐knock‐outexperimentshaveshownthatthetwo

mammalianInsiggenes,Insig1andInsig2,functionredundantlytoregulatesterol

biosynthesisandintheirabsence,miceaccumulatecholesterolandtriglycerides

(Engelkingetal.2005;Engelkingetal.2006).WecrossedtheInsig1(fl/fl);Insig2(‐/‐)

mouselinewiththeCol2a1‐CremouselinetoexciseInsig1inchondrocytes(Engelking

etal.2005;Ovchinnikovetal.2000).ToconfirmCre‐mediatedexcisioninchondrocytes,

articularcartilagewasharvestedfromkneejointsofInsig1(‐/‐);Insig2(‐/‐)mice,

Insig1(‐/fl);Insig2(‐/‐)mice,andInsig1(fl/fl);Insig2(‐/‐)mice.Westernblotanalysis

revealedInsig1proteintobereducedinInsig1(‐/‐);Insig2(‐/‐)cartilagebutnotInsig1(‐

/fl);Insig2(‐/‐)cartilage(Figure14),soallsubsequentanalyseswereperformedwith

double‐knock‐outInsig1(‐/‐);Insig2(‐/‐)mice(InsigDKO)and“control”littermates

[Insig1(fl/fl);Insig2(‐/‐)].ReductionofInsig1expressioninInsigDKOcartilagewasalso

confirmedbyreal‐timePCR(Figure15).

73  

Figure14.WesternblotofINSIG1inInsigDKOmice.ProteinanalysisforINSIG1in

murinecartilageresultingfromthecrossbetweenInsig1(fl/fl);Insig2(‐/‐)andCol2a1‐

Cre.ReductionofINSIG1wasobservedinInsig1(‐/‐)cartilage[Insig1(‐/‐);Insig2(‐/‐),

subsequentlydesignatedInsigDKO]butnotInsig1(‐/fl)cartilage[Insig1(‐/fl);Insig2(‐/‐

)],soallanalysesfocusedonInsig1(‐/‐)andInsig1(fl/fl)cartilage[Insig1(fl/fl);Insig2(‐

/‐),subsequentlydesignatedControl].ACTINisshownasaloadingcontrol.

Figure15.Real‐timePCRofInsig1inmurinecartilage.MicewithHhreduction

(Gli2+/‐),Hhactivation(Col2a1‐Gli2),and/orInsig1excision(InsigDKO)wereassayed

forexpressionofInsig1.ExpressionintheControlgroupwasarbitrarilydefinedas‘1’

anddatafromothergroupsgivenasthemean.Errorbarsare95%confidenceintervals

(n=3;*P<0.05).

74  

TheroleofInsigistotetherthemulti‐proteincomplexcontainingtheSrebf

transcriptionfactorstotheERmembrane(Figure4).IntheabsenceofInsig,Srebf

activitycanbeexpectedtoincrease,sinceitisfreeforprocessingandtranslocationto

thenucleus(Engelkingetal.2005).Theresultofthisisincreasedexpressionof

cholesterolbiosyntheticgenes,andsubsequentincreasedproductionofcholesterol.As

afunctionalassaytomeasuretotalsterolandlipidaccumulationinInsigDKOmice,Oil‐

Red‐Ostaining(Tsezouetal.2010)wasperformedonprimaryculturedchondrocytes

(Figure16,compareInsigDKOtoControl)(Gossetetal.2008).Anincreaseinred

stainingwasapparentuponvisualinspection,andwasalsoconfirmedby

spectrophotometricreadingsofalcohol‐extractedstain(Figure17,compareInsigDKO

toControl).Theseresultsverifyaccumulationofsterolandlipidsinthechondrocytesas

aresultofInsig1excision.

75  

Figure16.Visualizingtotallipidandsterolaccumulationinchondrocytes.

RepresentativeimagesofculturedprimarychondrocytesstainedwithOil‐Red‐O(red

colour)toshowtotalsterolandlipidaccumulationaccordingtogenotype.Scalebar,

100μm.

76  

Figure17.Quantifyingtotallipidandsterolaccumulationinchondrocytes.

Spectrophotometricquantificationofalcohol‐extractedOil‐Red‐Ostainfromcultured

murinechondrocytesshowninFigure16,normalizedtocrystalvioletstain.Errorbars

are95%confidenceintervals(n=3;*P<0.05).

ToaddressthepossibilityofamutualregulatoryrelationshipbetweenHh

signalingandcholesterolhomeostasis,InsigDKOcartilagewasprobedforexpressionof

Hhtargetgenes.IfcholesterollevelsimpactHhpathwayactivity,thenInsigDKO

chondrocyteswithsterolandlipidaccumulationareexpectedtoshowaltered

expressionofHhtargetgenes.Resultsfromreal‐timePCRshowednochangetotheHh

targetgenesGli1,Ptch1,andHhip(Figure18,InsigDKO).ThisdemonstratesthatHh

pathwayactivityisnotalteredbycholesterolaccumulationinthechondrocytes.

77  

Figure18.Real‐timePCRforHhtargetgenesinmice.GeneexpressionforHh

targetsGli1,Ptch1,andHhipinthearticularcartilageofmicewithcholesterol

accumulation(InsigDKO),reductionofHhsignaling(Gli2+/‐;InsigDKO),oractivationof

Hhsignaling(Col2a1‐Gli2;InsigDKO).ExpressioninControlmicewasarbitrarilydefined

as‘1’(dashedline)anddataforeachgenotypegivenasthemean.Errorbarsare95%

confidenceintervals(n=3;*P<0.05).

78  

HedgehogsignalingregulatescholesterolbiosynthesisinchondrocytesToestablishwhetherHhsignalingregulatesexpressionofInsig1inmurine

cartilage,weperformedWesternblotandreal‐timePCRexperimentsusingour

transgenicmicewithHhmodulation.WefirstconfirmedeffectivemodulationofHh

signalinginourmicebyreal‐timePCRforHhtargetgenes(Figure18,Gli2+/‐;InsigDKO

andCol2a1‐Gli2;InsigDKO).ConsistentwithHhmodulationinhumancartilage,an

inverserelationshipbetweenHhsignalingandInsig1expressionwasfoundinmurine

cartilage.ReductionofHhsignalinginGli2+/‐cartilage(Moetal.1997)increasedgene

andproteinexpressionofInsig1,whileactivationofHhsignalinginCol2a1‐Gli2

cartilage(Hopyanetal.2002)decreasedgeneandproteinexpressionofInsig1(Figure

15andFigure19).Real‐timePCRwasalsousedtoconfirmexcisionofInsig1inthe

compoundmutantsCol2a1‐Gli2;InsigDKO,andGli2+/‐;InsigDKO(Figure15).

Figure19.WesternblotofINSIG1inmicewithHhmodulation.Proteinanalysisfor

INSIG1inmurinecartilagewithHhreduction(Gli2+/‐)orHhactivation(Col2a1‐Gli2).

ACTINisshownasaloadingcontrol.

79  

WeinvestigatedtheeffectofHhmodulationonoverallsterolandlipidlevelby

performingOil‐Red‐Ostainingofchondrocytesfromeachmouseline.Wehypothesized

thatHhreductionlowerscholesterollevels,basedontheobservedincreaseinthe

negativeregulatorInsig1,andtheincreaseintranscriptionofcholesterolbiosynthetic

genes,whichisthetypicalresponsetolowintracellularcholesterol.Primary

chondrocytecultureswereestablishedaspreviouslydescribedfromeachofthe

geneticallymodifiedmicewithmodulatedHhsignalingand/orcholesterolbiosynthesis

(Gossetetal.2008).Aspredicted,Oil‐Red‐Ostainingfortotalsterolandlipid

accumulationshowedlowerlevelswithHhreduction(Gli2+/‐)andhigherlevelswithHh

activation(Col2a1‐Gli2;Figure16).Consistentwithremovalofthenegativeregulator

Insig1,InsigDKOchondrocytesshowedlipidandsterolaccumulation,anddespitethe

absenceofthisregulator,Hhsignalingstillmodulatedsterolandlipidlevels(Figure

16).WithHhactivation(Col2a1‐Gli2;InsigDKO),accumulationincreased,andwithHh

reduction(Gli2+/‐;InsigDKO),accumulationdecreased.Thesefindingswereapparent

uponvisualinspectionandwereconfirmedbyspectrophotometricquantification

(Figure17).

Todeterminewhetherthesterolaccumulationcouldbeattributedtocholesterol

biosynthesis,radiotracerexperimentswereconductedwiththehelpofSarahFarrand

MarkNaplesinthelaboratoryofDr.KhosrowAdeli.Primarychondrocyteculturesfrom

eachofthesixmouselineswereincubatedwith50µCi/mL3H‐aceticacidsodiumsalt

overnighttomeasurecholesterogenesisfromacetate.Lipidwasextractedfromthe

chondrocytesandsubjectedtothinlayerchromatographytoseparatecholesterol.

ResultsfromthisexperimentweresimilartothoseofOil‐Red‐O,showingdecreased

80  

cholesterolsynthesiswithHhreduction(Gli2+/‐),andincreasedcholesterolsynthesis

withHhactivation(Col2a1‐Gli2)andInsig1removal(InsigDKO).AgainHhsignalinghad

effectsindependentofInsig1(Col2a1‐Gli2;InsigDKOandGli2+/‐;InsigDKO),

demonstratinganovelroleforHhsignalingintheregulationofintracellularcholesterol

biosynthesisinchondrocytes(Figure20).

Figure20.Quantifyingcholesterolbiosynthesisinchondrocytes.Cholesterol

biosynthesisasmeasuredby3H‐aceticacidsodiumsaltincorporationinpooled

primarychondrocytesfromeachgenotypeshowninFigure16.Measuredintriplicate

andreportedasthemeanrelativetoControlincountsperminute(cpm).Errorbarsare

SEM.

81  

Cholesterolaccumulationinchondrocytesimpairsgrowthofthelongbones Tobeginassessingtheeffectofcholesterolaccumulationinchondrocytebiology,

grossphenotypicobservationsweremade.Visuallyapparentwasthesmallersizeof

InsigDKOmice(Figure21).Weightmeasurementstakenat24weeksand52weeks

confirmedthatInsigDKOmiceweresmallerinsizethantheircontrollittermates

(Figure21).ThesefindingswereconsistentwiththosereportedbyEngelkingetal.in

2006,whofoundthatubiquitousInsigDKOmiceweresmallerthantheircontrol

littermatesat18.5dayspostcoitum(dpc)(Engelkingetal.2006).Todetermine

whetherthissizedeficiencyalsoappliedtotheskeleton,measurementsofthelong

bonesweretakenat16weeks,24weeks,and52weeks.Boththefemurandtibiaof

InsigDKOmicewereshorterthanthoseofcontrolmice,atalltimepoints(Figure22).

Thesedataindicatethatcholesterolaccumulationinthechondrocytesimpairsgrowth

ofthelongbones.

82  

Figure21.GrosssizeofInsigDKOmice.(a)InsigDKOmicearevisiblysmallerthan

theirControllittermatesat52weeks.(b)InsigDKOmice(greybars)weighsignificantly

lessthantheirControllittermates(blackbars)at24weeksand52weeks.Errorbars

are95%confidenceintervals(n=5‐9;*P<0.05).

83  

Figure22.BonelengthinInsigDKOmice.(a)Thefemurand(b)thetibiaofInsigDKO

mice(greybars)aresignificantlyshorterthanthoseofControlmice(blackbars)at16

weeks,24weeks,and52weeks.Errorbarsare95%confidenceintervals(n=10‐32;*P

<0.05).

84  

ToelucidatethecauseofthegrowthimpairmentinInsigDKOmice,growthplate

analyseswereconductedsincethegrowthplateisresponsibleforelongationofthe

longbones.Analysisofthegrowthplatein17.5dpcembryossuggeststhatthe

proliferativezoneisexpandedandthehypertrophiczoneiscondensedinInsigDKO

mice(Figure23).Usedtomarkhypertrophicchondrocytes,typeXcollagenstaining

confirmedreductioninthehypertrophiczoneofthegrowthplate(Figure23).This

deficiencyinchondrocytedifferentiationlikelycontributedtotheimpairedgrowthof

thelongbonesinInsigDKOmice.Inaddition,InsigDKOmiceexhibiteddelayed

developmentofthesecondaryossificationcentre(SOC).By1weekofage,theSOCis

apparentbyradiographyincontrolmice,butnotInsigDKOmice(Figure24).Despite

this,totalskeletonpreparationsdidnotshowovertdifferencesinbone(Alizarenred

staining)norcartilage(Alcianbluestaining)distributioninInsigDKOmiceascompared

tocontrolmice(Figure25).Theseobservationssuggestthatcholesterolaccumulation

inchondrocytesdelaysgrowthofthelongbonesbydelayinggrowthplatechondrocyte

differentiation.

85  

Figure23.GrowthplateanalysesinInsigDKOmice.Thetibiaeof17.5dpcmicewere

stainedwithanantibodyagainsttypeXcollagen(Col10)toidentifythehypertrophic

zoneinthegrowthplate.InsigDKOmicehadashorterhypertrophiczonethanControl

littermates.Errorbaris95%confidenceinterval(n=3;*P<0.05).

86  

Figure24.SecondaryossificationcentredevelopmentinInsigDKOmice.InsigDKO

miceshowedadelayindevelopmentofthesecondaryossificationcentre,visibleby

radiographyat7daysinControlmice(redarrow).

87  

Figure25.SkeletalanalysesinInsigDKOmice.Totalskeletonswerestainedwith

AlcianbluetoidentifycartilageandAlizarenredtoidentifybone.Therewerenoovert

differencesbetweenInsigDKOandControlmiceat2days.

88  

Discussion

Thereismountingevidencetosupportanimportantroleforlipidandsterol

homeostasisinchondrocytebiologyandpathology.Disruptedsterolequilibriuminthe

cartilagemaydirectlyorindirectlyimpactdevelopmentalsignalingpathways,joint

architecture,chondrocytemetabolism,matrixsynthesisordegradation,bone

remodeling,andotherbiologicalprocesses(Eaton2008;Girkontaiteetal.1996;Ijiriet

al.2008;Kanbeetal.2006;Woodsetal.2009;S.WuandDeLuca2004;H.Zhangetal.

2002).Herewegeneratedmicewithchondrocyte‐specificcholesterolaccumulationin

ordertostudythesepossibilities.Asthefirstreportedmousewithcholesterol

accumulationspecifictothecartilage,thisisausefultoolforfurtheringour

understandingofcholesterolhomeostasisinchondrocytebiology.Usingthismodel,we

reporttwoimportantfindingspertainingtochondrocytes;first,thatcholesterol

accumulationisregulatedbyHhsignaling,andsecond,thatcholesterolaccumulation

resultsingrowthimpairmentinthelongbones.

GeneexpressionanalysesinhumanosteoarthriticcartilagedemonstratethatHh

signalingregulatesexpressionofcholesterolhomeostaticgenes.Weshowthatthis

relationshipalsoexistsinmurinecartilage,asHhsignalingalteredexpressionofInsig1

andeffectivelymodulatedlipidaccumulationandcholesterolbiosynthesis.Combined

withtheinsilicodatapresentedinChapterTwoshowingconservationofGliconsensus

bindingsitesbetweenhumanandmouse(Figure11),theseresultssuggestthatHh

signalingmayhavearoleinregulatingcholesterolbiosynthesisacrossmammals.

BecausethisisthefirstreportofHhsignalingregulatingcholesterolhomeostasis,the

universalityofthisrelationshipacrossspeciesremainstobeexplored.

89  

OurfindingsthatHhsignalingwasabletomodulatecholesterolaccumulation

withorwithoutInsig1supportourinitialresultsthatHhsignalingregulatesmultiple

mediatorsofcholesterolhomeostasis.OurdatasuggestthatthereareInsig1‐dependent

andInsig1‐independentmechanismsthroughwhichHhsignalingregulatescholesterol

homeostasis.WeshowthatHhmodulationinverselyalteredgeneandprotein

expressionofInsig1,andthereweresubsequenteffectsontotallipidaccumulationand

cholesterolproduction.BecauseInsigisamajordriverofcholesterolhomeostasis,its

modulationbyHhsignalingwaslikelymediatingthiseffect.However,weshowthatHh

modulationcontinuedtohaveeffectsontotallipidaccumulationandcholesterol

productionintheabsenceofInsig1.ThishighlightstheroleofHhsignalinginregulating

cholesterolhomeostaticgenesotherthanInsig1,suchthatalterationstooverall

cholesterolhomeostasiscanstillbeachievedwithoutInsig1.InChapterTwo,wepoint

toSREBF2asalikelymediatorofHhsignalingoncholesterolhomeostasis.

ThemiceexaminedherecarrygermlinedisruptionofInsig2,whosefunctionis

redundanttothatofInsig1intheliver.Engelkingetal.haveshownthatlossofeither

Insig1aloneorInsig2aloneintheliverresultsinnormallevelsofcholesteroland

triglycerides,andthatlossofeithergenedoesnotcauseanincreaseinexpressionofthe

othergene(Engelkingetal.2005).Despitethis,Insig2hasbeenshowntohaveunique

functionsindifferenttissues,forexample,polymorphismsinadiposetissuesis

associatedwithobesity(Krapivneretal.2008).Potentialcartilage‐specificfunctionsof

Insig2remainssubjectforfutureinvestigation,butthegermlinedisruptionexamined

hereisoneexplanationfortheweightdiscrepancybetweenControlandInsigDKOmice.

90  

AlthoughtheroleofHhsignalinginregulatingcholesterolhomeostasishasnot

beendocumented,thereverseinteractionhasbeenexaminedbyseveralgroups(Eaton

2008;Gilletal.2008;Riobo2012).Hhligandsaremodifiedwithcholesterolmoieties,

theroleofwhichisunclear,butthoughttobeinvolvedinHhligandsecretionand

trafficking.Insupportofthis,thetransmembraneproteinswhichsecreteHhligand

(Dispatched)anddetectHhligand(Patched)containsterol‐sensingdomains,muchlike

thedomainsfoundinthecholesterolbiosyntheticgenesHMGCoAreductase(HMGCR)

andsterol‐regulatory‐element‐bindingproteincleavage‐activatingprotein(SCAP)

(Eaton2008).Takenwiththecurrentfindingswhichidentifycholesterolbiosynthetic

genesasHhsignalingtargets,amutualregulatoryrelationshipbetweenHhsignaling

andcholesterolbiosynthesisisplausible.

OurfindingsdonotsupportthehypothesisthatHhsignalingregulates

cholesterollevelsinordertoregulateactivityofitsownpathway,asInsigDKOmicedid

notshowalterationstoHhsignaling.Thesefindingsareconsistentwiththoseof

Engelkingetal.,whoreportednodifferencesingeneexpressionlevelsofShh,Smo,

Ptch1,orGli1inthepalatetissuesat13.5dpcintotalInsigDKOembryos(Engelkinget

al.2006).BecausetheexactrelationshipbetweensterollevelsandHhactivityremains

unresolved,thisinteractionmeritsfurtherinvestigation.Onepossibilityisthatthe

accumulationofcholesterolandothersterolintermediatesgeneratedinInsigDKOmice

aredifferentfromthosewhichareresponsibleforalteringHhactivity.Othergroups

havedescribedaroleforspecificoxysterolsinregulatingHhsignaling(Corcoranand

Scott2006;Dwyeretal.2007;Gilletal.2008).Inthecurrentstudy,wedonotanalyze

thecompletesterolprofilethatisgeneratedinInsigDKOmice,ratherwetakea

91  

candidateapproachandquantifychangestocholesterolspecifically.Assuch,we

concludethatcholesterolaccumulationinchondrocytesdoesnotalterHhactivity.

BecauseHhtargetgeneexpressionremainedunalteredinourmicewith

cholesterolaccumulation,itisunlikelythatthephenotypicchangesareattributableto

perturbationsinHhsignalinginthecartilage.Weobservedadelayingrowthofthelong

bonesthatcanbeattributedtoimpairedgrowthplatechondrocytedifferentiation,yet

overallskeletonsappearednormal.Thissuggeststhatthecholesterolaccumulationin

InsigDKOmiceimpactedthecontrolledprocessofchondrocytedifferentiationbutnot

skeletalpatterning.Impairedgrowthofthelongboneshasalsobeenreportedtoresult

fromreducedlevelsofcholesterolinthebones(S.WuandDeLuca2004),soitmaybe

thatafinebalanceofcholesterolisrequiredfornormalchondrocytedifferentiation.

Whencholesterolisloweredwithstatintreatment,anincreaseinboneformationis

observed,butresultsdependonthemethodofadministration,dosage,andcarrierused

(Mundyetal.1999;J.B.Park2009;Woodsetal.2009).

Whethertheeffectsofcholesteroldysregulationonthegrowthplateare

mediatedbyHhsignaling,whichisknowntoregulategrowthplatechondrocyte

differentiation(Kobayashietal.2002),isunclear.Onepossibilityisthatcholesterol

levelsalterHhsignaltrafficking,ultimatelyimpactingsignaltransductionand

chondrocytedifferentiation.Insupportofthis,mutantswithdeficitsineitherHh

signalingorcholesterolhomeostasisshowsimilarskeletaldefects(Eaton2008),butthe

preciseinterplay,ifany,issubjectforfuturestudy.

92  

MaterialsandMethodsGeneticallymodifiedmice

WecrossedCol2a1‐Cremice(Ovchinnikovetal.2000)withInsig1(fl/fl);Insig2(‐

/‐)mice(Engelkingetal.2005)toexciseInsig1inCol2a1‐expressingcellsandgenerate

micewithchondrocyte‐specific(Grantetal.2000)cholesterolaccumulation.Mice

expressingCrearereferredtoasInsigDKO[Insig1(‐/‐);Insig2(‐/‐);Cre]andare

comparedtotheirCre‐negative[Insig1(fl/fl);Insig2(‐/‐);“control”]littermates.Cre‐

mediatedrecombinationwasconfirmedbyreal‐timePCRandWesternblotanalysis.To

activateHhsignalling,InsigDKOmicewerecrossedwithCol2a1‐Gli2mice(Hopyanetal.

2002),theprogenyofwhichweredesignatedCol2a1‐Gli2;InsigDKO.ToreduceHh

signalling,InsigDKOmicewerecrossedwithGli2zfdmice(Gli2+/‐)(Moetal.1997),the

progenyforwhichweredesignatedGli2+/‐;InsigDKO.Allanimalstudieswereapproved

bytheTorontoCentreforPhenogenomics.

Geneandproteinanalysis

RNAextractionwasconductedusingTRIzol®Reagent(Invitrogen,CA)

accordingtothemanufacturer’sprotocol.Real‐timePCRexperimentswereconducted

usingTaqManassaysfromAppliedBiosystems,asdescribedintheMaterialsand

MethodssectionofChapterTwo.Resultswerenormalizedtoanendogenouscontrol

(Gapdh4352932EorACTB4352933E),andanalyzedaccordingtothecomparativeCT

method(∆∆CT).ExcisionormodulationofInsig1wasconfirmedusing

Mm00463389_m1.HhpathwaymodulationwasverifiedusingGli1(Mm00494645_m1),

Ptch1(Mm00436026_m1),andHhip(Mm00469580_m1).Proteinextractionwas

93  

conductedusingReporterLysisBuffer(Promega),accordingtothemanufacturer’s

instructions.WesternblotanalyseswereperformedasdescribedintheMaterialsand

MethodssectionofChapterTwo,withtheassistanceofMushriqAl‐Jazrawe.Whole‐cell

proteinlysatesfromprimarymousechondrocyteswereassayedusingantibodies

againstINSIG1(1:100,sc‐25124‐R,SantaCruz)andACTIN(1:5000,A5441,Sigma).

Lipidandcholesterolmeasurement

Primarymurinechondrocytecultureswereestablishedusingarticularcartilage

fromthekneesof5dayoldmice(Gossetetal.2008).Briefly,dissectedcartilagewas

digestedovernightincollagenasetoliberatechondrocytes.Cellswerefilteredand

culturedfor2‐3days,untilusedforexperimentation.Fortotalsterolandlipidlevels,

primarychondrocyteswerefixedwith10%phosphate‐bufferedformalinfor10

minutes.CellswerestainedwithOil‐Red‐Oandextractedstainwasquantifiedby

spectrophotometry(OD500),withreadingsnormalizedtocrystalvioletstain(OD540).

Forcholesterolsynthesis,primarymousechondrocyteswereincubatedwith50µCi/mL

3H‐aceticacidsodiumsaltovernight.Lipidextractedfromthecellsunderwentthin

layerchromatographyforseparationofcomponents,includingcholesterol.

Incorporated3Hwasmeasuredintriplicateandreportedastherelativechangein

countsperminute(cpm).

Skeletalanalyses

Aftersacrifice,mousejointswereharvestedandfixedin10%phosphate‐

bufferedformalinfor4‐7days,dependingonage.Radiographsofjointsweretaken

94  

usingtheFaxitronMX20X‐raysystem.Totalskeletonswereharvestedandfixedin95%

ethanoluntilstainedforcartilagewithAlcianblueandbonewithAlizarenred.Bone

samplesweredecalcifiedin20%EDTA(pH8.0),dehydrated,andembeddedinto

paraffinforsectioningaspreviouslydescribed(Linetal.2009).Forgrowthplate

analyses,immunohistochemistrywasperformedusingantibodiesagainsttypeX

collagen(X53,Quartett,Germany).Theseexperimentswereconductedwiththe

assistanceofHeatherWhetstone.Briefly,5µMserialsectionsweredeparaffinizedwith

xylene,andrehydratedthroughanalcoholgradient(100%,95%,80%,70%),ending

withwater.Followingdigestion,endogenousperoxideactivitywasblockedby3%

peroxideinmethanolfor15minutesatroomtemperature.Non‐specificbindingwas

blockedwith2%normalhorseserum(Vectorlabs)inPBSfor30minutes.Primary

antibodywasincubatedovernight,thenbiotinylatedsecondaryantibodyand

avidin‐linkedperoxidase(VectastainUniversalEliteABCkit,Vectorlabs,Burlingame,

CA)wereusedtodetectprimaryantibodybinding.

Statisticalanalyses

Valuesarereportedasthemeananderrorbarsrepresent95%confidence

intervals.Student’sttestwasusedtodeterminestatisticalsignificancewithalphasetto

0.05.

95  

ChapterFour:

Cholesterolmodulationcanaltertheseverityofosteoarthritis

CholesterolhomeostasismediatesHedgehogsignalinginosteoarthritis

ShabanaAmandaAli,MushriqAl‐Jazrawe,HeatherWhetstone,RaymondPoon,HenryMa,SarahFarr,MarkNaples,KhosrowAdeli,BenjaminAlman

Manuscriptunderpreparation

96  

Summary InChapterTwoandChapterThree,wedemonstratethatHhsignaling

regulatescholesterolbiosynthesisinchondrocytesandthatdysregulationcanimpact

growthofthelongbones.Hereweassesstheeffectofcholesterolaccumulationinthe

articularcartilageandtheresultingimplicationsforosteoarthritispathogenesis.Using

histology,radiography,andgeneexpressionanalyses,weshowthatInsigDKOmice

developtypicalfeaturesofOA.Thisphenotypecanbemodulatedbygeneticreduction

ofHhsignalingviaGli2+/‐,whichnormalizescholesterolaccumulation.MarkersofOA

canalsobereducedbypharmacologicnormalizationofcholesterollevels,withstatin

treatmentinvivoinmurinecartilage,andinvitroinhumancartilage.Wesuggesta

potentialmechanismofactioninwhichstatintreatmentreducesexpressionof

ADAMTS5,themajorproteaseresponsibleforcartilagedegradationinOA.

97  

Introduction ActivationofHedgehogsignalinghasbeenshowntopredisposetothe

developmentofosteoarthritis,yetthemechanismsthroughwhichthisoccursremain

unknown(Linetal.2009).InChapterTwo,weidentifycholesterolhomeostaticgenes

tobetargetsofHhsignaling.InChapterThree,weshowthatHhsignalingmodulates

cholesterolaccumulationinchondrocytes,withimplicationsforgrowthplate

chondrocytedifferentiation.Hereweseektodeterminewhethercholesterol

accumulationinthechondrocytespredisposestoosteoarthritisdevelopment,andto

verifywhethercholesterolaccumulationmediatestheeffectsofHhsignalingon

osteoarthritisdevelopment.

AsdescribedinChapterOne:Experimentalmodelsofosteoarthritis,there

aremanyestablishedmodelsofosteoarthritiswhichallowresearcherstocapturethe

heterogeneityofthediseaseasitisobservedinpatientpopulations.Genetically‐

inducedosteoarthritishasbeendemonstratedthroughmodificationofvariousgenes

andpathways(Y.Lietal.2007),includingtheHhsignalingpathway,inwhichactivation

predisposestoosteoarthritis(Linetal.2009).Age‐relatedosteoarthritisiscaptured

whenanimalsareallowedtoreachadvancedageanddevelopspontaneous

osteoarthritis(Miyakietal.2010;Pouletetal.2014).Mechanically‐induced

osteoarthritisiscapturedbysurgicalprocedureswhichcreateinstabilityinthejoint

(Kamekuraetal.2005);thismodelsosteoarthritisthatresultsfromtraumainpatients.

InthecurrentstudyweusegeneticmodificationbyInsig1excisionand/orGli2

overexpressioninCol2a1‐expressingcells,agingofupto24weeks,andmechanical

98  

instabilitybysurgicalexcisionofthemedialmeniscustodeterminewhethercholesterol

modulationinthechondrocytescanaltertheseverityofosteoarthritis.

Toassessdevelopmentoftheosteoarthriticphenotype,thereareestablished

radiographicfeatures,histologicchanges,andgeneexpressionmarkers.Radiographs

aremostcommonlyusedtodiagnoseOAinthepatientpopulation.IndicatorsofOA

includesubchondralbonesclerosis(thickening)andirregularityofthebonecontour

(KellgrenandLawrence1957).Histologicanalysesaretypicallyconductedfollowing

totaljointreplacementsurgeries,toconfirmOAdiagnosis.Gradingscalesareusedto

identifythestageandseverityofOAbasedonpredefinedcriteria(Glassonetal.2010;

Mainil‐Varletetal.2003).IndicatorsofOAincludecartilagefibrillationanderosion,and

chondrocytehypertrophy.Genechangestypicallyincludeupregulatedexpressionofthe

proteaseswhichmediatecartilagedegradation,suchasMMP13andADAMTS5(Glasson

etal.2005).Weusetheseradiographic,histologic,andgeneexpressionmarkersto

assesstheseverityofosteoarthritisinourmicewithmodulatedcholesterollevels.

ModulationofcholesterolisachievedbycholesterolaccumulationinInsigDKOmice

andCol2a1‐Gli2mice,orbycholesterolreductioninGli2+/‐miceorwithstatin

treatment.Wealsoassesswhetherpharmacologiccholesterolinhibitionwithstatin

treatmentcanattenuatetheexpressionofOAmarkersinhumancartilage.

99  

Results Cholesterolaccumulationinchondrocytespredisposestoosteoarthritis

TodeterminewhethercholesterolaccumulationinthecartilageimpactsOA

development,kneesof24‐week‐oldInsigDKOmiceandrelevantcontrolswere

examinedfortypicalmarkersofOA(Poole1999).Methodsusedincludehistology,

radiography,andgeneexpressionanalysis.Examinationofthearticularcartilagein

InsigDKOmicerevealedalterationstosubchondralbone,cartilagemineralization,

irregularcelldistribution,andcartilageerosion(Figure26).Thishistologicevaluation

wasbasedoncriteriausedforscoringfeaturesofosteoarthritis,assetoutbythe

InternationalCartilageRepairSociety(ICRS)(Mainil‐Varletetal.2003).OverallICRS

scoreswerereducedfrom17.3incontrolmiceto11.3inInsigDKOmice(P<0.05),

wherelowerscoresrepresentmoresevereosteoarthritis(Mainil‐Varletetal.2003)

(Table5).InsigDKOmiceexhibitedincreasedchondrocytehypertrophyinthe

superficialzone,asmarkedbytypeXcollagenstaining(Figure27).Radiographic

resultsshowedcartilagemineralizationasmentionedabove,butalsoirregularityinthe

bonecontourwithchangestothesubchondralbone,includingsclerosis(Figure28).

InsigDKOmicehadincreasedexpressionofMmp13andAdamts5,proteasesknownto

mediatecartilagedegradation(Glassonetal.2005)(Figure29).Althoughthe

phenotypeisnotsevere,itisrobust,consistentwithosteoarthritisdevelopmentin

InsigDKOmice.

100  

Figure26.HistologicanalysesofInsigDKOcartilage.Representativehistological

sectionsshowinghaematoxylinandeosinstainingofthekneejointsin24‐week‐old

mice.Boxesdelineatemagnifiedregionsshownbelowtohighlightchangesin

chondrocytemorphologyanddistribution.InsigDKOmiceshowcharacteristicOA

changes,includingcartilageerosion.Scalebar,100μm.

101  

ICRS Cartilage Scoring Scale Control Gli2+/- InsigDKO

Gli2+/-; InsigDKO

1) Surface 3.0 ± 0.0 3.0 ± 0.0 2.3 ± 0.9 3.0 ± 0.0 2) Matrix 3.0 ± 0.0 2.7 ± 0.7 2.5 ± 1.0 3.0 ± 0.0 3) Cell Distribution 2.5 ± 0.6 2.0 ± 0.0 2.0 ± 0.8 2.3 ± 0.5 4) Cell Population Viability 3.0 ± 0.0 2.7 ± 0.7 2.8 ± 0.5 2.8 ± 0.5 5) Subchondral Bone 2.8 ± 0.5 3.0 ± 0.0 0.5 ± 0.6 2.5 ± 0.6 6) Cartilage Mineralization 3.0 ± 0.0 3.0 ± 0.0 1.3 ± 0.5 2.5 ± 0.6

ICRS Summary Score 17.3 16.3 11.3 16.0 P-value (U-test) P=0.190 P=0.020 P=0.178

Table5.ICRSScoringofGli2+/‐,InsigDKO,andGli2+/‐;InsigDKOcartilage.The

InternationalCartilageRepairSociety(ICRS)scorewasusedtogradehistologic

sectionsinablindedmanner.Themeanand95%confidenceintervalaregivenforeach

criterion,andasummaryscoreisprovidedwithMann‐WhitneyUstatisticalanalysisto

determinesignificantdifferences.Kneejointsfrom24‐week‐oldmiceweregradedfor

eachgenotypeshowninFigure26.StatisticalanalysescomparedeachofGli2+/‐,

InsigDKO,andGli2+/‐;InsigDKOtoControl.OnlythescorebetweenInsigDKOand

Controlwassignificant(P<0.05).

102  

Figure27.TypeXcollagenstainingofInsigDKOcartilage.Representative

histologicalsectionsshowingtypeXcollagenimmunohistochemistryinthearticular

cartilageofthefemurat24weeksforeachgenotypeshowninFigure26.InsigDKOmice

showincreasedchondrocytehypertrophyinthesuperficialzone.Scalebar,100μm.

Figure28.RadiographsofInsigDKOknees.Representativeradiographicimagesof

mousekneescorrespondingtothegenotypesshowninFigure26,showingalateral

viewat16weeksofage.InsigDKOmiceshowirregularityinthebonecontourand

increasedsubchondralsclerosis.

103  

Figure29.Real‐timePCRofOAmarkersinInsigDKOcartilage.Geneexpressionof

proteasesknowntomediatecartilagedegradationinosteoarthritis,Mmp13and

Adamts5,fromcartilagemicrodissectedfromthekneesof24‐week‐oldmice.

ExpressionofControlwasarbitrarilydefinedas‘1’,anddataforeachgenotypegivenas

themean.Errorbarsare95%confidenceintervals(n=4,*P<0.05).

ThecharacteristicOAchangesobservedbyhistology,radiography,andgene

expressioninInsigDKOmicewereallrescuedwhencholesterolwasloweredthrough

geneticreductionofHhsignaling(Gli2+/‐;InsigDKO).ResultsinChapterThreeshow

thatGli2+/‐;InsigDKOmicehavecholesterollevelsthatarecomparabletocontrolmice,a

rescueofthecholesterolaccumulationseeninInsigDKOmice.Demonstratedby

previousstudiesandconfirmedherewiththeGli2+/‐mice,Hhreductionprotectsagainst

osteoarthritisdevelopment(Linetal.2009;J.Zhouetal.2014),apotentialmechanism

throughwhichisbyloweringcholesterol.InGli2+/‐;InsigDKOmice,cartilage

mineralizationandcartilageerosionwerereduced,andthesubchondralboneandcell

104  

distributionwascomparabletothatofcontrolmice(Figure26).OverallICRSscores

wereimprovedfrom11.3inInsigDKOmiceto16.0inGli2+/‐;InsigDKOmice,whichwas

notsignificantlydifferentfromthescoreof17.3incontrolmice(P<0.05;Table5).

HypertrophyinthesuperficialzoneofGli2+/‐;InsigDKOcartilagewasalsoreduced,as

markedbytypeXcollagenstaining(Figure27).Radiographswerecomparabletothose

ofcontrolmice(Figure28)andexpressionofMmp13andAdamts5wasreduced

(Figure29).Takentogether,theseresultssuggestthatreductionofHhsignalingin

Gli2+/‐;InsigDKOmicenormalizedcholesterolaccumulationandattenuatedthe

osteoarthritisphenotype.

105  

Cholesterolinhibitionprotectsagainstosteoarthritisinmice

Toassesswhetherpharmacologicsterolnormalizationcouldmitigate

osteoarthritisprogression,miceweretreatedwith3mg/kg/daylovastatin,bysurgical

implantationofaslow‐releasedrugpellet(InnovativeResearchofAmerica,FL).The

dosageisphysiologicallyrelevantwithnotoxicityeffectsbeingreported(MacDonaldet

al.1988;Reagan‐Shawetal.2008).Pelletswereplacedadjacenttothesynovial

membraneofthekneetoinhibitcholesterolproductionfor8weeks.Weexaminedmice

whichreliablydeveloposteoarthritis,includingmicewithcholesterolaccumulation

(InsigDKO),Hhactivation[Col2a1‐Gli2(Linetal.2009)andCol2a1‐Gli2;InsigDKO],and

mechanicaljointinstability[medialmeniscectomysurgery(Kamekuraetal.2005;Lin

etal.2009)performedoncontrolandInsigDKOmice].

Effectivemodulationofthecholesterolpathwaywasconfirmedbyanincreasein

HMGCRexpressioninthearticularchondrocytes,anincreasewhichoccurswhen

intracellularcholesterolislow,asexpectedwithstatintreatment(Figure30).There

werenodetectablealterationstosystemiclevelsofcholesterol;serumlevelsremained

unchangedinstatin‐treatedmiceascomparedtoplacebo‐treatedmiceafterthe8‐week

period(Figure31).Thissuggeststhatthestatintreatmentwaslocallyeffectiveinthe

articularcartilageofthesynovialjointwithouthavingasignificantsystemiceffect.

106  

Figure30.Verificationofstatintreatmenteffectivenessinthecartilage.Increased

HMGCRexpressionisusedtoindicatedecreasedintracellularcholesterolwhichisthe

anticipatedeffectofstatintreatment.(a)Representativehistologicalsectionsshowing

HMGCRimmunohistochemistry(brown)inthearticularcartilageat16weeksfor

InsigDKOmicetreatedwithplaceboorstatin.Scalebar,50μm.(b)Real‐timePCRof

HMGCRinhumanosteoarthriticarticularcartilageexplantstreatedwithstatin.

Expressioninthecontrolgroupwasarbitrarilydefinedas‘1’anddatafromthestatin‐

treatedgroupgivenasthemean.Errorbaris95%confidenceinterval(n=4;*P<0.05).

107  

Figure31.Serumcholesterollevelfollowingstatintreatment.Systemiclevelsof

cholesterolintheserumofmicethatwereimplantedwithaslow‐releasePlaceboor

StatinpelletweremeasuredusingtheAmplexRedassay.Nostatisticallysignificant

differencesweredetectedafterthe8‐weektreatmentperiod.Errorbarsare95%

confidenceintervals(n=3).

108  

Ineachmousewithosteoarthritis,statintreatmentrescuedtheOAphenotype.

Examinationofthehistologyrevealedreducedcartilagefibrillationanderosion,with

normalizedcelldistribution(Figure32andFigure33).Therewerenoobvious

differencesinControl(Figure32)orSham‐operated(Figure33)micethatwere

treatedwithstatin.InmicewithanOAphenotype,statintreatmentsignificantly

improvedoverallICRSscores(Table6andTable7).Forexample,InsigDKO+Surgery

micescored11.3withplaceboand16.7withstatintreatment(P<0.05),wherehigher

scoresrepresentlesssevereosteoarthritis(Mainil‐Varletetal.2003)(Table7).

Furthermore,statintreatmentattenuatedchondrocytehypertrophyinthesuperficial

zone,asindicatedbyreducedtypeXcollagenstaining(Figure34andFigure35).

Thesedatasuggestthatstatintreatmentcanprotectagainstosteoarthritisdevelopment

inmice.

109  

Figure32.StatintreatmentinControl,Col2a1‐Gli2,InsigDKO,andCol2a1‐

Gli2;InsigDKOmice.Representative histological sections showing haematoxylin and eosin

staining of the knee joints in 16-week-old mice that were implanted with a slow-release

Placebo or Statin pellet at 8 weeks of age. Statin treatment reduces features of OA. Scale bar,

100 μm.

110  

Figure33.Statintreatmentinmicewithsurgically‐inducedOA.Representative

histologicalsectionsshowinghaematoxylinandeosinstainingofthekneejointsin16‐

week‐oldmicethatwereimplantedwithaslow‐releasePlaceboorStatinpellet.Mice

weresubjecttoexcisionofthemedialmeniscus(Surgery)orshamoperations(Sham)

at8weeksofage.StatintreatmentreducesfeaturesofOA.Scalebar,100μm.

111  

ICRS Cartilage Scoring Col2a1-Gli2 InsigDKO Col2a1-Gli2;

InsigDKO Scale Placebo Statin Placebo Statin Placebo Statin

1) Surface 2.7 ± 0.7 3.0 ± 0.0 2.8 ± 0.5 3.0 ± 0.0 2.3 ± 0.7 3.0 ± 0.0 2) Matrix 2.7 ± 0.7 2.7 ± 0.7 2.8 ± 0.5 3.0 ± 0.0 2.0 ± 0.0 3.0 ± 0.0 3) Cell Distribution 2.0 ± 0.0 2.7 ± 0.7 2.3 ± 0.9 2.7 ± 0.7 2.0 ± 0.0 2.7 ± 0.7 4) Cell Population Viability 2.7 ± 0.7 3.0 ± 0.0 2.8 ± 0.5 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0 5) Subchondral Bone 2.7 ± 0.7 3.0 ± 0.0 1.5 ± 0.6 3.0 ± 0.0 2.0 ± 0.0 2.7 ± 0.7 6) Cartilage Mineralization 2.0 ± 1.1 3.0 ± 0.0 1.8 ± 0.5 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0

ICRS Summary Score 14.7 17.3 13.8 17.7 13.7 17.3 P-value (U-test) P=0.043 P=0.026 P=0.046

Table6.ICRSScoringofCol2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKO

cartilage.TheInternationalCartilageRepairSociety(ICRS)scorewasusedtograde

histologicsectionsinablindedmanner.Themeanand95%confidenceintervalare

givenforeachcriterion,andasummaryscoreisprovidedwithMann‐WhitneyU

statisticalanalysistodeterminesignificantdifferences.Kneejointsfrom16‐week‐old

miceweregradedforgenotypesshowninFigure32.Statisticalanalysescompared

placebotostatintreatmentforeachofCol2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKO.

Statintreatmentwasfoundtohaveasignificantdifferenceinallgroups(P<0.05).

112  

Surgery

Control InsigDKO ICRS Cartilage Scoring Scale Placebo Statin Placebo Statin

1) Surface 2.0 ± 0.0 2.7 ± 0.7 1.3 ± 0.7 2.7 ± 0.7 2) Matrix 2.7 ± 0.7 3.0 ± 0.0 2.0 ± 1.1 3.0 ± 0.0 3) Cell Distribution 1.7 ± 0.7 2.7 ± 0.7 1.3 ± 0.7 2.3 ± 0.7 4) Cell Population Viability 2.0 ± 0.0 2.7 ± 0.7 2.3 ± 0.7 2.7 ± 0.7 5) Subchondral Bone 1.7 ± 0.7 2.3 ± 0.7 1.7 ± 0.7 3.0 ± 0.0 6) Cartilage Mineralization 2.7 ± 0.7 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0

ICRS Summary Score 12.7 16.3 11.3 16.7 P-value (U-test) P=0.046 P=0.046

Table7.ICRSScoringofcartilageaftersurgically‐inducedOA.TheInternational

CartilageRepairSociety(ICRS)scorewasusedtogradehistologicsectionsinablinded

manner.Themeanand95%confidenceintervalaregivenforeachcriterion,anda

summaryscoreisprovidedwithMann‐WhitneyUstatisticalanalysistodetermine

significantdifferences.Kneejointsfrom16‐week‐oldmiceweregradedforgenotypes

showninFigure33.StatisticalanalysescomparedplacebotostatintreatmentinControl

andInsigDKOmicethatunderwentexcisionofthemedialmeniscus(Surgery).Statin

treatmentwasfoundtohaveasignificantdifferenceinbothgroups(P<0.05).

113  

Figure34.TypeXcollagenstainingofCol2a1‐Gli2,InsigDKO,andCol2a1‐

Gli2;InsigDKOcartilage.RepresentativehistologicalsectionsshowingtypeXcollagen

immunohistochemistryinthearticularcartilageofthefemurat16weeksforeach

genotype/treatmentgroupshowninFigure32.StatintreatmentreducestypeX

collagenstaininginthesuperficialzone.Scalebar,100μm.

114  

Figure35.TypeXcollagenstainingofcartilageaftersurgically‐inducedOA.

RepresentativehistologicalsectionsshowingtypeXcollagenimmunohistochemistryin

thearticularcartilageofthefemurat16weeksforeachgenotype/treatmentgroup

showninFigure33.StatintreatmentreducestypeXcollagenstaininginthesuperficial

zone.Scalebar,100μm.

115  

StatintreatmentreducesOAmarkersinhumancartilage

Toinvestigatetheutilityofcholesterolinhibitionasapotentialtherapeutic

interventioninhumanosteoarthritis,articularcartilageexplantsweretreatedwith10

µMlovastatin.Thisconcentrationwaschosenbecauseitiswidelyusedinexperiments,

showinganeffectonthecholesterolpathwaywithouttoxicityeffects(Jakobisiaketal.

1991;MacDonaldetal.1988).Weconfirmedeffectivemodulationofthecholesterol

pathwaywithanincreaseinHMGCRexpressioninthecartilage,anincreasewhich

occurswhenintracellularcholesterolislow,asexpectedwithstatintreatment(Figure

30).

Treatmentofhumanosteoarthriticcartilagewithstatinresultedin

downregulatedexpressionoftheproteasesMMP13andADAMTS5(Figure36).These

proteasesareknowntomediatecartilagedegradationinOA(Glassonetal.2005).To

exploretherelationshipbetweenstatintreatmentandADAMTS5expression,human

osteoarthriticchondrocytesweretransfectedwithanADAMTS5luciferasereporter

constructandtreatedwithlovastatin.ADAMTS5promoteractivitywasreduced

followinglovastatintreatment(Figure37).Theseexperimentswereconductedwith

theassistanceofRaymondPoonandHenryMa.Becausecholesterollevelsareknownto

regulateSrebfs(BrownandGoldstein1997),weexaminedtheADAMTS5promoterfor

potentialSrebfbindingsites.Insilicoanalyses(usingtheMulantool‐

http://mulan.dcode.org)(Ovcharenkoetal.2005)revealedaSREbindingsitethatis

conservedbetweenhumanandmouse,suggestingthatthisgeneisapotentialtargetof

theSrebftranscriptionfactors(Figure38).Thesedataindicatethatthecholesterol

pathwaycanimpactosteoarthritisbymodulatingexpressionofADAMTS5.

116  

Figure36.Real‐timePCRofOAmarkersafterstatintreatment.Geneexpression

showingareductioninOAmarkersMMP13andADAMTS5inhumanosteoarthritic

cartilageexplantstreatedwithstatin(greybars).Expressioninthecontrolgroup(black

bars)wasarbitrarilydefinedas‘1’anddatafromthestatin‐treatedgroupgivenasthe

mean.Errorbarsare95%confidenceintervals(n=4;*P<0.05).

117  

Figure37.ADAMTS5reporterconstructassay.LuciferaseactivityfromtheADAMTS5

promoterconstructtransfectedintoprimaryhumanOAchondrocytes,treatedwith

controlorstatin.Measuredintriplicateandreportedasrelativelightunits(RLU).Error

barsareSEM.

Figure38.ADAMTS5promoteranalysis.SchematicoftheADAMTS5promoter

showingaSREBFconsensusbindingsite(redtick)thatisconservedbetweenmouse

andhuman(fromMULANanalysis,http://mulan.dcode.org).

118  

Discussion Theimportanceofcholesterolhomeostasisinarticularchondrocytefunctionand

articularcartilageintegrityisdemonstratedhere,asmicewithcholesterol

accumulationdevelopcharacteristicfeaturesofOA.Theroleofcholesterol

dysregulationinosteoarthriticcartilagehasnotbeenthoroughlyexplored.

Epidemiologicalstudiesdescribesystemiccholesterollevelscorrelatingwith

radiographicOA(Al‐Arfaj2003;Sturmeretal.1998),butthesefindingsare

complicatedbythefactthatserumlevelsofcholesteroldonotnecessarilycorrelate

withintra‐articularlevelsofcholesterol(Preteetal.1993).Weexamineknownmodels

ofosteoarthritis(Col2a1‐Gli2andmedialmeniscectomy)aswellasourmicewith

cartilage‐specificcholesterolaccumulation,andfindthatstatintreatmentcanrescue

theOAphenotypeineach.Thereareseveralimplicationsofthesefindings.

FindingthatstatintreatmentrescuedtheOAphenotypeinCol2a1‐Gli2mice

suggeststhattheHhactivationinthesemicecausesanaccumulationofcholesterol

whichisnormalizedbystatintreatment.Insupportofthis,weshowthatHhactivation

resultsincholesterolaccumulationinchondrocytesinChapterThree.Findingthat

statintreatmentrescuedtheOAphenotypeinducedbymedialmeniscectomyincontrol

micesuggeststhatthereisanabnormallevelofcholesterolaccumulationinthesemice

aswell,alevelthatissubsequentlynormalizedbystatintreatment.Onepossibilityis

thatcholesterolaccumulationinthesecontrolmiceresultsfromactivationofHh

signaling,whichwehavepreviouslyshowntobecomeactiveinmechanicalmodelsof

OA(Linetal.2009).Hhisknowntobeamechanoresponsivegene,wheremechanical

stress(suchasthatwhichresultsfollowingmedialmeniscectomy)inducesthe

119  

expressionofIhhinchondrocytes(Q.Wuetal.2001).OnceHhbecomesactiveinthe

chondrocytes,itmayinduceexpressionofcholesterolbiosyntheticgenes,cause

cholesterolaccumulation,andinduceOApathogenesis.

FindingthatstatintreatmentrescuedtheOAphenotypeinInsigDKOmice

suggeststhattheOAchangesareattributabletocholesterolaccumulation,andnotHh

modulation.Sincereal‐timePCRresultsshowednochangetoHhtargetgenesin

InsigDKOcartilage,theOAphenotypecanbeattributedtocholesterolaccumulationand

notperturbationstoHhsignaling(seeChapterThreeforfurtherdiscussion).Despite

hypothesessurroundingamutualregulatoryrelationshipbetweenHhsignalingandthe

cholesterolpathway,ourdatasupportaroleforHhsignalinginupstreamregulationof

cholesterolhomeostasisonly.WeshowthatHhreductioninGli2+/‐;InsigDKOmice

reducesthecholesterolaccumulationseeninInsigDKOmice,andeffectivelyrescuesthe

OAphenotype.AsdescribedinChapterThree,thissupportsanInsig1‐independent

mechanismthroughwhichHhsignalingregulatescholesterolhomeostasis,a

mechanismthatislikelydependentonHhregulationofSrebf2.

FindingthatstatintreatmentrescuedtheOAphenotypeinInsigDKOmicealso

suggeststhattheOAchangesareattributabletocholesterolaccumulationandnot

deformityofthejointduringdevelopment.OAisknowntoresultfromskeletal

dysplasia(Sahlmanetal.2004)sothepossibilityexiststhatcholesterolaccumulationin

InsigDKOmicecompromisesnormalskeletalandjointdevelopment,which

subsequentlypredisposestoOA.Thispossibilityispartiallysupportedbyourfindings

inChapterThree,whichshowimpairedgrowthofthelongbonesinInsigDKOmice;

butpartiallynegatedbyourfindingsinChapterThree,whichshownormaltotal

120  

skeletonsinInsigDKOmice.OurbestevidencetonegatethepossibilitythattheOA

phenotypeinInsigDKOmiceresultsfromskeletaldysplasiaisprovidedbythestatin

rescue.PharmacologiccholesterolinhibitionreducedtheOAfeaturesinInsigDKOmice

after8weeks.ThisissupportedbyfindingsfromWuetal.,whoshowthatcholesterol

inhibitioninratlongbonesreducedchondrocytehypertrophyinvitro(S.WuandDe

Luca2004).Ifthephenotypewerebeingcausedsolelybyjointdeformity,reductionin

cholesterollevelswouldnothaveanimpact.Rescuebystatintreatmentsuggeststhat

theOAphenotypewascausedatleastinpartbycholesterolaccumulationintheadult

cartilage.

Theimplicationsofthestatineffectinhumanosteoarthriticcartilagearealso

far‐reaching.Reducingexpressionoftheproteasesthatareresponsibleforcartilage

degradationisaviablestrategyforpreventingthecartilageerosionthatis

characteristicofosteoarthritis.Thisisofparticularimportancesincethereare

currentlynoeffectivetherapeuticsforpreventingcartilagedegradation.Wehave

previouslydemonstratedthatHhsignalingisactivatedinhumanosteoarthritic

cartilage(Linetal.2009),sothemechanismofactioninhumancartilagemaybethe

sameasthatinmousecartilage,wherebyactivationofHhsignalingcausescholesterol

accumulationandexacerbatesOAprogression.InstancesofOAresultingfromskeletal

deformityortraumainpatients(HunterandEckstein2009)maybecomparabletoour

medialmeniscectomy,wherecompromisedjointarchitectureandabnormaljoint

loadingcausesmechanotransductionofHhsignalingandsubsequentaccumulationof

cholesterol.Ourdatasuggestthatpatientswhosufferthesedeformitiesortraumasmay

benefitfromlocalstatintreatmenttopreventcartilageloss.

121  

Resultsfromourstatinexperimentshighlightapotentialmechanismthrough

whichcholesterolaccumulationmaybemediatingOAprogression:regulationof

ADAMTS5,themajorproteaseresponsibleforcartilagedegradation(Glassonetal.

2005).InsilicoanalysesrevealedaSREbindingsiteintheproximalpromoterregionof

theADAMTS5genethatisconservedbetweenhumansandmice.TheSrebf

transcriptionfactorsareknowntobindtotheSREsiteandmediatetranscriptionof

targetgenes.TheSrebftranscriptionfactorsareresponsivetointracellularcholesterol

levels.Whencholesterolishigh,thesefactorsaretetheredtotheERmembrane,and

transcriptionoftargetgenesisprevented(GoedekeandFernandez‐Hernando2012).

HereweshowcholesterolaccumulationpositivelycorrelateswithOAprogression,soif

Srebf2istetheredtotheERmembrane,itcannolongerpreventrepressionofADAMTS5

transcription,andlevelsofthisproteaseincrease.Inthealternativescenariowhen

statintreatmentisappliedandintracellularcholesterolislowered,processedSrebf2

translocatestothenucleustorepressexpressionofADAMTS5andpreventcartilage

degradation.TheSrebftranscriptionfactorshavebeenshowntofunctionwithco‐

regulatorstonegativelyregulatetargetgeneexpressioninthismanner(Bennettetal.

1999;Edwardsetal.2000).

AlthoughtheexactmechanismofSrebf‐mediatedregulationofADAMTS5

remainstobeelucidated,wehypothesizethatthelevelofintracellularcholesterolisin

partresponsibleforregulatingSrebf2andtheresultingOAphenotype.Whether

cholesterolaccumulationhasadditionaleffectsincontributingtoOApathogenesis

remainstobedetermined,andisdiscussedfurtherinChapterFive.

122  

MaterialsandMethodsOAassessment

Radiography

MousekneejointswereassessedforthedevelopmentofOAbyradiography,

histology,andreal‐timePCR.Forradiographs,miceweresacrificed,theirjoints

harvested,fixedin10%phosphate‐bufferedformalinfor4‐7days(dependingonage)

andimagedusingtheFaxitronMX20X‐raysystem.Lateralandanterior‐posterior

radiographsweretakenandassessedforsubchondralbonesclerosis(localized

whitening),irregularityinbonecontour,andmineralizationofthecartilage.These

featureswereusedtoevaluatesamplesforICRSscoring(seebelow).

Histology

Forhistology,kneejointsweredecalcifiedin20%ethylenediaminetetraacetic

acid(EDTA)atpH7.4,dehydratedinserialconcentrationsofethanol,andembedded

intoparaffinwax.ForOAassessment,5μmserialsectionsweremade,and

representativesectionsstainedwithhematoxylinandeosin(H&E).For

immunohistochemistry,sectionsweredeparaffinized,rehydrated,enzyme‐digested,

andstainedwithtypeXcollagen(X53,Quartett,Germany)orHMGCoA‐reductase

(ab174830,abcam;usedtoverifyeffectivenessofstatintreatmentinmurine

chondrocytes,seeLovastatintreatment).AdescriptionofthisisgivenintheMaterials

andMethodssectionofChapterThree.Theseexperimentswereconductedwiththe

assistanceofHeatherWhetstone.

123  

GradingforOAseveritywasperformedinablindedmannerusingtheICRS

scoringsystem(Mainil‐Varletetal.2003),asdescribedbyLinetal.previously(Linetal.

2009).Thisscoringsystemincludessixcategorieswhichcanbeassignedvaluesof0for

mostseverephenotypeto3forleastseverephenotype.Thecategoriesincludesurface

continuity,matrixcomposition,celldistribution,cellviability,subchondralbone

integrity,andcartilagemineralization.Theauthorsrecommendthateachofthese

categoriesbeconsideredseparately,buthereweprovideanoverallsummaryscoreto

easeinterpretationofphenotypeseverity.Assuch,foroverallscores,themostsevere

possiblescoreis0whiletheleastseverepossiblescoreis18(Mainil‐Varletetal.2003).

StatisticalsignificancewasdeterminedusingaMann‐WhitneyUtest,withthe

assistanceofMushriqAl‐Jazrawe.

TheICRSscoringsystemwaschosenoverotherscoringsystems,suchasthe

OARSIscale(Glassonetal.2010),forthreereasons.First,tocomparescoresto

previouslypublishedICRSscoresfromourlabgroup(Linetal.2009),facilitatingdirect

comparisonoftheseverityoftheOAphenotype.Second,toassesstherepairthatwas

anticipatedtoresultfromHhreductioninGli2+/‐;InsigDKOmiceandfromstatin

treatmentinmice.TheICRSscaleisuniquelyfocusedoncapturingcartilagerepair.

Third,tocircumventthelackofSafranin‐O‐stainedhistology,whichispartoftheOARSI

assessment.Despitetrouble‐shootingefforts,Safranin‐O,Alcianblue,andToluidine

bluestainingofcartilageproteoglycanwasnotachievedinInsigDKOnorcontrol

sections.

124  

Geneexpression

Forreal‐timePCR,experimentswereconductedusingTaqManassaysfrom

AppliedBiosystems,asdescribedintheMaterialsandMethodssectionofChapterTwo.

Formousecartilage,RNAextractionwasconductedusingTRIzol®Reagent(Invitrogen,

CA)accordingtothemanufacturer’sinstructions.Forhumancartilage,RNAextraction

wasconductedusingthemodifiedprotocoldescribedinChapterTwo.Real‐timePCR

resultswerenormalizedtoanendogenouscontrol(Gapdh4352932EorACTB

4352933Eformousesamples;ACTBHs99999903_m1orGAPDHHs99999905_m1for

humansamples),andanalyzedaccordingtothecomparativeCTmethod(∆∆CT).

MarkersofOAincludedMmp13Mm01168713_m1andAdamts5Mm00478620_m1for

mouse;MMP13Hs00233992_m1andADAMTS5Hs00199841_m1forhuman.Toverify

cholesterolinhibitionbystatintreatmentinhumancartilage,HMGCR

(Hs00168352_m1)wasused(seeLovastatintreatment).

Medialmeniscectomysurgery

Todestabilizethejointandrecapitulatemechanically‐inducedOA,we

performedmedialmeniscectomy,orsurgicalexcisionofthemedialmeniscusas

previouslydescribed(Kamekuraetal.2005;Linetal.2009).Briefly,anesthetizedmice

hadtheirkneesshavedandsurgicallypreparedundersterileconditions.UsingaNo.15

scalpel,amedialincisionwasmadealongthepatellatendonandthenthroughthejoint

capsule,revealingthemedialmeniscustobeexcised.Theincisionwasclosedusingtwo

sutures,andcoveredwithantibioticcream.Analgesicwasadministeredsubcutaneously

priortosurgeryandfor72hourspost‐operatively,twicedaily.Shamcontrolsreceived

125  

anidenticalprocedureexceptthemedialmeniscuswasleftintact.Nomiceperished

duringsurgerynorpost‐operatively.Insum,themedialmeniscuswasremovedfrom

theleftkneeof8‐week‐oldmice,andOAdevelopedpredictably8weekspost‐

operatively.AllanimalstudieswereapprovedbytheTorontoCentrefor

Phenogenomics.

Lovastatintreatment

Mouse

Toreducesterollevelsinvivo,miceweretreatedwith3mg/kg/daylovastatinby

surgicalimplantationofslow‐releasedrugpellets(InnovativeResearchofAmerica,FL).

At8weeksofage,pelletswereplacedadjacenttothesynovialmembraneoftheknee.

Pelletswere3mmindiameter,appropriateforimplantationin25grammice.Pellets

wereimplantedinControl,Col2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKOmice,and

formechanically‐inducedOA,inControlandInsigDKOmice.Effectivestatintreatment

inthecartilagewasconfirmedbyimmunohistochemistryforHMGCoA‐reductase(see

OAassessment)inthechondrocytes.

SerumlevelsofcholesterolwereassayedusingtheAmplexRedcholesterolassay

kit(MolecularProbes).Approximately1mLofbloodwascollectedfromeachmouseby

cardiacpunctureatthetimeofsacrifice.Sampleswereallowedtocoagulateandserum

wascollected.Theassaywasconductedaccordingtothemanufacturer’sprotocol.

Briefly,thisisafluorometricassaywhichdetectsproductsofcholesterol,usingH2O2

detection(bytheAmplexRedreagent)asareadout(M.Zhouetal.1997).

126  

Human

Toreducesterollevelsinvitro,humancartilageexplantswereestablishedand

treatmentappliedasdescribedinChapterTwo.Explantsweretreatedovernightwith

10µMlovastatinhydroxyacid(CaymanChemicalCompany,MI).Effectivestatin

treatmentwasconfirmedbyreal‐timePCRforHMGCR(seeOAassessment)inthe

cartilage.

ADAMTS5reporterconstruct

Primarycultureswereestablishedfromhumanosteoarthriticcartilageusinga

methodpreviouslydescribed(Oteroetal.2012),withtheassistanceofHenryMa.

ChondrocytesweretransfectedwithanADAMTS5reporterconstructornegative

controlvector(GeneCopoeia,MD)usingtheNeon®TransfectionSystem(Life

Technologies,ON).Theconstructcontainedaluciferasereporterunderthecontrolof

humanADAMTS5regulatoryelements,andaβ‐galreporterfornormalizationofthe

transfectionefficiency.Promoteractivitywasassayedfollowingovernighttreatment

with10µmlovastatinandisreportedasrelativelightunits(RLU).Theseexperiments

wereconductedwiththeassistanceofRaymondPoon.

127  

ChapterFive:

Conclusionsandfutureresearch

128  

Conclusions

ExaminingtheexpressionofgenesregulatedbyHhsignalinginhuman

osteoarthriticcartilageledustoidentifyamechanismthroughwhichHhsignaling

impactsosteoarthritisseverity(Figure39).OurdatasupportthehypothesisthatHh

signalingregulatescholesterolbiosynthesisinchondrocytes,andthatmodulating

cholesterolhomeostasiscanimpacttheseverityofOA.HigherlevelsofHhsignalingare

associatedwithaccumulationofintracellularcholesterolinchondrocytes.In

geneticallymodifiedmice,weshowthatcholesterolaccumulationcausesimpaired

growthofthelongbones;italsocausesanosteoarthritisphenotypewhichcanbe

modulatedbyHhsignaling.Reducingcholesterolaccumulationattenuatesthe

expressionofosteoarthritismarkersinvivoinmurinecartilageandinvitroinhuman

cartilage.Theprotectionagainstcartilagedegradationismediatedinpartbyreduction

inADAMTS5expression.ThesefindingsdemonstratethatHhsignalingregulates

cholesterolbiosynthesisinchondrocytes,andprovidesupportfortheuseof

cholesterol‐loweringagentstoslowarticularcartilagedegenerationinOA.

OurkeyfindingsshowthatHhsignalingregulatesexpressionofcholesterol

biosyntheticgenesinchondrocytes;thatcholesterolaccumulationinchondrocytes

impairsgrowthofthelongbonesandpredisposestoosteoarthritis;andthatcholesterol

inhibitioninthecartilageattenuatestheseverityofosteoarthritis.Thebroader

relevanceofthesefindingsandtheircontributiontotheadvancementofscientific

knowledgecanbefacilitatedwithadditionalexperiments.Furthermore,torealizethe

potentialofthisbiomedicalstudy,additionalstudiesarerequiredtotranslatethese

basicscientificfindingstoclinicalpractice.

129  

Figure39.Workingmodel:cholesterolhomeostasismediatesHedgehogsignaling

inosteoarthritis.Intracellularcholesterolconcentrationisgovernedinpartby

endogenouscholesterolbiosynthesiswhichisregulatedbyINSIGs(insulin‐induced

genes)andSREBFs(sterolregulatoryelement‐bindingfactors),inaproteincomplex

foundintheER(Figure4).Onceprocessed,theSREBFstranslocatetothenucleusto

regulateexpressionoftargetgenesviatheSrebindingsite.TheHedgehogsignaling

pathwayismediatedbyprocessingoftheGLItranscriptionfactorswhichalso

translocatetothenucleustoregulateexpressionoftargetgenesviatheconservedGli

bindingsite(Figure3).PutativetargetgenesofbothSREBFandGLIincludecholesterol

homeostaticgenes(choles.genes)suchasINSIG1,andgenesinvolvedinosteoarthritis

(OAgenes)pathogenesissuchasADAMTS5.PM=plasmamembrane.ER=endoplasmic

reticulum.+=homeostasis,‐=decrease,++=increase.

130  

FutureResearch

Hhsignalingregulatesexpressionofcholesterolhomeostaticgenesinchondrocytes. DoesHhsignalingregulatecholesterolhomeostaticgenesviaSrebf2?

Weperformedmicroarrayanalysesinhumanosteoarthriticcartilagetoidentify

Hhsignalingtargetgenes.Amongthemanygenesandpathwaysthatwereidentified,

wechosetopursuecholesterolbiosynthesisduetoitsessentialroleincellular

homeostasis.WeobservedanInsig1‐independentmechanismthroughwhichHh

signalingregulatescholesterolhomeostasis,andshowthatHhsignalingmayregulate

cholesterolhomeostaticgenesviaGli‐mediatedtargetingofSrebf2.Toconfirmthat

Srebf2isresponsibleformediatingtheeffectofHhsignalingoncholesterol

homeostasis,chondrocyte‐specificSrebf2‐knockoutexperimentsarerequired.IfSrebf2

isresponsible,thencholesterollevelsinSrebf2‐knockoutchondrocyteswillbe

unchangedwithHhmodulation.Ifcholesterollevelscanstillbealteredintheabsence

ofSrebf2,additionalpathwayswhichgoverncholesterolhomeostasis,suchasLXR

activation(Gentilietal.2005),canbeexplored.

WhatotherpathwaysareregulatedbyHhsignalinginchondrocytes?

Otherpathwayswhichwereidentifiedbyourmicroarrayanalyses,suchaslipid

metabolismandsteroidbiosynthesis,mayalsocontributetoOApathogenesisunderthe

regulationofHhsignaling.Thiscanbeinvestigatedusingthesameexperimentaldesign

asthecurrentstudy.Transgenicmicewhichlackcriticalregulatorsinpathwaysof

interestcanbeusedtodeterminethecontributionofthosepathwaystochondrocyte

biologyandosteoarthritisdevelopment.Theinter‐dependencyofpathwaysalsomerits

131  

furtherinvestigationsinceforexample,sterolintermediatesproducedinthe

cholesterolbiosyntheticpathwayfeedintothesteroidbiosyntheticpathway(Jefcoateet

al.1992).

WhatothersterolintermediatesareregulatedbyHhsignaling?

WhileweconfirmapositiverelationshipbetweenHhsignalingandcholesterol

levels,wedonotidentifyothersterolintermediatesthatwereregulatedbyHh

signaling.Previousstudieshaveshowntheretobedifferencesinlipidcomposition

betweengrowthplateandarticularchondrocytes(LeLousetal.1981),soitis

reasonabletoexpectdifferencesinosteoarthriticchondrocytes,potentiallygovernedby

Hhsignaling.Specifyingtheexactsterolintermediateswillallowrefinementof

hypothesesregardingpathwaycross‐talk,aswellashypothesesregardingthespecific

roleofthosesterolintermediatesinchondrocytebiology.

Establishingasterolprofileisrelevantincontinuingtoexplorethepotential

mutualregulatoryrelationshipbetweenHhsignalingandcholesterolbiosynthesis.

OxysterolshavebeenshowntoregulateHhsignalinginsometissues(Corcoranand

Scott2006;Dwyeretal.2007).IflevelsoftheseareregulatedbyHhsignalingviathe

cholesterolbiosyntheticpathwayinchondrocytes,amutualregulatoryrelationship

mayemerge.Massspectrometryexperimentswillproveusefulintestingthis

hypothesisbydeterminingwhetherspecificoxysterolsareregulatedbyHhsignalingin

chondrocytes.

DoesHhsignalingregulatecholesterolhomeostasisinothersystems?

WeshowforthefirsttimearoleforHhsignalinginregulatingcholesterol

biosynthesis.Weinvestigatethisrelationshipincartilageandosteoarthritis,butitmay

132  

alsotranslatetoothertissuetypesandothercartilagepathologies.Regardingother

tissues,simplestainssuchasOil‐Red‐OcanbeusedtocrudelyassesswhetherHh

modulationalterslipidcontent.Regardingothercartilagepathologies,thecontribution

ofcholesteroldysregulationtochondrosarcoma(L.Hoetal.2009),whichalsoresults

fromactivationofHhsignaling,meritsfurtherinvestigation.Thiscanbeexploredusing

themicewithcartilage‐specificcholesterolaccumulationandHhmodulationdescribed

inChapterThree.

133  

Cholesterolaccumulationinchondrocytesimpairsgrowthofthelongbonesandpredisposestoosteoarthritis. DoesInsig2havechondrocyte‐specificroles?

Toelucidatetheroleofcholesterolaccumulationinchondrocytebiology,we

excisedInsig1specificallyinCol2a1‐expressingcells,inabackgroundInsig2knockout.

Thesemiceshoweddelayedgrowthofthelongbonesandapredispositionto

osteoarthritis.Themiceexaminedinthisstudy[including“control”Insig1(fl/fl);Insig2(‐

/‐)mice]carrygermlinedisruptionofInsig2,whosefunctionisredundanttothatof

Insig1intheliver(Engelkingetal.2005).LossofeitherInsig1aloneorInsig2alonein

theliverresultsinnormallevelsofcholesterolandtriglycerides,andlossofeithergene

doesnotcauseacompensatingincreaseinexpressionoftheothergene(Engelkingetal.

2005).Despitethis,Insig2canserveuniquerolesindifferenttissues,forexample,itisa

coloncancerbiomarker(C.G.Lietal.2008),andpolymorphismsinInsig2inadipose

tissuesisassociatedwithobesity(Krapivneretal.2008).Toassesspotential

chondrocyte‐specificfunctions,reintroductionoftheInsig2geneinInsigDKOcartilage

isrequired.Differences,ifany,betweenInsig1(fl/fl);Insig2(‐/‐)cartilageand

Insig1(fl/fl);Insig2(+/+)cartilagewouldhighlightchondrocyte‐specificfunctionsof

Insig2.

IstheOAphenotypeinInsigDKOmiceduetodevelopmentalperturbations?BecauseInsigDKOmiceexhibitedcholesterolaccumulationinchondrocytes

throughoutdevelopment,wewereunabletodefinitivelyascertainwhetherthe

phenotypesobservedwereduetodevelopmentalperturbationsinchondrocytebiology

andsubsequentboneformation,ortothecholesterolaccumulationitself.Futurework

134  

toaddressthisincludesuseofaninducibleCol2a1promoter(GroverandRoughley

2006)todriveCreexpressionandexciseInsig1inadultcartilage.Ifcholesterol

accumulationthatisinducedinadultcartilagealsocreatesanosteoarthriticphenotype

asweobservehere,thenthecontributionofdevelopmentalabnormalitiescanbe

excluded.Phenotypicanalysesofthesemicecanbeconductedinasimilarmanneras

describedinChapterFour,usingradiography,histology,andgeneexpressiontoassess

typicalfeaturesofosteoarthritis.

WheredoescholesterolaccumulateinInsigDKOchondrocytes?

Removalofthemajornegativeregulatorofcholesterolbiosynthesiscaused

robustcholesterolaccumulationinthechondrocytes.Thisallowedustoexaminethe

effectsoftissue‐specificcholesteroldysregulationonosteoarthritisoutcomes,offering

specificityoverstudieswhichexaminesystemiccholesteroldysregulation.Toachieve

greaterspecificity,theprecisesub‐cellularlocalizationoftheexcesscholesterolinour

InsigDKOmiceisrequired.Techniquesascrudeassub‐cellularfractionationoras

sophisticatedaselectronmicroscopycouldbeusedtodeterminelocalization.This

wouldinformhypothesessurroundingthemechanismsthroughwhichcholesterol

accumulationresultsinosteoarthriticchanges.Forexample,accumulationinthe

plasmamembranemayfacilitatelipidraftformationandpotentiatesignaltransduction

ofpathwayswhichmediateOApathogenesis.

HowdoescholesterolaccumulationexacerbateOApathogenesis?

Additionalstudiesarerequiredtoelucidatethemechanismthroughwhich

cholesterolaccumulationcontributestoOApathogenesis.Becausecholesterolisa

componentofthelipidbilayer,alterationsinfree(unesterified)cholesterolmay

135  

compromisemembranefluidityanddisruptmembranedomains,ultimatelyperturbing

cellhomeostasisbydisruptingfunctionofintegralmembraneproteinsand/orsignaling

pathways(Tabas2002).Forexample,lipidraftshavebeenshowntofacilitatehigh

activationlevelsofHedgehogsignalingbyconcentratingSmoothened,thepathway

transducer(Shietal.2013).ActivationofHedgehogsignalingmayincreasecholesterol

accumulationwhichpotentiatestransductionoftheHhsignalpastahomeostatic

threshold,pushingthechondrocyteintoanOA‐profile.

ERstresshasbeenexploredasacontributortoOA(PattersonandDealy2014).

ExistingdatasuggestthatchondrocytescanrecoverfromERstressthatisinducedby

accumulationofmisfoldedproteins,butthosechondrocytesexhibitalteredphenotypes

andbehaviorwhichultimatelycompromisestissuestructure(Tsangetal.2007).Patra

etal.showthatlackofS1P(site‐1protease,whichregulatesSREBFprocessingandthe

unfoldedproteinresponse)inthecartilagecompromisestypeIIcollagenformationdue

toretentionofsubunitsintheER,ultimatelyaffectingendochondralbonedevelopment

(Patraetal.2014).CholesterolaccumulationinourInsigDKOmicemayinducetheER

stressresponsewhichcompromiseschondrocytefunctionandpromotesOA(Patterson

andDealy2014).ERstresscanbeinvestigatedbyprobingvalidatedgenemarkerssuch

asPERK(EIF2AK3;eukaryotictranslationinitiationfactor2‐alphakinase3)andIRE1

(ERN1;endoplasmicreticulumtonucleussignaling1),amongothers(RonandWalter

2007),inourInsigDKOchondrocytes.

DeterminingwhetherlipidraftformationorERstressarealteredbyHh

signaling‐inducedcholesterolmodulationwillclarifyourunderstandingofthe

contributionofcholesteroltoOApathogenesis.Onemechanismexploredinthecurrent

136  

studyistheroleforcholesterol‐mediatedregulationofADAMTS5expression.Alternate

mechanismsareequallypossible,suchasinductionofchondrocytehypertrophyby

cholesterol‐mediatedactivationofthelipidregulatorRor‐alpha(Woodsetal.2009).

Barteretal.highlighttheroleofproteingeranylgeranylation,showingthatstatin

treatment(andsubsequentreductionoftheisoprenoidintermediategeranylgeranyl

pyrophosphate)canreduceproteases,whichareknowncontributorstocartilage

degradationandOApathogenesis(Barteretal.2010).Asdescribedabove,additional

informationregardingtheexactsterolintermediatesandtheirsubcellularlocalization

willbeusefulinrefininghypothesesanddirectingfuturestudiestoelucidatethe

contributionofcholesteroltoOApathogenesis.

137  

Cholesterolinhibitioninthecartilageattenuatestheseverityofosteoarthritis.Inmousecartilageinvivo,loweringcholesterolbyHhreductionorstatin

treatmentattenuatestheseverityofosteoarthritis.Inhumancartilageinvitro,lowering

cholesterolbystatintreatmentreducesexpressionofosteoarthritismarkers.

Cholesterolinhibition,therefore,isaviablestrategyformitigatingthecartilage

degradationthatischaracteristicofosteoarthritis.Currenttherapeuticsfor

osteoarthritisarelargelylimitedtoanalgesics,withnopharmacologicavailablefor

slowingcartilagedegradation.Perhapsthemostimportantimplicationofourresults

pertainingtostatintreatmentisthepotentialtranslationtotheclinicfortreating

osteoarthritis.Becausestatinsarewidelyusedtolowercholesterolincardiovascular

diseases,thereisexistingdataontheefficacyandsafetyofthesedrugs,whichremoves

abarriertotranslation(Lvetal.2014).

CancholesterolinhibitionattenuatetheseverityofotherdiseaseswhichresultfromHhactivation?OurfindingthatstatintreatmentrescuedtheOAphenotypeinmicewithHh

activation(Col2a1‐Gli2)pointstothepossibilitythatstatintreatmentmaybeusefulin

attenuatingotherdiseaseswhichresultfromHhactivation.Medulloblastomaandbasal

cellcarcinoma,forexample,canresultfromHhactivation(Goodrichetal.1997;

Grachtchouketal.2000).Becausethemedulloblastomapatientpopulationispediatric,

andHhsignalingiscrucialtothenormaldevelopmentofseveraltissues,

pharmacologicswhichinhibitHhsignalingarenotanidealtherapy(JiangandHui

2008).Asdescribedabove,itisimportanttoexplorewhethertherelationshipbetween

Hhsignalingandcholesterolregulationtranslatestoothertissues,suchasthebrainand

138  

skin.Ifthisisindeedthecase,statintreatmentmaybeefficaciousandrelativelysafein

treatingmedulloblastoma,basalcellcarcinoma,andotherdiseaseswhichareknownto

resultfromHhactivation.

IslocaladministrationofstatinrequiredtoimproveOAoutcomes?

Phase2clinicaltrialsarecurrentlybeingconductedbyStanfordUniversityto

assessthebenefitsofstatintreatmentonOAoutcomes(Hydroxychloroquine/

AtorvastatinintheTreatmentofOsteoarthritisoftheKnee;

http://clinicaltrials.gov/show/NCT01645176).Ananticipatedlimitationofthisclinical

trialisthemethodofdrugdelivery,forwhichtheychoseoraladministration.Studies

whichexaminethebioavailabilityandefficacyofstatinsinthesynovialjointtissuesof

patientsfollowingoraladministrationhaveyettobeconducted.Existingstudies

examiningtheeffectofstatintreatmentonOAoutcomesshowinconsistentresults

(Kadametal.2013;Valdesetal.2014;W.Weietal.2014)whichmaybeexplainedin

partbymethodofdrugdeliveryandresultingbioavailabilityofthedruginthejoint.

Sinceourtreatmentswereappliedlocally,ourfindingssupporttheuseofintra‐

articularstatininjectiontoreducecartilagedegradationinOApatients.Further

researchintostatinpharmacokineticsincartilagearerequired,inadditiontomore

carefullydesignedclinicaltrialstotranslatethesebasicfindingsintotheclinicalsetting.

139  

Relevance

IdentifyingHhtargetgenesandnetworksimprovesourunderstandingof

cartilagebiologyandpathology,andproposespotentialmechanismsbywhichHh

activationcontributestoOApathogenesis.Withhumanandanimalsamples,weshow

thatHhsignalingregulatescholesterolhomeostasisandthatcholesterolmodulation

canalterOApathogenesis.Theseresultshighlighttheneedtofurtherexplorethe

complexinteractionsamongHhsignaling,cholesterolhomeostasis,andchondrocyte

biology.Theabilitytopharmacologicallycorrectsterolimbalancesinthecartilageto

preventOApathologyrepresentsapromisingtherapeuticintervention.

140  

References

Abramson,S.B.,Attur,M.,andYazici,Y.(2006),'Prospectsfordiseasemodificationin

osteoarthritis',NatClinPractRheumatol,2(6),304‐12.

Aigner,T.,etal.(2007),'Mechanismsofdisease:roleofchondrocytesinthe

pathogenesisofosteoarthritis‐‐structure,chaosandsenescence',NatClinPract

Rheumatol,3(7),391‐9.

Aigner,T.,etal.(2006),'Large‐scalegeneexpressionprofilingrevealsmajor

pathogeneticpathwaysofcartilagedegenerationinosteoarthritis',Arthritis

Rheum,54(11),3533‐44.

Al‐Arfaj,A.S.(2003),'Radiographicosteoarthritisandserumcholesterol',SaudiMedJ,

24(7),745‐7.

Ala‐Kokko,L.,etal.(1990),'SinglebasemutationinthetypeIIprocollagengene

(COL2A1)asacauseofprimaryosteoarthritisassociatedwithamild

chondrodysplasia',ProcNatlAcadSciUSA,87(17),6565‐8.

Ali,S.A.andAlman,B.(2012),'RNAextractionfromhumanarticularcartilageby

chondrocyteisolation',AnalBiochem,429,39‐41.

Altman,R.D.,etal.(2006),'Commentary:osteoarthritisofthekneeandglucosamine',

OsteoarthritisCartilage,14(10),963‐6.

Aspden,R.M.,Scheven,B.A.,andHutchison,J.D.(2001),'Osteoarthritisasasystemic

disorderincludingstromalcelldifferentiationandlipidmetabolism',Lancet,357

(9262),1118‐20.

Baker,J.F.,Walsh,P.,andMulhall,K.J.(2011),'Statins:apotentialroleinthe

managementofosteoarthritis?',JointBoneSpine,78(1),31‐4.

Barter,M.J.,etal.(2010),'Lipophilicstatinspreventmatrixmetalloproteinase‐

mediatedcartilagecollagenbreakdownbyinhibitingprotein

geranylgeranylation',AnnRheumDis,69(12),2189‐98.

Bennett,M.K.,etal.(1999),'Co‐stimulationofpromoterforlowdensitylipoprotein

receptorgenebysterolregulatoryelement‐bindingproteinandSp1is

specificallydisruptedbytheyinyang1protein',JBiolChem,274(19),13025‐32.

141  

Bombardier,C.,Hawker,G.,andMosher,M.(2011),'TheImpactofArthritisinCanada:

TodayandOvertheNext30years',(ArthritisAllianceofCanada).

Brazma,A.,etal.(2001),'Minimuminformationaboutamicroarrayexperiment

(MIAME)‐towardstandardsformicroarraydata',NatGenet,29(4),365‐71.

Brighton,C.T.,Wang,W.,andClark,C.C.(2008),'Theeffectofelectricalfieldsongene

andproteinexpressioninhumanosteoarthriticcartilageexplants',JBoneJoint

SurgAm,90(4),833‐48.

Brown,M.S.andGoldstein,J.L.(1996),'MichaelS.Brown,MDandJosephL.Goldstein,

MD.1985Nobellaureatesinmedicine',JInvestigMed,44(2),14‐23.

Brown,M.S.andGoldstein,J.L.(1997),'TheSREBPpathway:regulationofcholesterol

metabolismbyproteolysisofamembrane‐boundtranscriptionfactor',Cell,89

(3),331‐40.

Bumcrot,D.(2005),'RNAinterferencemodulatorsofhedgehogsignalinganduses

thereof',(GooglePatents).

Carney,S.L.andMuir,H.(1988),'Thestructureandfunctionofcartilageproteoglycans',

PhysiolRev,68(3),858‐910.

Carr,A.J.,etal.(2012),'Kneereplacement',Lancet,379(9823),1331‐40.

Chen,J.K.,etal.(2002),'SmallmoleculemodulationofSmoothenedactivity',ProcNatl

AcadSciUSA,99(22),14071‐6.

Chou,C.H.,etal.(2013),'Directassessmentofarticularcartilageandunderlying

subchondralbonerevealsaprogressivegeneexpressionchangeinhuman

osteoarthriticknees',OsteoarthritisCartilage,21(3),450‐61.

Cillero‐Pastor,B.,etal.(2012),'Time‐of‐flightsecondaryionmassspectrometry‐based

moleculardistributiondistinguishinghealthyandosteoarthritichuman

cartilage',AnalChem,84(21),8909‐16.

Cooper,M.K.,etal.(2003),'AdefectiveresponsetoHedgehogsignalingindisordersof

cholesterolbiosynthesis',NatGenet,33(4),508‐13.

Corcoran,R.B.andScott,M.P.(2006),'OxysterolsstimulateSonichedgehogsignal

transductionandproliferationofmedulloblastomacells',ProcNatlAcadSciUS

A,103(22),8408‐13.

142  

Craft,A.M.,etal.(2013),'Specificationofchondrocytesandcartilagetissuesfrom

embryonicstemcells',Development,140(12),2597‐610.

Cram,P.,etal.(2012),'Totalkneearthroplastyvolume,utilization,andoutcomes

amongMedicarebeneficiaries,1991‐2010',JAMA,308(12),1227‐36.

Dell'accio,F.,etal.(2008),'Identificationofthemolecularresponseofarticularcartilage

toinjury,bymicroarrayscreening:Wnt‐16expressionandsignalingafterinjury

andinosteoarthritis',ArthritisRheum,58(5),1410‐21.

Donahue,L.R.,etal.(2003),'AmissensemutationinthemouseCol2a1genecauses

spondyloepiphysealdysplasiacongenita,hearingloss,andretinoschisis',JBone

MinerRes,18(9),1612‐21.

Downey,T.(2006),'AnalysisofamultifactormicroarraystudyusingPartekgenomics

solution',MethodsEnzymol,411,256‐70.

Dwyer,J.R.,etal.(2007),'Oxysterolsarenovelactivatorsofthehedgehogsignaling

pathwayinpluripotentmesenchymalcells',JBiolChem,282(12),8959‐68.

Eaton,S.(2008),'MultiplerolesforlipidsintheHedgehogsignallingpathway',NatRev

MolCellBiol,9(6),437‐45.

Edwards,P.A.,etal.(2000),'RegulationofgeneexpressionbySREBPandSCAP',

BiochimBiophysActa,1529(1‐3),103‐13.

Engelking,L.J.,etal.(2006),'SeverefacialcleftinginInsig‐deficientmouseembryos

causedbysterolaccumulationandreversedbylovastatin',JClinInvest,116(9),

2356‐65.

Engelking,L.J.,etal.(2005),'Schoenheimereffectexplained‐‐feedbackregulationof

cholesterolsynthesisinmicemediatedbyInsigproteins',JClinInvest,115(9),

2489‐98.

Felson,D.T.,etal.(1987),'Theprevalenceofkneeosteoarthritisintheelderly.The

FraminghamOsteoarthritisStudy',ArthritisRheum,30(8),914‐8.

Felson,D.T.,etal.(2000),'Osteoarthritis:newinsights.Part1:thediseaseanditsrisk

factors',AnnInternMed,133(8),635‐46.

Flurkey,K.,Currer,J.M.,andHarrison,D.E.(2007),TheMouseinAgingResearch,ed.J.G.

Fox(TheMouseinBiomedicalResearch2ndEdition;Burlington,MA:American

CollegeLaboratoryAnimalMedicine(Elsevier)).

143  

Galois,L.,etal.(2004),'Dose‐responserelationshipforexerciseonseverityof

experimentalosteoarthritisinrats:apilotstudy',OsteoarthritisCartilage,12

(10),779‐86.

Gehrsitz,A.,etal.(2001),'IsolationofRNAfromsmallhumanarticularcartilage

specimensallowsquantificationofmRNAexpressionlevelsinlocalarticular

cartilagedefects',JOrthopRes,19(3),478‐81.

Gentili,C.,etal.(2005),'Cholesterolsecretionandhomeostasisinchondrocytes:aliver

XreceptorandretinoidXreceptorheterodimermediatesapolipoproteinA1

expression',MatrixBiol,24(1),35‐44.

Geyer,M.,etal.(2009),'Differentialtranscriptomeanalysisofintraarticularlesionalvs

intactcartilagerevealsnewcandidategenesinosteoarthritispathophysiology',

OsteoarthritisCartilage,17(3),328‐35.

Gierman,L.M.,etal.(2014),'Osteoarthritisdevelopmentisinducedbyincreased

dietarycholesterolandcanbeinhibitedbyatorvastatininAPOE*3Leiden.CETP

mice‐‐atranslationalmodelforatherosclerosis',AnnRheumDis,73(5),921‐7.

Gill,S.,Chow,R.,andBrown,A.J.(2008),'Sterolregulatorsofcholesterolhomeostasis

andbeyond:theoxysterolhypothesisrevisitedandrevised',ProgLipidRes,47

(6),391‐404.

Girkontaite,I.,etal.(1996),'ImmunolocalizationoftypeXcollageninnormalfetaland

adultosteoarthriticcartilagewithmonoclonalantibodies',MatrixBiol,15(4),

231‐8.

Glasson,S.S.,etal.(2010),'TheOARSIhistopathologyinitiative‐recommendationsfor

histologicalassessmentsofosteoarthritisinthemouse',OsteoarthritisCartilage,

18Suppl3,S17‐23.

Glasson,S.S.,etal.(2005),'DeletionofactiveADAMTS5preventscartilagedegradation

inamurinemodelofosteoarthritis',Nature,434(7033),644‐8.

Goedeke,L.andFernandez‐Hernando,C.(2012),'Regulationofcholesterol

homeostasis',CellMolLifeSci,69(6),915‐30.

Gofflot,F.,etal.(2003),'Molecularmechanismsunderlyinglimbanomaliesassociated

withcholesteroldeficiencyduringgestation:implicationsofHedgehog

signaling',HumMolGenet,12(10),1187‐98.

144  

Goldring,M.B.(2005),'Humanchondrocyteculturesasmodelsofcartilage‐specific

generegulation',MethodsMolMed,107,69‐95.

Goldring,M.B.,Tsuchimochi,K.,andIjiri,K.(2006),'Thecontrolofchondrogenesis',J

CellBiochem,97(1),33‐44.

Gong,Y.,etal.(2006),'Sterol‐regulatedubiquitinationanddegradationofInsig‐1

createsaconvergentmechanismforfeedbackcontrolofcholesterolsynthesis

anduptake',CellMetab,3(1),15‐24.

Goodrich,L.V.,etal.(1997),'Alteredneuralcellfatesandmedulloblastomainmouse

patchedmutants',Science,277(5329),1109‐13.

Gosset,M.,etal.(2008),'Primarycultureandphenotypingofmurinechondrocytes',Nat

Protoc,3(8),1253‐60.

Grachtchouk,M.,etal.(2000),'BasalcellcarcinomasinmiceoverexpressingGli2in

skin',NatGenet,24(3),216‐7.

Grant,T.D.,etal.(2000),'Col2‐GFPreportermarkschondrocytelineageand

chondrogenesisduringmouseskeletaldevelopment',DevDyn,218(2),394‐400.

Griffin,T.M.,etal.(2010),'Diet‐inducedobesitydifferentiallyregulatesbehavioral,

biomechanical,andmolecularriskfactorsforosteoarthritisinmice',ArthritisRes

Ther,12(4),R130.

Grover,J.andRoughley,P.J.(2006),'GenerationofatransgenicmouseinwhichCre

recombinaseisexpressedundercontrolofthetypeIIcollagenpromoterand

doxycyclineadministration',MatrixBiol,25(3),158‐65.

Hassett,G.,etal.(2006),'Therelationbetweenprogressiveosteoarthritisoftheknee

andlongtermprogressionofosteoarthritisofthehand,hip,andlumbarspine',

AnnRheumDis,65(5),623‐8.

Hayman,D.M.,etal.(2006),'Theeffectsofisolationonchondrocytegeneexpression',

TissueEng,12(9),2573‐81.

Ho,L.,etal.(2009),'Gli2andp53cooperatetoregulateIGFBP‐3‐mediatedchondrocyte

apoptosisintheprogressionfrombenigntomalignantcartilagetumors',Cancer

Cell,16(2),126‐36.

Ho,M.L.,etal.(2013),'Treatmentofearly‐stageosteoarthritis',(GooglePatents).

145  

Hopyan,S.,etal.(2002),'AmutantPTH/PTHrPtypeIreceptorinenchondromatosis',

NatGenet,30(3),306‐10.

Horton,J.D.,Goldstein,J.L.,andBrown,M.S.(2002),'SREBPs:activatorsofthe

completeprogramofcholesterolandfattyacidsynthesisintheliver',JClin

Invest,109(9),1125‐31.

Horton,J.D.,etal.(2003),'Combinedanalysisofoligonucleotidemicroarraydatafrom

transgenicandknockoutmiceidentifiesdirectSREBPtargetgenes',ProcNatl

AcadSciUSA,100(21),12027‐32.

Hunter,D.J.andEckstein,F.(2009),'Exerciseandosteoarthritis',JAnat,214(2),197‐

207.

Hunter,D.J.,Schofield,D.,andCallander,E.(2014),'Theindividualandsocioeconomic

impactofosteoarthritis',NatRevRheumatol.

Ijiri,K.,etal.(2008),'DifferentialexpressionofGADD45betainnormaland

osteoarthriticcartilage:potentialroleinhomeostasisofarticularchondrocytes',

ArthritisRheum,58(7),2075‐87.

Issa,R.I.andGriffin,T.M.(2012),'Pathobiologyofobesityandosteoarthritis:

integratingbiomechanicsandinflammation',PathobiolAgingAgeRelatDis,2

(2012).

Iversen,M.D.(2010),'ManagingHipandKneeOsteoarthritiswithExercise:Whatisthe

BestPrescription?',TherAdvMusculoskeletDis,2(5),279‐90.

Jakobisiak,M.,etal.(1991),'Cellcycle‐specificeffectsoflovastatin',ProcNatlAcadSciU

SA,88(9),3628‐32.

Janusz,M.J.,etal.(2002),'Inductionofosteoarthritisintheratbysurgicaltearofthe

meniscus:Inhibitionofjointdamagebyamatrixmetalloproteinaseinhibitor',

OsteoarthritisCartilage,10(10),785‐91.

Jefcoate,C.R.,etal.(1992),'Regulationofcholesterolmovementtomitochondrial

cytochromeP450sccinsteroidhormonesynthesis',JSteroidBiochemMolBiol,

43(8),751‐67.

Jiang,J.andHui,C.C.(2008),'Hedgehogsignalingindevelopmentandcancer',DevCell,

15(6),801‐12.

Jira,P.(2013),'Cholesterolmetabolismdeficiency',HandbClinNeurol,113,1845‐50.

146  

Jungmann,P.M.,etal.(2013),'Associationofmetabolicriskfactorswithcartilage

degradationassessedbyT2relaxationtimeattheknee:datafromthe

osteoarthritisinitiative',ArthritisCareRes(Hoboken),65(12),1942‐50.

Kadam,U.T.,Blagojevic,M.,andBelcher,J.(2013),'Statinuseandclinicalosteoarthritis

inthegeneralpopulation:alongitudinalstudy',JGenInternMed,28(7),943‐9.

Kamekura,S.,etal.(2005),'Osteoarthritisdevelopmentinnovelexperimentalmouse

modelsinducedbykneejointinstability',OsteoarthritisCartilage,13(7),632‐41.

Kanbe,K.,etal.(2006),'Identificationofclockasamechanosensitivegenebylarge‐

scaleDNAmicroarrayanalysis:downregulationinosteoarthriticcartilage',Mod

Rheumatol,16(3),131‐6.

Kapoor,M.,etal.(2012),'GlucosaminesulfatereducesprostaglandinE(2)productionin

osteoarthriticchondrocytesthroughinhibitionofmicrosomalPGEsynthase‐1',J

Rheumatol,39(3),635‐44.

Karlen,Y.,etal.(2007),'StatisticalsignificanceofquantitativePCR',BMCBioinformatics,

8,131.

Karlsson,C.,etal.(2010),'Genome‐wideexpressionprofilingrevealsnewcandidate

genesassociatedwithosteoarthritis',OsteoarthritisCartilage,18(4),581‐92.

Karvonen‐Gutierrez,C.A.,etal.(2013),'Associationofleptinlevelswithradiographic

kneeosteoarthritisamongacohortofmidlifewomen',ArthritisCareRes

(Hoboken),65(6),936‐44.

Kato,H.,etal.(2007),'Large‐scalegeneexpressionprofiles,differentiallyrepresented

inosteoarthriticsynoviumofthekneejointusingcDNAmicroarraytechnology',

Biomarkers,12(4),384‐402.

Kellgren,J.H.andLawrence,J.S.(1957),'Radiologicalassessmentofosteo‐arthrosis',

AnnRheumDis,16(4),494‐502.

Kobayashi,T.,etal.(2005),'Indianhedgehogstimulatesperiarticularchondrocyte

differentiationtoregulategrowthplatelengthindependentlyofPTHrP',JClin

Invest,115(7),1734‐42.

Kobayashi,T.,etal.(2002),'PTHrPandIndianhedgehogcontroldifferentiationof

growthplatechondrocytesatmultiplesteps',Development,129(12),2977‐86.

147  

Koide,T.,Hayata,T.,andCho,K.W.(2006),'NegativeregulationofHedgehogsignaling

bythecholesterogenicenzyme7‐dehydrocholesterolreductase',Development,

133(12),2395‐405.

Kosinska,M.K.,etal.(2013),'Alipidomicstudyofphospholipidclassesandspeciesin

humansynovialfluid',ArthritisRheum,65(9),2323‐33.

Kostopoulou,F.,etal.(2012),'CentralroleofSREBP‐2inthepathogenesisof

osteoarthritis',PLoSOne,7(5),e35753.

Krapivner,S.,etal.(2008),'Insulin‐inducedgene2involvementinhumanadipocyte

metabolismandbodyweightregulation',JClinEndocrinolMetab,93(5),1995‐

2001.

Kronenberg,H.M.(2003),'Developmentalregulationofthegrowthplate',Nature,423

(6937),332‐6.

Kruegel,J.,Miosge,N.,andKoelling,S.(2008),'TheRoleofProgenitorCellsin

Osteoarthritis',CurrentRheumatologyReviews,4(3),210‐13.

Lai,L.P.andMitchell,J.(2005),'Indianhedgehog:itsrolesandregulationin

endochondralbonedevelopment',JCellBiochem,96(6),1163‐73.

LeLous,M.,Corvol,M.T.,andMaroteaux,P.(1981),'Lipidcompositionoftwotypesof

chondrocytesinprimaryculture',CalcifTissueInt,33(4),403‐7.

Li,C.G.,etal.(2008),'Insig2isassociatedwithcolontumorigenesisandinhibitsBax‐

mediatedapoptosis',IntJCancer,123(2),273‐82.

Li,Y.,Xu,L.,andOlsen,B.R.(2007),'Lessonsfromgeneticformsofosteoarthritisforthe

pathogenesisofthedisease',OsteoarthritisCartilage,15(10),1101‐5.

Liddle,A.D.,Pegg,E.C.,andPandit,H.(2013),'Kneereplacementforosteoarthritis',

Maturitas,75(2),131‐6.

Lin,A.C.,etal.(2009),'Modulatinghedgehogsignalingcanattenuatetheseverityof

osteoarthritis',NatMed,15(12),1421‐5.

Livak,K.J.andSchmittgen,T.D.(2001),'Analysisofrelativegeneexpressiondatausing

real‐timequantitativePCRandthe2(‐DeltaDeltaC(T))Method',Methods,25(4),

402‐8.

Loven,J.,etal.(2012),'Revisitingglobalgeneexpressionanalysis',Cell,151(3),476‐82.

148  

Lv,H.L.,etal.(2014),'Long‐termefficacyandsafetyofstatintreatmentbeyondsix

years:Ameta‐analysisofrandomizedcontrolledtrialswithextendedfollow‐up',

PharmacolRes,81,64‐73.

Lyons,T.J.,etal.(2006),'Thenormalhumanchondro‐osseousjunctionalregion:

evidenceforcontactofuncalcifiedcartilagewithsubchondralboneandmarrow

spaces',BMCMusculoskeletDisord,7,52.

MacDonald,J.S.,etal.(1988),'Preclinicalevaluationoflovastatin',AmJCardiol,62(15),

16J‐27J.

Mainil‐Varlet,P.,etal.(2003),'Histologicalassessmentofcartilagerepair:areportby

theHistologyEndpointCommitteeoftheInternationalCartilageRepairSociety

(ICRS)',JBoneJointSurgAm,85‐ASuppl2,45‐57.

Mak,K.K.,etal.(2008),'IndianhedgehogsignalsindependentlyofPTHrPtopromote

chondrocytehypertrophy',Development,135(11),1947‐56.

Mankin,H.J.,etal.(1971),'Biochemicalandmetabolicabnormalitiesinarticular

cartilagefromosteo‐arthritichumanhips.II.Correlationofmorphologywith

biochemicalandmetabolicdata',JBoneJointSurgAm,53(3),523‐37.

McKenna,L.A.,etal.(2000),'Effectiveisolationofhigh‐qualitytotalRNAfromhuman

adultarticularcartilage',AnalBiochem,286(1),80‐5.

McMahon,A.P.(2000),'MoresurprisesintheHedgehogsignalingpathway',Cell,100

(2),185‐8.

Messier,S.P.,etal.(2013a),'StrengthTrainingforArthritisTrial(START):designand

rationale',BMCMusculoskeletDisord,14(1),208.

Messier,S.P.,etal.(2013b),'Effectsofintensivedietandexerciseonkneejointloads,

inflammation,andclinicaloutcomesamongoverweightandobeseadultswith

kneeosteoarthritis:theIDEArandomizedclinicaltrial',JAMA,310(12),1263‐73.

Miyaki,S.,etal.(2010),'MicroRNA‐140playsdualrolesinbothcartilagedevelopment

andhomeostasis',GenesDev,24(11),1173‐85.

Mo,R.,etal.(1997),'SpecificandredundantfunctionsofGli2andGli3zincfingergenes

inskeletalpatterninganddevelopment',Development,124(1),113‐23.

Moseley,J.B.,etal.(2002),'Acontrolledtrialofarthroscopicsurgeryforosteoarthritis

oftheknee',NEnglJMed,347(2),81‐8.

149  

Mundy,G.,etal.(1999),'Stimulationofboneformationinvitroandinrodentsby

statins',Science,286(5446),1946‐9.

Muraoka,T.,etal.(2007),'Roleofsubchondralboneinosteoarthritisdevelopment:a

comparativestudyoftwostrainsofguineapigswithandwithoutspontaneously

occurringosteoarthritis',ArthritisRheum,56(10),3366‐74.

Nagy,A.(2000),'Crerecombinase:theuniversalreagentforgenometailoring',Genesis,

26(2),99‐109.

Neogi,T.andZhang,Y.(2013),'Epidemiologyofosteoarthritis',RheumDisClinNorth

Am,39(1),1‐19.

Ng,D.S.andHegele,R.A.(1993),'High‐density‐lipoproteincholesteroland

atherosclerosis',CMAJ,149(12),1807‐8.

Olex,A.L.,etal.(2014),'Integrationofgeneexpressiondatawithnetwork‐based

analysistoidentifysignalingandmetabolicpathwaysregulatedduringthe

developmentofosteoarthritis',Gene.

Otero,M.,etal.(2012),'Humanchondrocyteculturesasmodelsofcartilage‐specific

generegulation',MethodsMolBiol,806,301‐36.

Ovcharenko,I.,etal.(2005),'Mulan:multiple‐sequencelocalalignmentand

visualizationforstudyingfunctionandevolution',GenomeRes,15(1),184‐94.

Ovchinnikov,D.A.,etal.(2000),'Col2a1‐directedexpressionofCrerecombinasein

differentiatingchondrocytesintransgenicmice',Genesis,26(2),145‐6.

Park,H.L.,etal.(2000),'MouseGli1mutantsareviablebuthavedefectsinSHH

signalingincombinationwithaGli2mutation',Development,127(8),1593‐605.

Park,J.B.(2009),'Theuseofsimvastatininboneregeneration',MedOralPatolOralCir

Bucal,14(9),e485‐8.

Patra,D.,etal.(2014),'Cartilage‐specificablationofsite‐1proteaseinmiceresultsin

theendoplasmicreticulumentrapmentoftypeIIbprocollagenanddown‐

regulationofcholesterolandlipidhomeostasis',PLoSOne,9(8),e105674.

Patterson,S.E.andDealy,C.N.(2014),'Mechanismsandmodelsofendoplasmic

reticulumstressinchondrodysplasia',DevDyn.

Perez‐Novo,C.A.,etal.(2005),'ImpactofRNAqualityonreferencegeneexpression

stability',Biotechniques,39(1),52,54,56.

150  

Pfander,D.,Swoboda,B.,andKirsch,T.(2001),'Expressionofearlyandlate

differentiationmarkers(proliferatingcellnuclearantigen,syndecan‐3,annexin

VI,andalkalinephosphatase)byhumanosteoarthriticchondrocytes',AmJ

Pathol,159(5),1777‐83.

Poole,A.R.(1999),'Anintroductiontothepathophysiologyofosteoarthritis',Front

Biosci,4,D662‐70.

Porter,F.D.(2003),'Humanmalformationsyndromesduetoinbornerrorsof

cholesterolsynthesis',CurrOpinPediatr,15(6),607‐13.

Poulet,B.,etal.(2014),'Modificationsingaitaspredictorsofnaturalosteoarthritis

progressioninStr/ortmice',ArthritisRheumatol.

Pradervand,S.,etal.(2008),'AffymetrixWhole‐TranscriptHumanGene1.0STarrayis

highlyconcordantwithstandard3'expressionarrays',Biotechniques,44(6),

759‐62.

Prete,P.E.,Gurakar‐Osborne,A.,andKashyap,M.L.(1993),'Synovialfluidlipoproteins:

reviewofcurrentconceptsandnewdirections',SeminArthritisRheum,23(2),

79‐89.

Proffen,B.,Vavken,P.,andDorotka,R.(2013),'Surgicalmanagementofosteoarthritis',

WienMedWochenschr,163(9‐10),243‐50.

Provot,S.andSchipani,E.(2005),'Molecularmechanismsofendochondralbone

development',BiochemBiophysResCommun,328(3),658‐65.

Rahman,M.M.,etal.(2013),'Riskofcardiovasculardiseaseinpatientswith

osteoarthritis:aprospectivelongitudinalstudy',ArthritisCareRes(Hoboken),65

(12),1951‐8.

Ramos,Y.F.,etal.(2013),'Genesexpressedinbloodlinkosteoarthritiswithapoptotic

pathways',AnnRheumDis.

Reagan‐Shaw,S.,Nihal,M.,andAhmad,N.(2008),'Dosetranslationfromanimalto

humanstudiesrevisited',FASEBJ,22(3),659‐61.

Riobo,N.A.(2012),'CholesterolanditsderivativesinSonicHedgehogsignalingand

cancer',CurrOpinPharmacol,12(6),736‐41.

151  

Rockel,J.S.andAlman,B.A.(2011),'Don'thedgeyourbets:hedgehogsignalingasa

centralmediatorofendochondralbonedevelopmentandcartilagediseases',J

OrthopRes,29(6),810‐5.

Ron,D.andWalter,P.(2007),'Signalintegrationintheendoplasmicreticulumunfolded

proteinresponse',NatRevMolCellBiol,8(7),519‐29.

Roughley,P.J.andLee,E.R.(1994),'Cartilageproteoglycans:structureandpotential

functions',MicroscResTech,28(5),385‐97.

Ruettger,A.,etal.(2010),'Comparisonofdifferentmethodsforpreparationand

characterizationoftotalRNAfromcartilagesamplestouncoverosteoarthritisin

vivo',BMCResNotes,3,7.

RuiziAltaba,A.(1999),'Gliproteinsencodecontext‐dependentpositiveandnegative

functions:implicationsfordevelopmentanddisease',Development,126(14),

3205‐16.

Sahlman,J.,etal.(2004),'AhumanCOL2A1genewithanArg519Cysmutationcauses

osteochondrodysplasiaintransgenicmice',ArthritisRheum,50(10),3153‐60.

Sanchez,C.,etal.(2005),'Subchondralboneosteoblastsinducephenotypicchangesin

humanosteoarthriticchondrocytes',OsteoarthritisCartilage,13(11),988‐97.

Sandell,L.J.andAdler,P.(1999),'Developmentalpatternsofcartilage',FrontBiosci,4,

D731‐42.

Sandell,L.J.andAigner,T.(2001),'Articularcartilageandchangesinarthritis.An

introduction:cellbiologyofosteoarthritis',ArthritisRes,3(2),107‐13.

Schroeder,A.,etal.(2006),'TheRIN:anRNAintegritynumberforassigningintegrity

valuestoRNAmeasurements',BMCMolBiol,7,3.

Seeuws,S.,etal.(2010),'Amultiparameterapproachtomonitordiseaseactivityin

collagen‐inducedarthritis',ArthritisResTher,12(4),R160.

Sever,N.,etal.(2003),'AccelerateddegradationofHMGCoAreductasemediatedby

bindingofinsig‐1toitssterol‐sensingdomain',MolCell,11(1),25‐33.

Shi,D.,etal.(2013),'Smoothenedoligomerization/higherorderclusteringinlipidrafts

isessentialforhighHedgehogactivitytransduction',JBiolChem,288(18),

12605‐14.

152  

Simopoulou,T.,etal.(2010),'Protectiveeffectofatorvastatininculturedosteoarthritic

chondrocytes',JOrthopRes,28(1),110‐5.

Stottmann,R.W.,etal.(2011),'Cholesterolmetabolismisrequiredforintracellular

hedgehogsignaltransductioninvivo',PLoSGenet,7(9),e1002224.

Sturmer,T.,etal.(1998),'Serumcholesterolandosteoarthritis.Thebaseline

examinationoftheUlmOsteoarthritisStudy',JRheumatol,25(9),1827‐32.

Sudo,A.,etal.(2008),'Prevalenceandriskfactorsforkneeosteoarthritisinelderly

Japanesemenandwomen',JOrthopSci,13(5),413‐8.

Tabas,I.(2002),'Consequencesofcellularcholesterolaccumulation:basicconceptsand

physiologicalimplications',JClinInvest,110(7),905‐11.

Tchetina,E.V.,Squires,G.,andPoole,A.R.(2005),'IncreasedtypeIIcollagen

degradationandveryearlyfocalcartilagedegenerationisassociatedwith

upregulationofchondrocytedifferentiationrelatedgenesinearlyhuman

articularcartilagelesions',JRheumatol,32(5),876‐86.

Tsang,K.Y.,etal.(2007),'Survivingendoplasmicreticulumstressiscoupledtoaltered

chondrocytedifferentiationandfunction',PLoSBiol,5(3),e44.

Tsezou,A.,etal.(2010),'Impairedexpressionofgenesregulatingcholesteroleffluxin

humanosteoarthriticchondrocytes',JOrthopRes,28(8),1033‐9.

Uitterlinden,E.J.,etal.(2007),'Glucosaminereducesanabolicaswellascatabolic

processesinbovinechondrocytesculturedinalginate',OsteoarthritisCartilage,

15(11),1267‐74.

Valdes,A.M.,etal.(2014),'Useofstatinsisassociatedwithalowerprevalenceof

generalisedosteoarthritis',AnnRheumDis,73(5),943‐5.

vandenBerg,W.B.(2008),'Lessonsfromanimalmodelsofosteoarthritis',Curr

RheumatolRep,10(1),26‐9.

Villalvilla,A.,etal.(2013),'Lipidtransportandmetabolisminhealthyandosteoarthritic

cartilage',IntJMolSci,14(10),20793‐808.

Vlad,S.C.,etal.(2007),'Glucosamineforpaininosteoarthritis:whydotrialresults

differ?',ArthritisRheum,56(7),2267‐77.

153  

vonderMark,K.,etal.(1977),'Relationshipbetweencellshapeandtypeofcollagen

synthesisedaschondrocyteslosetheircartilagephenotypeinculture',Nature,

267(5611),531‐2.

Vortkamp,A.,etal.(1996),'RegulationofrateofcartilagedifferentiationbyIndian

hedgehogandPTH‐relatedprotein',Science,273(5275),613‐22.

Vos,T.,etal.(2012),'Yearslivedwithdisability(YLDs)for1160sequelaeof289

diseasesandinjuries1990‐2010:asystematicanalysisfortheGlobalBurdenof

DiseaseStudy2010',Lancet,380(9859),2163‐96.

Wang,Y.,McMahon,A.P.,andAllen,B.L.(2007),'ShiftingparadigmsinHedgehog

signaling',CurrOpinCellBiol,19(2),159‐65.

Wei,L.(2012),'IndianHedgehog(Ihh)asaMarkertoPredictOsteoarthritis(OA)and

MethodsforthePreventionandTreatmentofOA',(GooglePatents).

Wei,W.,etal.(2014),'Statinsandfibratesdonotaffectdevelopmentofspontaneous

cartilagedamageinSTR/Ortmice',OsteoarthritisCartilage,22(2),293‐301.

Williams,J.A.,etal.(2003),'Identificationofasmallmoleculeinhibitorofthehedgehog

signalingpathway:effectsonbasalcellcarcinoma‐likelesions',ProcNatlAcad

SciUSA,100(8),4616‐21.

Woods,A.,etal.(2009),'Controlofchondrocytegeneexpressionbyactindynamics:a

novelroleofcholesterol/Ror‐alphasignallinginendochondralbonegrowth',J

CellMolMed,13(9B),3497‐516.

Woolf,A.D.andPfleger,B.(2003),'Burdenofmajormusculoskeletalconditions',Bull

WorldHealthOrgan,81(9),646‐56.

Wu,Q.,Zhang,Y.,andChen,Q.(2001),'Indianhedgehogisanessentialcomponentof

mechanotransductioncomplextostimulatechondrocyteproliferation',JBiol

Chem,276(38),35290‐6.

Wu,S.andDeLuca,F.(2004),'Roleofcholesterolintheregulationofgrowthplate

chondrogenesisandlongitudinalbonegrowth',JBiolChem,279(6),4642‐7.

Zhang,H.,Liew,C.C.,andMarshall,K.W.(2002),'Microarrayanalysisrevealsthe

involvementofbeta‐2microglobulin(B2M)inhumanosteoarthritis',

OsteoarthritisCartilage,10(12),950‐60.

154  

Zhang,R.,etal.(2012),'Geneexpressionanalysesofsubchondralboneinearly

experimentalosteoarthritisbymicroarray',PLoSOne,7(2),e32356.

Zhao,C.andDahlman‐Wright,K.(2010),'LiverXreceptorincholesterolmetabolism',J

Endocrinol,204(3),233‐40.

Zhou,J.,etal.(2014),'DisruptingtheIndianhedgehogsignalingpathwayinvivo

attenuatessurgicallyinducedosteoarthritisprogressioninCol2a1‐CreERT2;

Ihhfl/flmice',ArthritisResTher,16(1),R11.

Zhou,M.,etal.(1997),'Astablenonfluorescentderivativeofresorufinforthe

fluorometricdeterminationoftracehydrogenperoxide:applicationsindetecting

theactivityofphagocyteNADPHoxidaseandotheroxidases',AnalBiochem,253

(2),162‐8.

155  

Appendix:Microarraygenelist

TranscriptsClusterId

Foldchange([Treatment1]vs[Control1])

Foldchange([Treatment2]vs[Control2])

Foldchange([Treatment3]vs[Control3])

AVERAGEFoldchange Genesymbol

7896754 ‐1.239296 ‐1.028587 ‐1.2576101 ‐1.187896859 ‐1.1681848 ‐1.1480374 ‐1.0222958 ‐1.117897520 ‐1.1661352 ‐1.0919778 ‐1.179166 ‐1.157898371 ‐1.1814516 ‐1.1686509 ‐1.0819559 ‐1.14 LOC6446347898736 ‐1.0208108 ‐1.2438325 ‐1.1888715 ‐1.15 HSPC1577899534 ‐1.0573555 ‐1.115906 ‐1.2213967 ‐1.13 EPB417900635 ‐1.0297656 ‐1.1724788 ‐1.176768 ‐1.13 hCG_231777901565 ‐1.1557424 ‐1.2017087 ‐1.0000958 ‐1.12 DIO17901951 ‐1.1134347 ‐1.0275791 ‐1.1232364 ‐1.09 PGM17902425 ‐1.1350677 ‐1.1362531 ‐1.0528945 ‐1.11 ST6GALNAC37902861 ‐1.1970338 ‐1.0311054 ‐1.2001237 ‐1.14 LRRC8B7903203 ‐1.1277745 ‐1.0156893 ‐1.1988994 ‐1.11 SNX77903391 ‐1.1768523 ‐1.2460194 ‐1.0103074 ‐1.147905033 ‐1.124537 ‐1.1265637 ‐1.1901158 ‐1.157905035 ‐1.2440093 ‐1.1450274 ‐1.0724686 ‐1.157905308 ‐1.1481949 ‐1.0208989 ‐1.1683198 ‐1.11 BNIPL7905481 ‐1.0264015 ‐1.1864425 ‐1.214625 ‐1.147906330 ‐1.0612032 ‐1.1251994 ‐1.1199013 ‐1.10 CD1D7906954 ‐1.331442 ‐1.1345092 ‐1.1225402 ‐1.20 PBX17908861 ‐1.2182635 ‐1.136407 ‐1.091596 ‐1.15 OCR17909422 ‐1.3482366 ‐1.2777729 ‐1.0128561 ‐1.21 LOC6425877909990 ‐1.2309741 ‐1.0400394 ‐1.2586567 ‐1.187910010 ‐1.0530959 ‐1.3169386 ‐1.2339808 ‐1.207910186 ‐1.2155724 ‐1.1121147 ‐1.1317921 ‐1.157910257 ‐1.1233639 ‐1.1575162 ‐1.0553399 ‐1.11 GJC27910377 ‐1.6061957 ‐1.4717146 ‐1.040646 ‐1.377910727 ‐1.1677496 ‐1.0868012 ‐1.1296215 ‐1.13 ACTN27911049 ‐1.2393565 ‐1.1588407 ‐1.0629451 ‐1.15 C1orf1007911349 ‐1.0628706 ‐1.1624547 ‐1.1226256 ‐1.127911413 ‐1.2520198 ‐1.1395662 ‐1.054782 ‐1.15 TNFRSF47911578 ‐1.217956 ‐1.1199672 ‐1.0720618 ‐1.14 CDC2L2|CDC2L17912861 ‐1.1867154 ‐1.1722811 ‐1.0707278 ‐1.14 LOC6446347915184 ‐1.1162472 ‐1.1842861 ‐1.018505 ‐1.11 RHBDL27915229 ‐1.1994841 ‐1.1562371 ‐1.0787473 ‐1.14 HEYL7915252 ‐1.0718507 ‐1.2693925 ‐1.1473501 ‐1.16 BMP8B7915331 ‐1.0315639 ‐1.2819028 ‐1.2351078 ‐1.187915896 ‐1.2836509 ‐1.155666 ‐1.3532797 ‐1.26 CYP4Z2P7916112 ‐1.3307526 ‐1.0329095 ‐1.2239927 ‐1.20 RAB3B7916743 ‐1.133749 ‐1.173384 ‐1.0798877 ‐1.137917634 ‐1.0058423 ‐1.12566 ‐1.1890664 ‐1.11 HFM17917695 ‐1.13969 ‐1.0306325 ‐1.1260939 ‐1.107918048 ‐1.1583831 ‐1.1604854 ‐1.0550547 ‐1.127919155 ‐1.1688854 ‐1.8558902 ‐1.1456141 ‐1.397919299 ‐1.221169 ‐1.0376744 ‐1.1911544 ‐1.15 LOC1001302367919380 ‐1.2176822 ‐1.0196455 ‐1.1871936 ‐1.14 LOC100130236

156  

7919390 ‐1.1749278 ‐1.1701393 ‐1.1066655 ‐1.157919405 ‐1.174467 ‐1.8295774 ‐1.1409572 ‐1.387920193 ‐1.1835285 ‐1.2680614 ‐1.1082168 ‐1.19 LCE1C7920285 ‐1.2409769 ‐1.3551095 ‐1.3021888 ‐1.30 S100A27920567 ‐1.0126534 ‐1.1684762 ‐1.1436824 ‐1.11 PMVK7921031 ‐1.2109495 ‐1.1401324 ‐1.0691005 ‐1.147922389 ‐1.0073905 ‐1.127707 ‐1.1371604 ‐1.097922404 ‐1.0381347 ‐1.2563999 ‐1.3448906 ‐1.21 SNORD80|GAS57923037 ‐1.1190828 ‐1.1477737 ‐1.1112567 ‐1.137923974 ‐1.1193688 ‐1.0059763 ‐1.1330968 ‐1.09 7924445 ‐1.1240531 ‐1.0341871 ‐1.202964 ‐1.127924819 ‐1.2243763 ‐1.1744118 ‐1.1711066 ‐1.197925062 ‐1.1920491 ‐1.1615798 ‐1.0512623 ‐1.13 SIPA1L27925182 ‐1.0364329 ‐1.202894 ‐1.2015712 ‐1.15 SNORA14B7925242 ‐1.0110915 ‐1.1201626 ‐1.1419328 ‐1.097925318 ‐1.5950552 ‐1.3424733 ‐1.4629917 ‐1.477925718 ‐1.1497971 ‐1.0367104 ‐1.1552188 ‐1.11 OR2B117925846 ‐1.0420406 ‐1.1636237 ‐1.1852438 ‐1.13 C10orf1107926297 ‐1.3219967 ‐1.2300143 ‐1.1535416 ‐1.247927186 ‐1.116926 ‐1.2489287 ‐1.0269914 ‐1.13 RASSF47927681 ‐1.117609 ‐1.1308718 ‐1.0092411 ‐1.09 BICC17928514 ‐1.2361337 ‐1.1492356 ‐1.0223653 ‐1.147929032 ‐1.0980208 ‐1.1755128 ‐1.1332781 ‐1.14 FAS7929533 ‐1.2300106 ‐1.0713491 ‐1.208209 ‐1.17 CC2D2B|LOC1001317207929653 ‐1.1190768 ‐1.321037 ‐1.1438507 ‐1.19 ANKRD27929779 ‐1.1462467 ‐1.0364449 ‐1.1603695 ‐1.11 ABCC27931108 ‐1.1349638 ‐1.1980145 ‐1.0000949 ‐1.11 DMBT17931204 ‐1.124299 ‐1.1153541 ‐1.1310067 ‐1.12 LHPP7931838 ‐1.1907548 ‐1.0391468 ‐1.2228552 ‐1.157933008 ‐1.0160373 ‐1.1437327 ‐1.2838147 ‐1.157933209 ‐1.1713217 ‐1.1251779 ‐1.0760181 ‐1.12 C10orf257933665 ‐1.1675456 ‐1.1430777 ‐1.0292453 ‐1.11 MBL27935679 ‐1.097733 ‐1.1846622 ‐1.1227124 ‐1.14 CPN17937950 ‐1.1202292 ‐1.299221 ‐1.1406789 ‐1.19 OR51D17938758 ‐1.0261204 ‐1.29826 ‐1.239581 ‐1.19 SAA1|SAA27939052 ‐1.1756849 ‐1.0749184 ‐1.1778132 ‐1.14 FIBIN7939365 ‐1.1141043 ‐1.2389454 ‐1.0409287 ‐1.13 FJX17940822 ‐1.0201366 ‐1.1302205 ‐1.2145262 ‐1.127941749 ‐1.1619815 ‐1.1304215 ‐1.0315908 ‐1.11 SYT127944049 ‐1.2701621 ‐1.1039438 ‐1.1240085 ‐1.17 SIDT27944867 ‐1.2389716 ‐1.1459262 ‐1.337283 ‐1.247944970 ‐1.2426654 ‐1.0866597 ‐1.1966279 ‐1.187945663 ‐1.1985242 ‐1.048137 ‐1.2108531 ‐1.15 CTSD|LOC4027787946569 ‐1.146311 ‐1.2217132 ‐1.1331683 ‐1.17 RNF1417946977 ‐1.1775659 ‐1.2733252 ‐1.0118728 ‐1.15 SAA47946983 ‐1.5070485 ‐1.1571165 ‐1.1382565 ‐1.27 SAA2|SAA17947230 ‐1.072386 ‐1.1168576 ‐1.2412677 ‐1.14 BDNF7947462 ‐1.1457043 ‐1.2049994 ‐1.0657629 ‐1.14 ABTB27947551 ‐1.1180062 ‐1.2851707 ‐1.0894003 ‐1.167947742 ‐1.1297166 ‐1.0042957 ‐1.115263 ‐1.08

157  

7948133 ‐1.1340811 ‐1.2189684 ‐1.0596061 ‐1.14 OR5M37948344 ‐1.1773208 ‐1.1347039 ‐1.0178528 ‐1.11 GLYAT7948369 ‐1.2485758 ‐1.0573007 ‐1.1535907 ‐1.157948834 ‐1.3164091 ‐1.1715544 ‐1.0344025 ‐1.177948987 ‐1.0112492 ‐1.1268858 ‐1.1415362 ‐1.09 HRASLS37950036 ‐1.1263306 ‐1.1254836 ‐1.0950731 ‐1.12 FGF37950553 ‐1.09396 ‐1.2258033 ‐1.1205388 ‐1.157951004 ‐1.1444405 ‐1.0152844 ‐1.1616305 ‐1.11 C11orf757951429 ‐1.2179612 ‐1.1664883 ‐1.164392 ‐1.18 KBTBD37951668 ‐1.1966258 ‐2.26246 ‐1.0374669 ‐1.507952339 ‐1.2256712 ‐1.2279723 ‐1.0599637 ‐1.17 LOC853897952631 ‐1.1697 ‐1.1174647 ‐1.0379763 ‐1.11 P53AIP17953032 ‐1.1515633 ‐1.2214835 ‐1.0870336 ‐1.15 LRTM27953665 ‐1.1767784 ‐1.1308128 ‐1.1538184 ‐1.15 DPPA37954100 ‐1.2158893 ‐1.1309227 ‐1.0168245 ‐1.127955043 ‐1.029899 ‐1.1148893 ‐1.2694701 ‐1.147955694 ‐1.1697878 ‐1.0139592 ‐1.1990726 ‐1.13 IGFBP67955729 ‐1.2305291 ‐1.0022773 ‐1.2645892 ‐1.17 MFSD57957023 ‐1.1383976 ‐1.0393449 ‐1.1166416 ‐1.10 LYZ7957161 ‐1.1278166 ‐1.1962087 ‐1.1203734 ‐1.15 THAP27958207 ‐1.2753422 ‐1.2590711 ‐1.1286881 ‐1.227958453 ‐1.0169996 ‐1.2286311 ‐1.2156032 ‐1.157960338 ‐1.2524924 ‐1.1274483 ‐1.1650988 ‐1.187960436 ‐1.3187519 ‐1.0697742 ‐1.2069689 ‐1.20 FLJ448747961891 ‐1.1398716 ‐1.116494 ‐1.0586067 ‐1.10 BHLHB37962139 ‐1.2944684 ‐1.6259124 ‐1.2575294 ‐1.39 OVOS2|LOC1001328817962236 ‐1.1196722 ‐1.2289126 ‐1.1078498 ‐1.157963502 ‐1.1831151 ‐1.3382368 ‐1.0556839 ‐1.19 KRT777963689 ‐1.1279773 ‐1.1819038 ‐1.0707328 ‐1.13 NPFF7963946 ‐1.1534932 ‐1.0060945 ‐1.1139452 ‐1.09 MMP197964246 ‐1.1678979 ‐1.0677938 ‐1.2752719 ‐1.17 SNORD59B7964602 ‐1.1717365 ‐1.1507206 ‐1.1221786 ‐1.15 LRIG37965767 ‐1.0049801 ‐1.1923171 ‐1.1170946 ‐1.107966127 ‐1.1933887 ‐1.1227053 ‐1.0343099 ‐1.12 SELPLG7966225 ‐1.1991614 ‐1.3619182 ‐1.114256 ‐1.237966293 ‐1.1580918 ‐1.0965353 ‐1.1602176 ‐1.14 FLJ401427967107 ‐1.1083332 ‐1.3466547 ‐1.1840038 ‐1.21 C12orf277967193 ‐1.1167853 ‐1.2344662 ‐1.0399429 ‐1.13 MORN37967698 ‐1.122709 ‐1.2789763 ‐1.6820436 ‐1.367967872 ‐1.1310834 ‐1.0743521 ‐1.1848722 ‐1.137970388 ‐1.0185413 ‐1.1809233 ‐1.1150835 ‐1.107971526 ‐1.0544024 ‐1.1821325 ‐1.2638721 ‐1.17 HTR2A7972546 ‐1.1597128 ‐1.1144748 ‐1.021918 ‐1.107973652 ‐1.145908 ‐1.0977293 ‐1.1228678 ‐1.12 TSSK47974214 ‐1.0512942 ‐1.1815953 ‐1.2344646 ‐1.16 KLHDC17974335 ‐1.0273261 ‐1.1712015 ‐1.1275516 ‐1.117974687 ‐1.1308441 ‐1.1638987 ‐1.075547 ‐1.127975309 ‐1.1326947 ‐1.1706564 ‐1.2940979 ‐1.207976239 ‐1.1648555 ‐1.0687666 ‐1.2078676 ‐1.15 PTMAP77976810 ‐1.1919054 ‐1.1559659 ‐1.1252129 ‐1.16 SNORD113‐3

158  

7977761 ‐1.2426963 ‐1.1237935 ‐1.0531527 ‐1.14 SALL27978653 ‐1.1632799 ‐1.2946237 ‐1.0140741 ‐1.16 GARNL17978690 ‐1.1967659 ‐1.0102891 ‐1.2214823 ‐1.147980603 ‐1.2631735 ‐1.0843284 ‐1.1148542 ‐1.157980605 ‐1.0962623 ‐1.1209263 ‐1.3158514 ‐1.18 KCNK107980859 ‐1.0359589 ‐1.1238269 ‐1.1359125 ‐1.10 PP89617980889 ‐1.1272774 ‐1.162957 ‐1.0199138 ‐1.107981742 ‐1.1492268 ‐1.1603285 ‐1.0562541 ‐1.12 LOC909257981947 ‐1.1578717 ‐1.0548266 ‐1.2465389 ‐1.15 SNRPN7981972 ‐1.1312745 ‐1.6127473 ‐1.0674124 ‐1.27 SNRPN7982098 ‐1.160935 ‐1.0550139 ‐1.2484871 ‐1.15 SNRPN7982230 ‐1.0053731 ‐1.1952736 ‐1.1650352 ‐1.12 FAM7A1|GOLGA9P|FAM7A37982284 ‐1.1217887 ‐1.170737 ‐1.1689181 ‐1.15 GOLGA9P7982535 ‐1.054629 ‐1.1901615 ‐1.1304559 ‐1.13 C15orf417982876 ‐1.1565158 ‐1.0874544 ‐1.122222 ‐1.12 FAM92A1|FAM92A27983500 ‐1.1225733 ‐1.394044 ‐1.0550944 ‐1.197983630 ‐1.108441 ‐1.1242946 ‐1.1825603 ‐1.14 FGF77983771 ‐1.003591 ‐1.2217277 ‐1.2300467 ‐1.157984436 ‐1.2748382 ‐1.2105106 ‐1.0067405 ‐1.16 LBXCOR17985037 ‐1.226634 ‐1.3714616 ‐1.2055959 ‐1.277985221 ‐1.0213271 ‐1.1221585 ‐1.1226256 ‐1.09 LOC6469347985587 ‐1.1192448 ‐1.151928 ‐1.0999775 ‐1.12 SCAND27986561 ‐1.3620731 ‐1.1061167 ‐1.1974267 ‐1.227987066 ‐1.0151405 ‐1.2709823 ‐1.145822 ‐1.147987310 ‐1.0802622 ‐1.2658659 ‐1.1149721 ‐1.15 GJD2|GJA97988077 ‐1.1254036 ‐1.0084178 ‐1.169678 ‐1.10 LCMT27988424 ‐1.0237318 ‐1.4947435 ‐1.4157429 ‐1.317988440 ‐1.1148093 ‐1.0219171 ‐1.349166 ‐1.167989968 ‐1.2974492 ‐1.0074338 ‐1.1229858 ‐1.14 CALML47990452 ‐1.0519546 ‐1.2617414 ‐1.2692586 ‐1.19 LOC554175|LOC7298097991047 ‐1.1691381 ‐1.1899309 ‐1.1677655 ‐1.18 LOC1001318607991542 ‐1.2985655 ‐1.0675676 ‐1.1495607 ‐1.17 FLJ422897991815 ‐1.1650152 ‐1.1390272 ‐1.0242103 ‐1.11 PDIA27992863 ‐1.1628617 ‐1.1943543 ‐1.2081535 ‐1.19 OR1F17992877 ‐1.1656126 ‐1.1188745 ‐1.0911243 ‐1.13 ZNF75A|LOC1001285107993754 ‐1.1725152 ‐1.1423855 ‐1.0191922 ‐1.11 LOC1001294887995332 ‐1.1419132 ‐1.2413559 ‐1.0103017 ‐1.137995350 ‐1.0441247 ‐1.1841248 ‐1.2310412 ‐1.157996498 ‐1.1525491 ‐1.1862032 ‐1.0500996 ‐1.13 SLC9A57997372 ‐1.1426249 ‐1.1882234 ‐1.004234 ‐1.117997875 ‐1.053784 ‐1.1225687 ‐1.185921 ‐1.12 C16orf817998485 ‐1.107941 ‐1.225454 ‐1.1583207 ‐1.16 LOC2839517998927 ‐1.1506369 ‐1.1043873 ‐1.2603927 ‐1.177999886 ‐1.1707772 ‐1.1728144 ‐1.0082905 ‐1.127999981 ‐1.2192432 ‐1.0627617 ‐1.1467855 ‐1.14 ACSM18000738 ‐1.0783455 ‐1.1448008 ‐1.1565655 ‐1.13 KCTD138001102 ‐1.1731738 ‐1.2441498 ‐1.1090258 ‐1.188001371 ‐1.0241971 ‐1.1183219 ‐1.1471221 ‐1.108004184 ‐1.079198 ‐1.1509663 ‐1.1279597 ‐1.12 XAF18004309 ‐1.1344624 ‐1.1494528 ‐1.1473053 ‐1.14 SLC2A4

159  

8004408 ‐1.2454047 ‐1.1325206 ‐1.0803822 ‐1.15 FGF118005225 ‐1.1170769 ‐1.2349257 ‐1.1674564 ‐1.17 LOC1626328005685 ‐1.1663718 ‐1.0901207 ‐1.1965191 ‐1.158005753 ‐1.003376 ‐1.1873051 ‐1.4082209 ‐1.208005785 ‐1.1949079 ‐1.1610156 ‐1.0469292 ‐1.13 KSR18005809 ‐1.178125 ‐1.2208102 ‐1.2303269 ‐1.21 LGALS98005953 ‐1.0350748 ‐1.2790414 ‐1.2803051 ‐1.20 SNORD4A8006081 ‐1.1821449 ‐1.1199597 ‐1.0864234 ‐1.138006433 ‐1.1256804 ‐1.0086231 ‐1.1455703 ‐1.09 CCL28006865 ‐1.2422552 ‐1.194151 ‐1.0407062 ‐1.16 PPP1R1B8007263 ‐1.125596 ‐1.1608901 ‐1.1517155 ‐1.15 HSD17B18007603 ‐1.1137339 ‐1.1158684 ‐1 ‐1.088008350 ‐1.1626219 ‐1.1449513 ‐1.0000218 ‐1.10 MYCBPAP8008588 ‐1.0730299 ‐1.1852114 ‐1.1709827 ‐1.14 HLF8008965 ‐1.1299677 ‐1.0466642 ‐1.2586576 ‐1.158009844 ‐1.1973641 ‐1.1401241 ‐1.1038592 ‐1.15 LLGL28010078 ‐1.1731684 ‐1.248726 ‐1.1627487 ‐1.19 SNORD1C8010897 ‐1.1377529 ‐1.1930258 ‐1.0499513 ‐1.13 METRNL8011671 ‐1.2097199 ‐1.1669418 ‐1 ‐1.13 GGT68011732 ‐1.2225828 ‐1.0491917 ‐1.1758633 ‐1.15 LOC1001329788012309 ‐1.1198332 ‐1.1229507 ‐1.0949949 ‐1.11 ALOX12B8012951 ‐1.4136238 ‐1.0232565 ‐1.1511729 ‐1.20 CDRT1|TRIM168013356 ‐1.0691361 ‐1.2525641 ‐1.1390085 ‐1.158013527 ‐1.183047 ‐1.062078 ‐1.1270676 ‐1.128013529 ‐1.3972099 ‐1.0783712 ‐1.2473458 ‐1.248013987 ‐1.1142015 ‐1.4133202 ‐1.5968835 ‐1.378014704 ‐1.1859324 ‐1.2775048 ‐1.2140096 ‐1.238015681 ‐1.1925843 ‐1.2735271 ‐1.0437707 ‐1.17 CCR108015906 ‐1.1705462 ‐1.1195908 ‐1.1021883 ‐1.138016487 ‐1.1224438 ‐1.1857282 ‐1.0014987 ‐1.10 HOXB138016532 ‐1.1204137 ‐1.1956154 ‐1.0427014 ‐1.12 GNGT28016739 ‐1.1387917 ‐1.1250964 ‐1.120523 ‐1.13 TOB18017378 ‐1.1446226 ‐1.0477349 ‐1.1547782 ‐1.12 CYB5618018652 ‐1.361631 ‐1.1638983 ‐1.0695252 ‐1.20 RNF1578019643 ‐1.1769046 ‐1.1450365 ‐1.120059 ‐1.15 DOC2B8020123 ‐1.2161952 ‐1.1715344 ‐1.2168515 ‐1.20 TXNDC28020760 ‐1.2144672 ‐1.1808996 ‐1.1884449 ‐1.198021245 ‐1.1288146 ‐1.3623563 ‐1.0556556 ‐1.18 DCC8023528 ‐1.0272005 ‐1.3503045 ‐1.1576416 ‐1.18 ALPK28023926 ‐1.3542663 ‐1.1436003 ‐1.0449892 ‐1.18 PARD6G8025826 ‐1.1852858 ‐1.1862342 ‐1.0969282 ‐1.168026182 ‐1.2166154 ‐1.1255115 ‐1.0662421 ‐1.14 MGC32078026787 ‐1.1088161 ‐1.2473087 ‐1.2091366 ‐1.19 FAM129C8026926 ‐1.138226 ‐1.155456 ‐1.0498385 ‐1.11 MAST38027233 ‐1.1181829 ‐1.0932386 ‐1.1441342 ‐1.12 ZNF1018027377 ‐1.1775936 ‐1.120066 ‐1.0015619 ‐1.108027429 ‐1.2911018 ‐1.3320327 ‐1.0229461 ‐1.228028206 ‐1.2205637 ‐1.0949863 ‐1.3631274 ‐1.23 ZNF3458028309 ‐1.1849595 ‐1.2031429 ‐1.1301652 ‐1.178029375 ‐1.165125 ‐1.2562487 ‐1.0103853 ‐1.14

160  

8029914 ‐1.0083652 ‐1.1516474 ‐1.2063702 ‐1.12 GPR778030831 ‐1.056192 ‐1.2895998 ‐1.1884161 ‐1.18 ZNF1758032491 ‐1.1193259 ‐1.1811864 ‐1.0043415 ‐1.10 LMNB28034320 ‐1.1282841 ‐1.1623744 ‐1.1057303 ‐1.13 ZNF4338034326 ‐1.2614471 ‐1.0487839 ‐1.1463664 ‐1.158034349 ‐1.2122726 ‐1.027877 ‐1.2724067 ‐1.17 ZNF448034390 ‐1.1742053 ‐1.0433823 ‐1.3176584 ‐1.18 ZNF7998034694 ‐1.1095688 ‐1.1832575 ‐1.2473186 ‐1.188035847 ‐1.1244503 ‐1.0256978 ‐1.4222808 ‐1.19 ZNF6758036351 ‐1.1939071 ‐1.2281318 ‐1.0104808 ‐1.14 LOC3428928037231 ‐1.463514 ‐1.2127107 ‐1.0521618 ‐1.24 PSG38037259 ‐1.1891646 ‐1.1960526 ‐1.2747034 ‐1.22 PSG118037283 ‐1.1774278 ‐1.1716328 ‐1.2232862 ‐1.19 PSG48037621 ‐1.1437429 ‐1.0150472 ‐1.1588085 ‐1.11 EML28038314 ‐1.0293359 ‐1.1624051 ‐1.1162008 ‐1.10 FLJ104908038874 ‐1.2146318 ‐1.169089 ‐1.1225892 ‐1.17 FLJ304038038989 ‐1.1474153 ‐1.1355653 ‐1.1895802 ‐1.16 ZNF6008040365 ‐1.237787 ‐1.1225224 ‐1.1014553 ‐1.15 TRIB28040712 ‐1.1338233 ‐1.1207978 ‐1.016668 ‐1.09 CENPA8042038 ‐1.1501498 ‐1.0612081 ‐1.213359 ‐1.14 RPL23AP138042308 ‐1.1846063 ‐1.219864 ‐1.178802 ‐1.198042324 ‐1.0209745 ‐1.2211428 ‐1.2123812 ‐1.15 8042464 ‐1.016172 ‐1.2357031 ‐1.1922982 ‐1.158042532 ‐1.0403354 ‐1.2517078 ‐1.1173948 ‐1.14 VAX28043043 ‐1.1452181 ‐1.2406706 ‐1.0604295 ‐1.15 DNHL1|LOC2003838043071 ‐1.1253717 ‐1.0869683 ‐1.2295554 ‐1.15 LOC2003838043502 ‐1.041727 ‐1.1712826 ‐1.4214735 ‐1.218043512 ‐1.0799599 ‐1.1247827 ‐1.233348 ‐1.15 ZNF28044035 ‐1.1625024 ‐1.0278059 ‐1.2220144 ‐1.14 IL18R18044764 ‐1.0152888 ‐1.1281484 ‐1.1196922 ‐1.098045279 ‐1.0548402 ‐1.2509687 ‐1.2248049 ‐1.188046922 ‐1.1524653 ‐1.0906504 ‐1.1461868 ‐1.13 COL3A18047337 ‐1.2136518 ‐1.1245916 ‐1.0514058 ‐1.138047557 ‐1.1737839 ‐1.1763889 ‐1.1030033 ‐1.158049487 ‐1.2017003 ‐1.1330436 ‐1.1553528 ‐1.16 MLPH8050060 ‐1.0316539 ‐1.1242983 ‐1.2154266 ‐1.12 TSSC18050071 ‐1.0232592 ‐1.1345288 ‐1.2759494 ‐1.14 ADI18050687 ‐1.2850941 ‐1.1354996 ‐1.1409444 ‐1.198052784 ‐1.0668502 ‐1.2068669 ‐1.2600424 ‐1.18 NFU18052866 ‐1.0840428 ‐1.1528002 ‐1.1390525 ‐1.13 FAM136A8053231 ‐1.1174583 ‐1.2038665 ‐1.2263949 ‐1.18 LOXL38053379 ‐1.3621267 ‐1.140152 ‐1.0118667 ‐1.17 LOC1292938054295 ‐1.1482404 ‐1.0459356 ‐1.2824177 ‐1.168055643 ‐1.1755197 ‐1.1234406 ‐1.319307 ‐1.218056877 ‐1.1515645 ‐1.209793 ‐1.0940906 ‐1.15 CHRNA18056995 ‐1.1414709 ‐1.0475291 ‐1.6364006 ‐1.28 TTC30B8057599 ‐1.0779386 ‐1.1524447 ‐1.2133337 ‐1.15 TFPI8057817 ‐1.0712577 ‐1.1770087 ‐1.144253 ‐1.138058106 ‐1.0137728 ‐1.2836937 ‐1.1311256 ‐1.148059578 ‐1.3709984 ‐1.2950534 ‐1.0034155 ‐1.22

161  

8059864 ‐1.1359365 ‐1.1819448 ‐1.0493227 ‐1.12 GBX28059996 ‐1.1858007 ‐1.0751188 ‐1.1151752 ‐1.13 PER28060765 ‐1.1278725 ‐1.2077934 ‐1.0838586 ‐1.14 PRND8061404 ‐1.1667578 ‐1.0267144 ‐1.2232623 ‐1.148062864 ‐1.1299961 ‐1.1280899 ‐1.0402436 ‐1.10 WISP28063074 ‐1.161579 ‐1.3275356 ‐1.030984 ‐1.17 ZSWIM18063410 ‐1.0729451 ‐1.1434417 ‐1.1414875 ‐1.12 PARD6B8063531 ‐1.167091 ‐1.0631039 ‐1.1878158 ‐1.14 C20orf1078064868 ‐1.2455668 ‐1.2226378 ‐1.2192042 ‐1.23 RP5‐1022P6.28065194 ‐1.0284455 ‐1.4689733 ‐1.2417064 ‐1.25 8066745 ‐1.1293821 ‐1.0006018 ‐1.1949793 ‐1.11 ZNF3348067585 ‐1.1276138 ‐1.0314044 ‐1.1306652 ‐1.10 BHLHB48068383 ‐1.1109526 ‐1.2716205 ‐1.1471775 ‐1.18 CLIC68069804 ‐1.2963073 ‐1.1182044 ‐1.0640442 ‐1.16 KRTAP26‐18069811 ‐1.1415263 ‐1.1699111 ‐1.0471985 ‐1.12 KRTAP23‐18070097 ‐1.2714323 ‐1.1978985 ‐1.0551244 ‐1.17 C21orf558070681 ‐1.2725102 ‐1.0779703 ‐1.1427877 ‐1.16 C21orf848071168 ‐1.1572989 ‐1.0671101 ‐1.1214296 ‐1.12 DKFZP434P211|LOC6460748071272 ‐1.159128 ‐1.2243245 ‐1.1595939 ‐1.18 GP1BB8073015 ‐1.1491311 ‐1.0548085 ‐1.1289803 ‐1.11 KDELR38073088 ‐1.166444 ‐1.2652293 ‐1.041814 ‐1.16 APOBEC3G|APOBEC3F8074057 ‐1.1644158 ‐1.2275028 ‐1.1066564 ‐1.178074714 ‐1.1829728 ‐1.1847116 ‐1.0770009 ‐1.15 DKFZP434P211|LOC6460748075785 ‐1.140406 ‐1.116487 ‐1.0849069 ‐1.11 FOXRED28075924 ‐1.1899663 ‐1.1209122 ‐1.2011963 ‐1.17 MFNG8076113 ‐1.1582625 ‐1.1279755 ‐1.0208787 ‐1.10 RP1‐199H16.18077323 ‐1.0886214 ‐1.4730018 ‐1.2605501 ‐1.27 CNTN48077595 ‐1.1630408 ‐1.0093642 ‐1.128436 ‐1.10 BRPF18077635 ‐1.121752 ‐1.0662351 ‐1.1674826 ‐1.12 OGG18078155 ‐1.1356629 ‐1.1786535 ‐1.0015367 ‐1.11 GALNTL28079613 ‐1.1212101 ‐1.0444125 ‐1.12985 ‐1.10 8079988 ‐1.3122028 ‐1.1141529 ‐1.00804 ‐1.14 C3orf458081233 ‐1.2021098 ‐1.1782302 ‐1.2279524 ‐1.208081348 ‐1.0009781 ‐1.1224022 ‐1.3112178 ‐1.14 PCNP8081503 ‐1.046727 ‐1.1794485 ‐1.1418022 ‐1.12 DZIP38083061 ‐1.182843 ‐1.1338469 ‐1.2538469 ‐1.198083214 ‐1.176936 ‐1.204946 ‐1.0205301 ‐1.13 CHST28084217 ‐1.1498879 ‐1.2449409 ‐1.1839526 ‐1.198084766 ‐1.2596422 ‐1.0055674 ‐1.1228255 ‐1.13 TP638085732 ‐1.2460425 ‐1.1491847 ‐1.0675083 ‐1.15 EFHB8087825 ‐1.1354399 ‐1.1155609 ‐1.0376215 ‐1.10 ABHD14B8088285 ‐1.15899 ‐1.1783205 ‐1.0223178 ‐1.12 HESX18088952 ‐1.0175797 ‐1.1159927 ‐1.2305539 ‐1.128089993 ‐1.0024654 ‐1.1609001 ‐1.1301271 ‐1.10 WDR5B8090291 ‐1.2615534 ‐1.0849414 ‐1.2922174 ‐1.21 LOC2008108090559 ‐1.0148567 ‐1.1266266 ‐1.1708612 ‐1.10 RPL32P38090674 ‐1.0479083 ‐1.2538457 ‐1.169852 ‐1.168091537 ‐1.1301827 ‐1.2261946 ‐1.0756636 ‐1.14 IGSF108091922 ‐1.1779809 ‐1.2007847 ‐1.1210434 ‐1.17 WDR498092169 ‐1.5019922 ‐1.336261 ‐1.0817556 ‐1.31 TNFSF10

162  

8092541 ‐1.1631415 ‐1.1345767 ‐1.1707602 ‐1.16 LIPH8092596 ‐1.1804836 ‐1.1404588 ‐1.0028856 ‐1.11 DGKG8095251 ‐1.0698718 ‐1.1722296 ‐1.2220927 ‐1.158096411 ‐1.0418754 ‐1.1272963 ‐1.1589227 ‐1.11 TIGD28096955 ‐1.2390587 ‐1.2746465 ‐1.3130344 ‐1.288097447 ‐1.021497 ‐1.1401143 ‐1.1438525 ‐1.108098506 ‐1.1622338 ‐1.0544153 ‐1.1623598 ‐1.138098671 ‐1.1506845 ‐1.182949 ‐1.2312691 ‐1.19 F118099025 ‐1.1469216 ‐1.2548261 ‐1.0398202 ‐1.158099255 ‐1.1978335 ‐1.1434828 ‐1.0145589 ‐1.12 PSAPL18099570 ‐1.0125599 ‐1.1833833 ‐1.1953404 ‐1.13 C4orf308099922 ‐1.3966119 ‐1.489955 ‐1.2042067 ‐1.368100229 ‐1.1427068 ‐1.3490162 ‐1.1700697 ‐1.228100362 ‐1.0851167 ‐1.1568668 ‐1.1268337 ‐1.12 LNX18100519 ‐1.1381354 ‐1.0797079 ‐1.1146693 ‐1.11 TXNDC98100541 ‐1.0728738 ‐1.173856 ‐1.1154237 ‐1.12 IGFBP78100555 ‐1.0007436 ‐1.2045817 ‐1.1667786 ‐1.12 LOC3916568100699 ‐1.1165882 ‐1.1587764 ‐1.0265064 ‐1.10 SYT14L8101061 ‐1.0544167 ‐1.1955289 ‐1.1307522 ‐1.138101780 ‐1.1413743 ‐1.0857375 ‐1.153593 ‐1.13 PGDS8102781 ‐1.2576001 ‐1.1657729 ‐1.2119558 ‐1.21 LOC6461878103023 ‐1.114999 ‐1.089999 ‐1.1910535 ‐1.138103166 ‐1.1877081 ‐1.0424626 ‐1.1459823 ‐1.13 SH3D19|FLJ464818103516 ‐1.0159528 ‐1.1446517 ‐1.1705551 ‐1.118103853 ‐1.2490022 ‐1.1504657 ‐1.3950586 ‐1.26 MGC458008104348 ‐1.0803001 ‐1.1395029 ‐1.3162934 ‐1.188104617 ‐1.1307904 ‐1.1290511 ‐1.0110162 ‐1.09 LOC3917648104654 ‐1.2701516 ‐1.002062 ‐1.1434624 ‐1.148105989 ‐1.1451144 ‐1.4596436 ‐1.0017636 ‐1.20 FLJ40092|LOC4410818106743 ‐1.0679519 ‐1.1786653 ‐1.2343683 ‐1.16 VCAN8106776 ‐1.0397291 ‐1.1978922 ‐1.3132606 ‐1.18 COX7C8107162 ‐1.169216 ‐1.0817614 ‐1.1604179 ‐1.14 LOC1345058107857 ‐1.08141 ‐1.2999642 ‐1.1465683 ‐1.188108217 ‐1.8351796 ‐1.3044615 ‐1.069777 ‐1.40 TGFBI8108832 ‐1.0880775 ‐1.1365558 ‐1.1258345 ‐1.12 KIAA01418109486 ‐1.0083958 ‐1.408165 ‐1.2317425 ‐1.228109612 ‐1.1516181 ‐1.0644593 ‐1.1284639 ‐1.11 ADRA1B8109693 ‐1.1391666 ‐1.137372 ‐1.0107262 ‐1.10 8110668 ‐1.019779 ‐1.1163096 ‐1.1654474 ‐1.108112258 ‐1.2392946 ‐1.1847371 ‐1.1845342 ‐1.208112476 ‐1.0388913 ‐1.390674 ‐1.2577717 ‐1.23 FLJ40092|LOC4410818112615 ‐1.1377119 ‐1.1698558 ‐1.1605732 ‐1.16 ENC18113658 ‐1.1629851 ‐1.3860811 ‐1.1062201 ‐1.22 LOC1001320148113724 ‐1.0659521 ‐1.2787704 ‐1.3962063 ‐1.258114111 ‐1.7492881 ‐1.2535564 ‐1.0454283 ‐1.358114300 ‐1.2430623 ‐1.1569777 ‐1.0910265 ‐1.16 KLHL38114780 ‐1.246393 ‐1.0990406 ‐1.149239 ‐1.16 PCDH128117583 ‐1.0010923 ‐1.261282 ‐1.1729195 ‐1.15 HIST1H2AI|HIST1H3H8117760 ‐1.0341878 ‐1.2558444 ‐1.1446778 ‐1.14 HLA‐F8117869 ‐1.0342736 ‐1.1565961 ‐1.1581645 ‐1.12 TRIM39|TRIM39R

163  

8118322 ‐1.2387516 ‐1.054911 ‐1.1256804 ‐1.14 SNORD52|C6orf488119423 ‐1.1338044 ‐1.161085 ‐1.2753941 ‐1.19 LOC2214428119896 ‐1.1155329 ‐1.1786734 ‐1.011395 ‐1.108120057 ‐1.0891224 ‐1.2881671 ‐1.1179605 ‐1.178120247 ‐1.1962273 ‐1.1585102 ‐1.0011622 ‐1.128120271 ‐1.2747245 ‐1.0986568 ‐1.1650711 ‐1.18 FBXO98120460 ‐1.0332854 ‐1.204305 ‐1.211022 ‐1.15 LOC4422298121130 ‐1.1169311 ‐1.0591099 ‐1.1551149 ‐1.118121741 ‐1.0830096 ‐1.1472548 ‐1.1256161 ‐1.128123644 ‐1.069684 ‐1.1721658 ‐1.1153345 ‐1.12 TUBB2A8124553 ‐1.1262603 ‐1.0392927 ‐1.2153076 ‐1.13 ZKSCAN48125234 ‐1.0007002 ‐1.1907489 ‐1.1743116 ‐1.12 TNXB|TNXA8125289 ‐1.0670452 ‐1.2549875 ‐1.1605295 ‐1.16 TNXA|TNXB8125859 ‐1.0436913 ‐1.113749 ‐1.1736687 ‐1.11 TAF118126184 ‐1.3638836 ‐1.0601834 ‐1.1557374 ‐1.19 KIF68126442 ‐1.1755081 ‐1.1922375 ‐1.1053839 ‐1.16 TBCC8126484 ‐1.3382208 ‐1.1082193 ‐1.1804925 ‐1.218127660 ‐1.1673032 ‐1.1541712 ‐1.0719168 ‐1.138127932 ‐1.1354865 ‐1.0172201 ‐1.1865585 ‐1.11 TBX188129392 ‐1.1739697 ‐1.1553757 ‐1.0564686 ‐1.13 KIAA0408|C6orf1748129876 ‐1.1449543 ‐1.0052804 ‐1.2982544 ‐1.15 PBOV18130181 ‐1.121079 ‐1.1840845 ‐1.0383615 ‐1.118130660 ‐1.3080856 ‐1.2649312 ‐1.5105453 ‐1.368131919 ‐1.1996411 ‐1.1020441 ‐1.1206433 ‐1.14 NPY8132245 ‐1.1235428 ‐1.010256 ‐1.2548987 ‐1.13 FLJ207128132250 ‐1.172944 ‐1.0751791 ‐1.1522795 ‐1.13 BMPER8132830 ‐1.2432225 ‐1.6542674 ‐1.3796985 ‐1.438132843 ‐1.0427817 ‐1.7100819 ‐1.2685616 ‐1.34 FAM29A(HAUS6)8135224 ‐1.0270871 ‐1.2254953 ‐1.1621358 ‐1.14 NF‐E48135915 ‐1.1398461 ‐1.0279154 ‐1.1221076 ‐1.10 HIG28136063 ‐1.0105916 ‐1.1974611 ‐1.1231867 ‐1.118136078 ‐1.3011712 ‐1.3270377 ‐1.4341699 ‐1.35 8136658 ‐1.1611602 ‐1.2796263 ‐1.1266106 ‐1.198136954 ‐1.1303827 ‐1.0141361 ‐1.1445726 ‐1.10 FAM139B|LOC6527958137232 ‐1.1385602 ‐1.123844 ‐1.1237752 ‐1.13 GIMAP88137464 ‐1.2233617 ‐1.3429384 ‐1.1178629 ‐1.238137927 ‐1.3463244 ‐1.2116418 ‐1.2346703 ‐1.26 PAPOLB8137931 ‐1.1515138 ‐1.2659065 ‐1.193679 ‐1.20 MMD28141560 ‐1.2044296 ‐1.0075576 ‐1.1465406 ‐1.12 TFR28142763 ‐1.1865283 ‐1.2030901 ‐1.4484302 ‐1.288143154 ‐1.2802789 ‐1.3185332 ‐1.0211422 ‐1.21 DGKI8143463 ‐1.1763363 ‐1.1017003 ‐1.155456 ‐1.14 LOC1362428144230 ‐1.0584028 ‐1.1357356 ‐1.2626258 ‐1.15 ZNF5968144621 ‐1.1177125 ‐1.3881612 ‐1.1920708 ‐1.23 C8orf128144717 ‐1.0508242 ‐1.1644509 ‐1.1943152 ‐1.148144878 ‐1.1645075 ‐1.0226784 ‐1.2938935 ‐1.168146330 ‐1.1271794 ‐1.0504675 ‐1.1689004 ‐1.128146517 ‐1.039835 ‐1.2308658 ‐1.3265978 ‐1.20 CHCHD78146859 ‐1.0421897 ‐1.5340792 ‐1.2190603 ‐1.278148158 ‐1.1408248 ‐1.0781295 ‐1.3091106 ‐1.18 WDR67

164  

8148474 ‐1.0087148 ‐1.1766478 ‐1.1217109 ‐1.108149275 ‐1.3396361 ‐1.2699971 ‐1.535426 ‐1.388149809 ‐1.1729199 ‐1.2145904 ‐1.0561767 ‐1.158149865 ‐1.1649675 ‐1.193713 ‐1.0087445 ‐1.12 EBF28150687 ‐1.2286807 ‐1.22496 ‐1.0197637 ‐1.168151215 ‐1.125452 ‐1.032116 ‐1.1211839 ‐1.09 LOC7287748151223 ‐1.2021682 ‐1.161791 ‐1.0005025 ‐1.12 SLCO5A18151310 ‐1.1394893 ‐1.1520045 ‐1.029043 ‐1.11 EYA18152597 ‐1.1296767 ‐1.0231435 ‐1.2725624 ‐1.14 MRPL138153304 ‐1.1329118 ‐1.1144217 ‐1.0485513 ‐1.10 TSNARE18153320 ‐1.0592437 ‐1.2180276 ‐1.1353679 ‐1.148153363 ‐1.1578265 ‐1.1863923 ‐1.0210525 ‐1.12 CYP11B18154447 ‐1.0598923 ‐1.1965804 ‐1.1690933 ‐1.148154491 ‐1.1434791 ‐1.3207484 ‐1.3826722 ‐1.28 ADAMTSL18154512 ‐1.178788 ‐1.0925812 ‐1.1565659 ‐1.14 ADAMTSL18155508 ‐1.1404332 ‐1.0695829 ‐1.4007633 ‐1.208156523 ‐1.0563921 ‐1.1138076 ‐1.1858414 ‐1.128157027 ‐1.0185847 ‐1.258886 ‐1.1265827 ‐1.13 NIPSNAP3B8157731 ‐1.1228418 ‐1.1581258 ‐1.0029973 ‐1.09 CRB28158081 ‐1.074475 ‐1.2064265 ‐1.1591502 ‐1.15 C9orf1178158542 ‐1.1450524 ‐1.2212651 ‐1.0259508 ‐1.13 LOC1001285018159004 ‐1.1418691 ‐1.2781873 ‐1.1137958 ‐1.18 RPL7A|SNORD248160439 ‐1.258943 ‐1.2016276 ‐1.0595245 ‐1.178160459 ‐1.1193366 ‐1.1142873 ‐1.0563816 ‐1.10 ELAVL28160663 ‐1.1149685 ‐1.2610207 ‐1.1951705 ‐1.19 AQP78160900 ‐1.1776699 ‐1.1401237 ‐1.0365562 ‐1.12 C9orf144|RP11‐251H13.28161513 ‐1.1618793 ‐1.0299528 ‐1.1498747 ‐1.11 PGM5P28161520 ‐1.215008 ‐1.0410358 ‐1.1574442 ‐1.14 PGM5P28161943 ‐1.096678 ‐1.5495085 ‐1.179431 ‐1.28 LOC6447148162214 ‐1.1449199 ‐1.1378958 ‐1.0113769 ‐1.10 LOC6459618162929 ‐1.1644909 ‐1.1324682 ‐1.1883187 ‐1.16 OR13C48163533 ‐1.2028567 ‐1.194494 ‐1.0311501 ‐1.14 FLJ317138163618 ‐1.1829689 ‐1.3177207 ‐1.0297363 ‐1.18 TNFSF158163637 ‐1.2208376 ‐1.1397737 ‐1.0292698 ‐1.13 TNC8164438 ‐1.2453928 ‐1.1182964 ‐1.0166935 ‐1.138166565 ‐1.1291627 ‐1.183872 ‐1.0187716 ‐1.11 MAGEB188166619 ‐1.087334 ‐1.1201719 ‐1.1349844 ‐1.11 MAGEB1|MAGEB48167027 ‐1.1201622 ‐1.2636591 ‐1.018737 ‐1.13 RGN8167854 ‐1.2211274 ‐1.1944569 ‐1.1137935 ‐1.18 APEX28168079 ‐1.8974931 ‐2.0799263 ‐1.214796 ‐1.738168855 ‐1.1354032 ‐1.1902515 ‐1.0709162 ‐1.13 ARMCX48169002 ‐1.0021577 ‐1.1306237 ‐1.2562608 ‐1.138169634 ‐1.1175219 ‐1.1776197 ‐1.2227252 ‐1.178170015 ‐1.0306959 ‐1.1264628 ‐1.193451 ‐1.12 ZNF4498170247 ‐1.1763655 ‐1.2534027 ‐1.0686966 ‐1.17 CXorf188170282 ‐1.1169677 ‐1.1205648 ‐1.0308256 ‐1.09 SPANXN48171172 ‐1.465611 ‐1.4290162 ‐1.1539816 ‐1.35 MXRA58171449 ‐1.1684867 ‐1.1505685 ‐1.0259542 ‐1.12 ACE28172028 ‐1.0661342 ‐1.138698 ‐1.4076415 ‐1.208172191 ‐1.0958965 ‐1.2049588 ‐1.1820956 ‐1.16

165  

8172453 ‐1.1303902 ‐1.0334401 ‐1.251748 ‐1.14 TIMM17B8173208 ‐1.1529659 ‐1.1574439 ‐1.0678133 ‐1.13 SPIN48173366 ‐1.2473128 ‐1.2616489 ‐1.1043935 ‐1.20 P2RY48174119 ‐1.0489181 ‐1.238366 ‐1.1494266 ‐1.15 ZMAT18174513 ‐1.1753209 ‐1.1280079 ‐1.062383 ‐1.12 CHRDL18175537 ‐1.1147419 ‐1.2108519 ‐1.0806557 ‐1.14 CXorf188175900 ‐1.1410948 ‐1.1629108 ‐1.0659852 ‐1.12 ARHGAP48176427 ‐1.1547782 ‐1.1477331 ‐1.0614046 ‐1.128177114 ‐1.1616321 ‐1.1432308 ‐1.0622799 ‐1.128177269 ‐1.1526574 ‐1.1473463 ‐1.1023531 ‐1.138177717 ‐1.0475904 ‐1.1508293 ‐1.1399816 ‐1.11 HLA‐F8177770 ‐1.0348558 ‐1.1373097 ‐1.1595732 ‐1.11 TRIM39|TRIM39R8178193 ‐1.00785 ‐1.1741405 ‐1.228931 ‐1.14 HLA‐DRA8178712 ‐1.0386152 ‐1.2842276 ‐1.1668032 ‐1.16 TNXB|TNXA8179088 ‐1.0336882 ‐1.1408814 ‐1.1610003 ‐1.11 TRIM39|TRIM39R8179935 ‐1.0352781 ‐1.2866459 ‐1.1692953 ‐1.16 TNXB|TNXA7897076 1.1510614 1.0922247 1.1336693 1.137897295 1.1296413 1.1846787 1.1459758 1.15 TAS1R17897620 1.0159584 1.1886483 1.0875065 1.10 PGD7897648 1.0742676 1.0919988 1.0921133 1.09 PEX147897774 1.081394 1.2034408 1.1758256 1.15 CLCN67897955 1.0289028 1.2011805 1.171541 1.13 AADACL47897964 1.1254687 1.0040089 1.131356 1.097898355 1.2891978 1.2042652 1.2153044 1.247898516 1.009878 1.3230559 1.2712504 1.20 ACTL87898537 1.2447776 1.1138451 1.2788566 1.21 PAX77898549 1.2348049 1.3195752 1.1745745 1.24 MRTO47898663 1.0843025 1.100151 1.0889727 1.09 PINK17898739 1.2353425 1.0166035 1.1003829 1.12 CDC427898793 1.1355668 1.0965538 1.139344 1.12 C1QA7898856 1.2672035 1.2139896 1.2527413 1.247899075 1.085975 1.0914208 1.0482724 1.08 EXTL17899462 1.1110444 1.093446 1.0471518 1.08 RCC1|SNHG3‐RCC17900167 1.0893992 1.1468533 1.0830995 1.11 CDCA87900201 1.1359103 1.3685161 1.0178107 1.17 UTP11L7900228 1.1390303 1.1057076 1.0194008 1.09 NDUFS57901477 1.2765534 1.1397523 1.0855181 1.177901744 1.045477 1.1447905 1.1884142 1.137902205 1.2056524 1.1910024 1.2658346 1.22 IL12RB27902223 1.2519118 1.3212445 1.0250919 1.207902623 1.0964338 1.0446494 1.0905982 1.08 DNASE2B7902891 1.1400611 1.2392255 1.0652698 1.15 ZNF3267903113 1.0922941 1.1563128 1.1760311 1.147903117 1.1081648 1.1664362 1.0551777 1.117903294 1.0723974 1.1966654 1.1622926 1.14 HIAT17903945 1.0037584 1.1432108 1.1668503 1.10 CHIA7903972 1.1381075 1.0802768 1.106901 1.11 ATP5F17904166 1.1696506 1.1603684 1.1469288 1.167904244 1.0837214 1.0459411 1.0839715 1.07 C1orf1617904923 1.2396585 1.3253466 1.1765984 1.25

166  

7905145 1.0588506 1.1196725 1.1170189 1.107905500 1.0511688 1.2970569 1.1326501 1.16 LCE2D7905510 1.0440439 1.0986836 1.0889133 1.08 LCE4A7905831 1.0230776 1.1363654 1.1631527 1.11 FLAD17905848 1.1692652 1.0885009 1.0860368 1.11 LENEP7905862 1.1145992 1.1555128 1.2117912 1.16 DCST17905986 1.3869632 1.7196069 1.1069486 1.40 FDPS7906056 1.1189053 1.1951638 1.0007069 1.10 MSTO17906374 1.0072769 1.1109996 1.1015762 1.07 OR6K67906602 1.0904256 1.0040228 1.1228875 1.07 VANGL27906904 1.4218113 1.2711087 1.1200882 1.27 HSD17B7|HSD17B7P27906948 1.1174898 1.4249172 1.1140597 1.227907156 1.0840542 1.0169584 1.2204956 1.11 XCL17907213 1.1200727 1.1662153 1.1467499 1.14 SCYL1BP17907396 1.0090443 1.1240501 1.2126802 1.12 C1orf1057907655 1.3123606 1.1973704 1.2120551 1.247907972 1.1202178 1.142938 1.029045 1.10 C1orf1207908347 1.0988703 1.062123 1.1159657 1.09 OCLM7908931 1.0695376 1.3283019 1.1153204 1.17 OPTC7909146 1.0852911 1.2265384 1.0553892 1.12 FAM72A|FAM72B|GCUD27909400 1.1327516 1.2043886 1.0362749 1.12 CD467909839 1.1213129 1.3361613 1.2022796 1.227909866 1.1986332 1.101886 1.0296522 1.11 MOSC27909896 1.1353232 1.0421346 1.141205 1.117910054 1.0979434 1.0001305 1.1058396 1.07 DNAH14|C1orf677910140 1.0114939 1.0947012 1.1365994 1.087910217 1.1021385 1.1127702 1.0617039 1.09 WNT3A7910379 1.148149 1.3181341 1.0679494 1.18 DUSP5P7910383 1.1402104 1.0086511 1.0853363 1.087910694 1.0433437 1.1133544 1.1981535 1.12 EDARADD|ENO1P7911108 1.2828081 1.1870481 1.1030408 1.197911199 1.0091224 1.1293362 1.1507008 1.10 C1orf1507911213 1.1964147 1.1321957 1.0847105 1.147911258 1.1114 1.3891696 1.0549266 1.19 OR2L37911285 1.0558071 1.140611 1.1309208 1.11 OR2T5|OR2T297911333 1.1230642 1.1506171 1.0293548 1.10 MGC70863|FLJ450557911351 1.1160182 1.1789832 1.1202208 1.14 FLJ226397911444 1.1064351 1.1174395 1.0151646 1.08 UBE2J27911600 1.0068133 1.279063 1.0944381 1.13 NADK7912527 1.0142835 1.1646087 1.1273626 1.107912622 1.0601285 1.1916683 1.1488372 1.13 LRRC387912627 1.1789157 1.1858736 1.0238465 1.137912968 1.0082502 1.1276168 1.1568687 1.10 TAS1R27914015 1.1174606 1.14287 1.1878577 1.15 FAM46B7914326 1.079862 1.1228778 1.1078744 1.10 NKAIN17914334 1.1119776 1.2920896 1.1981182 1.20 WDR577914342 1.1617012 1.3512344 1.5707223 1.36 FABP37914550 1.1483771 1.0828764 1.0230229 1.08 ZBTB8OS7914555 1.0936936 1.0962142 1.0451782 1.087914630 1.050928 1.1656728 1.1586297 1.13 TRIM62

167  

7914748 1.0315499 1.1066 1.1204625 1.097914758 1.0936173 1.1922749 1.014112 1.10 DLGAP37914898 1.0482907 1.1133273 1.1456944 1.10 C1orf787914992 1.0759733 1.2026142 1.1429445 1.147915238 1.0408008 1.1267428 1.2073572 1.12 NT5C1A7915261 1.0372485 1.085276 1.1448836 1.09 TRIT17915275 1.3849834 1.3089769 1.042553 1.257915347 1.0418375 1.1573647 1.211245 1.14 CITED47916432 1.2098842 1.64858 1.2048577 1.35 DHCR247916541 1.0414288 1.1376148 1.3182639 1.17 DAB17917741 1.1115043 1.0984734 1.0592563 1.09 TMED57917902 1.0128384 1.1041617 1.1562387 1.097917942 1.6705587 1.1041701 1.1271787 1.30 FLJ354097918235 1.0988994 1.2054938 1.027409 1.11 C1orf627918550 1.0542278 1.159891 1.1082348 1.117918558 1.2091035 1.2154393 1.2108947 1.21 KCND37918755 1.2315357 1.0314361 1.1361754 1.13 DENND2C7919038 1.0523465 1.1045549 1.1197166 1.09 WARS27919578 1.054149 1.255575 1.2388761 1.18 ATG9B7919591 1.3053297 1.6244799 1.0333732 1.32 GCUD2|FAM72D|FAM72A7919598 1.003229 1.1634614 1.1458443 1.10 LOC4405707920971 1.2899604 1.2021253 1.0843492 1.19 C1orf857921014 1.1146063 1.1519368 1.0498892 1.11 MEF2D7921319 1.0344708 1.1278032 1.0983605 1.09 FCRL17922297 1.1045629 1.2463247 1.0308124 1.137922610 1.1290392 1.0493231 1.0866148 1.09 ABL27922994 1.0058644 1.1389682 1.093652 1.08 FAM5C7923183 1.0312009 1.1985829 1.2430952 1.16 ZNF2817923440 1.256046 1.0613726 1.1499282 1.167923705 1.0341591 1.0831965 1.2549328 1.127924150 1.0795887 1.1059997 1.1198283 1.10 TMEM2067924603 1.0399779 1.296999 1.2516326 1.20 LBR7924823 1.0687306 1.1632595 1.0935035 1.11 JMJD47924956 1.0293341 1.0849783 1.0909822 1.07 ABCB107925043 1.1420517 1.030057 1.1220131 1.10 EXOC87925229 1.092141 1.1935173 1.0363517 1.11 B3GALNT27925500 1.0417713 1.1028578 1.1574033 1.10 CHML7925525 1.08177 1.1719398 1.1723881 1.14 CEP1707925747 1.0585822 1.0971439 1.1531976 1.10 OR2T29|OR2T57925761 1.0779314 1.1119624 1.230066 1.14 OR14I17925763 1.1406046 1.0677868 1.1155417 1.11 SH3BP5L7926117 1.2071596 1.0595473 1.3675073 1.21 LOC3899367926817 1.10835 1.0796828 1.1206197 1.107927513 1.1023061 1.2055846 1.0079533 1.11 FAM21C|FAM21D|FAM21A7927560 1.0337442 1.1759382 1.0979863 1.10 FAM21A|FAM21B|FAM21C7927784 1.0175204 1.1400449 1.0830547 1.08 MGC144257927872 1.1065938 1.1735663 1.0344774 1.107928489 1.1326423 1.1186335 1.028298 1.097928524 1.0620534 1.1777104 1.0836878 1.11 VDAC27928695 1.1559491 1.0789545 1.2232057 1.15 C10orf58

168  

7928705 1.0606804 1.1212865 1.1762773 1.12 TSPAN147928838 1.0029606 1.3414345 1.2627882 1.20 LDB37929282 1.0360326 1.1672171 1.1315142 1.11 HHEX7929550 1.0913357 1.1902641 1.0615526 1.11 CCNJ7929677 1.1078944 1.2551439 1.3218403 1.23 PI4K2A7929744 1.1790128 1.0968956 1.1068331 1.13 NKX2‐37929816 1.5850128 1.9766299 1.3361237 1.63 SCD7929945 1.119577 1.126624 1.0864425 1.117929952 1.1187308 1.3101586 1 1.147930378 1.027271 1.0892463 1.2632704 1.137931159 1.1748859 1.0861951 1.0713869 1.11 PSTK7931346 1.196999 1.1290709 1.1652806 1.167931469 1.0455859 1.0909204 1.1101393 1.08 PWWP2B7931561 1.1201508 1.0559185 1.083648 1.09 ZNF5117931681 1.3009871 1.3304496 1.2147334 1.287931754 1.231239 1.4713528 1.2925997 1.33 IDI17932014 1.2965382 1.1418325 1.0369838 1.16 FLJ459837933129 1.1843921 1.1452696 1.0921273 1.14 LOC100129482|ZNF37B7933147 1.1312752 1.0122029 1.2116097 1.127933192 1.0854497 1.3804832 1.1720936 1.21 HNRNPA3P17933296 1.1154513 1.292157 1.1341951 1.187933421 1.1193206 1.2825233 1.1166298 1.177933638 1.0474097 1.1015114 1.1408814 1.10 FLJ319587933821 1.1985829 1.1087329 1.0333914 1.11 RHOBTB17933933 1.2579958 1.2026166 1.1425769 1.20 DNAJC127934074 1.0786033 1.1573533 1.144447 1.13 TACR27934244 1.1751884 1.1735554 1.0545021 1.13 DNAJB127934271 1.136074 1.0507158 1.087107 1.09 PLA2G12B7934544 1.0885196 1.0293219 1.0935862 1.07 COMTD17934613 1.1336018 1.1092608 1.0699803 1.107934698 1.0384477 1.0872359 1.1559892 1.09 SFTPA2B|SFTPA1B7934708 1.0388824 1.0823795 1.1559365 1.09 SFTPA2B|SFTPA1B7934812 1.1261479 1.0725825 1.0846485 1.09 WAPAL7934868 1.1136621 1.2912312 1.1214911 1.187935027 1.0178965 1.1333126 1.1398641 1.10 IDE7935139 1.3728848 1.0030696 1.1046494 1.16 PIPSL|LOC1001014387935421 1.2131793 1.091393 1.033727 1.11 FRAT27935588 1.0740135 1.1189808 1.1826713 1.13 HPS17935707 1.1037136 1.0849199 1.0068731 1.07 CHUK7935855 1.0938482 1.0066755 1.1499692 1.08 LBX17936050 1.0600826 1.4191047 1.1628467 1.21 CYP17A17936683 1.0954373 1.1046684 1.0176779 1.07 TIAL17937287 1.0887424 1.0952773 1.0305613 1.07 PSMD137937698 1.0787804 1.1278588 1.200434 1.14 LOC3998517938059 1.1703485 1.4165095 1.1346682 1.24 OR52N27938111 1.1257846 1.2397081 1.013732 1.13 FXC17938299 1.2610881 1.2292888 1.2756087 1.267938592 1.0952924 1.1346829 1.0691513 1.10 MLSTD27938683 1.1110739 1.04629 1.1285176 1.10 LOC4414537938702 1.3140308 1.1609738 1.0365065 1.17

169  

7938730 1.177552 1.0083178 1.0988424 1.09 MYOD17938746 1.1688286 1.0548737 1.1260433 1.12 MRGPRX47938762 1.1071415 1.0946639 1.1324207 1.11 GTF2H17938812 1.0423496 1.1353784 1.2142669 1.13 TMEM86A7939102 1.1423005 1.0973692 1.039132 1.09 ELP47939184 1.0911779 1.0858637 1.0518315 1.08 TCP11L17939368 1.0951831 1.0864446 1.0221207 1.07 TRIM447939418 1.0984491 1.0007416 1.1906186 1.107939465 1.111458 1.2245847 1.0204897 1.12 HSD17B127939640 1.2565863 1.0807068 1.1328729 1.167939657 1.216549 1.0448631 1.2185317 1.16 DGKZ7939751 1.1183647 1.0890802 1.0074081 1.07 NR1H37939922 1.0266309 1.2180333 1.0940413 1.117940187 1.1143329 1.0950109 1.1184886 1.11 OR4D117940565 1.3872017 1.7684774 1.2019056 1.45 FADS27940643 1.1741626 1.181732 1.0231256 1.13 ASRGL17941401 1.0975063 1.0172814 1.2586476 1.12 OVOL17941879 1.1098359 1.1003135 1.1989672 1.14 TBC1D10C7942261 1.0690874 1.0934864 1.2273028 1.13 KRTAP5‐97942379 1.0375736 1.1976575 1.0957266 1.117942812 1.3635063 1.2716907 1.0301949 1.227942964 1.2704325 1.4564365 1.0640963 1.26 TMEM1357943345 1.1077659 1.0215929 1.1010072 1.087943777 1.0491816 1.13531 1.153611 1.117944656 1.2045447 1.2950277 1.2545313 1.25 SC5DL7944763 1.1613625 1.253128 1.0034267 1.147944765 1.0282325 1.0983852 1.2368402 1.127944795 1.1469857 1.1459273 1.1776855 1.16 OR4D57944843 1.0414501 1.1149102 1.2490575 1.14 OR8A17945031 1.0249689 1.0884944 1.1658881 1.097945069 1.1631116 1.0412408 1.2805629 1.167945241 1.1454351 1.1015193 1.0055007 1.08 C11orf447945657 1.3039912 1.4724731 1.2182237 1.33 KRTAP5‐47945660 1.5319107 1.4647156 1.1134888 1.37 FAM99A|FAM99B7945781 1.0217073 1.1350085 1.1727331 1.11 PHLDA27945892 1.1008136 1.1695052 1.1381199 1.147946008 1.00011 1.1394441 1.1971904 1.117946071 1.129915 1.1219008 1.0397937 1.10 OR51B57946082 1.111807 1.0947167 1.0247531 1.08 UBQLNL7946278 1.1363707 1.0970964 1.0222907 1.09 OR2AG27946504 1.0945919 1.0172179 1.144433 1.09 TMEM41B7946849 1.060365 1.1535525 1.2143649 1.147947332 1.0968391 1.1908724 1.0286076 1.11 IMMP1L7947423 1.1941888 1.3714901 1.6846529 1.427947490 1.0571951 1.2150092 1.2498909 1.17 APIP7947563 1.129081 1.0794399 1.1245191 1.11 ALX47947969 1.0690955 1.1012129 1.0934912 1.09 FNBP47948148 1.3908156 1.4505529 1.096228 1.31 OR5M107948176 1.0941694 1.0229589 1.0897971 1.07 TNKS1BP17948314 1.194864 1.0590371 1.2500573 1.17 OR10Q1

170  

7948324 1.0332417 1.2115849 1.1202226 1.12 OR5B37948612 1.389518 1.7409058 1.272718 1.47 FADS1|FADS37950067 1.3876474 1.832188 1.1571846 1.46 DHCR77950136 1.1195134 1.0001894 1.2956498 1.14 PHOX2A7950162 1.0858828 1.1071364 1.1212779 1.10 PDE2A7950501 1.1048265 1.0198611 1.1152036 1.08 GDPD57950726 1.1184409 1.3252301 1.1168975 1.19 MGC338467951040 1.0746651 1.1269238 1.1651138 1.12 GPR837951157 1.1260823 1.1829653 1.083151 1.13 CCDC827951246 1.1862001 1.0646971 1.1608717 1.14 MMP87952022 1.0447702 1.0909963 1.1357466 1.09 AMICA17953590 1.1206003 1.0188628 1.2319261 1.127953765 1.0099763 1.2900819 1.2698985 1.19 FAM80B7953967 1.1011623 1.0506123 1.1270609 1.09 PRH2|PRB4|PRH17954938 1.1795764 1.0999851 1.1194687 1.137954985 1.076839 1.1501229 1.095421 1.11 TMEM1177954995 1.0341314 1.2999603 1.1168126 1.157955535 1.0911982 1.0106016 1.1407554 1.08 ACVR1B7955817 1.1311092 1.0987116 1.0532029 1.09 PCBP27955983 1.1368139 1.1433543 1.363487 1.21 OR10A77956005 1.0583957 1.1199627 1.106764 1.10 OR2AP17956522 1.0336608 1.1303023 1.405738 1.19 KIF5A7956741 1.0825801 1.1973653 1.1008457 1.137956987 1.0862237 1.091143 1.046039 1.07 PRO22687957052 1.0643302 1.1715544 1.0828124 1.11 CCT27957417 1.2373077 1.014112 1.0966556 1.12 TMTC27957499 1.1622119 1.1353277 1.1585225 1.157957514 1.0783886 1.1922607 1.0920544 1.12 PLEKHG77957570 1.1432176 1.0110654 1.1663827 1.11 PLXNC17957631 1.088002 1.3721077 1.1352185 1.207957649 1.1610371 1.224155 1.080859 1.16 SNRPF7957850 1.1165051 1.0808126 1.2338009 1.14 GAS2L37957960 1.1097082 1.0739101 1.0983471 1.097958051 1.0225999 1.1275226 1.1127305 1.09 ASCL17958147 1.1144184 1.0850046 1.0941907 1.10 TDG7958724 1.02681 1.0909945 1.1304996 1.08 LOC1001311387958884 1.1535778 1.2142336 1.2014108 1.19 OAS17959012 1.370048 1.1330421 1.3531141 1.297959251 1.0851576 1.2516458 1.0322863 1.12 P2RX77959322 1.1088755 1.1379831 1.0196953 1.09 PSMD97959386 1.0339738 1.1046001 1.1514525 1.10 LRRC437960730 1.2675259 1.4684078 1.0755075 1.27 MBOAT57960863 1.081137 1.1636052 1.1822531 1.147962137 1.2396445 1.2175374 1.3502492 1.277962146 1.032202 1.2679836 1.1003829 1.13 FAM60A7962246 1.0311723 1.0905982 1.1257217 1.087962479 1.4602299 1.4280101 1.5332865 1.477963203 1.0689492 1.1160101 1.0937091 1.09 LOC1210067963289 1.083213 1.0129496 1.1010953 1.07 BIN27963333 1.0512803 1.0989691 1.1194476 1.09 KRT80

171  

7963406 1.1009116 1.2505653 1.0876651 1.15 KRT6B7963664 1.0119687 1.1286112 1.1865722 1.11 SP77963817 1.144295 1.4497429 1.0557984 1.22 GTSF17964303 1.0955354 1.3246254 1.0217543 1.15 TAC37964870 1.1928493 1.1166136 1.0773939 1.137964907 1.1051848 1.1004604 1.0611215 1.09 PTPRR7965384 1.220489 1.0686723 1.0885041 1.13 C12orf127965436 1.1297354 1.098101 1.0283854 1.09 EEA17965469 1.1091039 1.4083502 1.1535625 1.227965573 1.1028498 1.036066 1.0910089 1.08 NTN47966072 1.025804 1.1294109 1.1071484 1.09 PRDM47966223 1.1547279 1.3182181 1.1657082 1.217966441 1.2536273 1.1330818 1.0028051 1.13 C12orf477967021 1.1387017 1.0625207 1.1101751 1.10 PXN7967028 1.2020141 1.3786241 1.090111 1.22 RNU4‐27967034 1.0327302 1.151292 1.2710814 1.15 PLA2G1B7967109 1.111036 1.0445648 1.2158339 1.12 C12orf437967544 1.0834718 1.333641 1.1164919 1.18 SCARB17968052 1.2335387 1.102786 1.0110296 1.12 C1QTNF97968197 1.2333361 1.1292721 1.0094873 1.127968746 1.160036 1.1529062 1.0059245 1.11 WBP47969241 1.3022971 1.095593 1.0411228 1.157969256 1.0910633 1.3244904 1.1587361 1.19 LOC220115|LOC1001340957969286 1.1325064 1.0711763 1.1349784 1.11 RP11‐431O22.27969569 1.0429975 1.1060424 1.2404983 1.13 SLITRK57969576 1.0354006 1.1117365 1.1242694 1.09 MIRHG17969881 1.0288134 1.2028539 1.1012675 1.11 TPP27969933 1.3986505 1.580853 1.2018008 1.397970542 1.3067088 1.1880211 1.3679639 1.297970949 1.0546083 1.196276 1.2209293 1.16 MAB21L17971013 1.0821723 1.0899265 1.1500072 1.117971124 1.006146 1.3127251 1.178963 1.17 UFM17971222 1.0251362 1.2545222 1.1603802 1.15 MTRF17971539 1.023081 1.1282327 1.2724336 1.147971573 1.0836498 1.1699401 1.0020424 1.09 RCBTB27971602 1.027613 1.1974037 1.1060293 1.11 RCBTB17972601 1.0371648 1.2099782 1.0865897 1.11 NALCN7972650 1.1956738 1.0518117 1.1586274 1.14 FGF147972663 1.1712626 1.1278383 1.1559732 1.157972674 1.170441 1.2206416 1.1669599 1.19 C13orf277972682 1.1372461 1.1010579 1.0048937 1.08 KDELC17973014 1.1571643 1.1704097 1.0060112 1.11 OR4K57973032 1.0701144 1.1795222 1.4005175 1.22 OR11H7P7973371 1.1175157 1.0799937 1.1743419 1.12 C14orf1197973618 1.0128793 1.117256 1.1019509 1.08 IRF97973894 1.084861 1.103896 1.1849309 1.12 LOC6443847973900 1.277512 1.2312207 1.0148386 1.17 C14orf197973943 1.112018 1.0763191 1.084629 1.09 INSM27974257 1.0565803 1.1360556 1.1161568 1.10 ATP5S7974341 1.194222 1.0767535 1.1049204 1.13 GNG2

172  

7974814 1.1724974 1.0842123 1.0359843 1.107975613 1.0894377 1.0432116 1.1125485 1.08 ACOT67976059 1.1047761 1.0872284 1.0662763 1.097976156 1.1090075 1.2369903 1.08833 1.147976241 1.106283 1.0497328 1.1598787 1.117976571 1.0948288 1.2413256 1.161264 1.17 C14orf1297976783 1.0099055 1.124259 1.1557459 1.10 DLK17976826 1.1132116 1.1682184 1.094083 1.13 SNORD114‐267977340 1.1935812 1.0985421 1.1226556 1.14 BTBD67977592 1.067013 1.109497 1.0887517 1.09 GAFA17977801 1.0938928 1.3290436 1.0973006 1.177977906 1.031997 1.1426003 1.1025349 1.09 ACIN17977933 1.12468 1.1275591 1.1175241 1.12 SLC7A87978628 1.0935526 1.1938313 1.0122821 1.10 PPP2R3C7978776 1.1141639 1.22823 1.0455937 1.13 C14orf1067979085 1.0848105 1.1967698 1.0032496 1.09 PYGL7979129 1.0666901 1.1493925 1.1010994 1.117979721 1.300675 1.3126569 1.01888 1.21 C14orf837979743 1.1067533 1.3534411 1.2476686 1.24 RDH117980003 1.1816949 1.2180598 1.1220624 1.177980304 1.5645561 1.2236837 1.2202382 1.347980309 1.0865674 1.3893831 1.0114367 1.16 C14orf17980485 1.0197796 1.1656303 1.416423 1.20 DIO27980496 1.0140839 1.2891518 1.1298579 1.14 C14orf1457980718 1.0863254 1.0807147 1.1453567 1.107980828 1.3185114 1.1558684 1.0060302 1.16 CCDC88C7981290 1.094767 1.0923302 1.0449519 1.08 WARS7981309 1.033831 1.1132084 1.1284893 1.09 BEGAIN7981320 1.1120496 1.109826 1.1998976 1.14 MEG37981346 1.1711774 1.2496294 1.0841571 1.17 RAGE7981951 1.3169165 1.2044766 1.2997162 1.27 SNRPN|SNORD116‐27982341 1.113481 1.1026537 1.0791156 1.10 CHRNA77982574 1.1251559 1.1889567 1.0456314 1.12 FAM98B7982663 1.1635729 1.0039536 1.1874813 1.12 BUB1B7982792 1.0273577 1.1636568 1.1217232 1.10 RAD517983051 1.020741 1.0987304 1.1223521 1.087983405 1.1588959 1.081942 1.2900145 1.18 DUOXA27984016 1.1804863 1.163158 1.1531154 1.17 FAM148A7984152 1.062806 1.2502553 1.1483809 1.15 FBXL227984952 1.0143697 1.0855261 1.1534745 1.08 C15orf397985025 1.180257 1.093124 1.0016623 1.09 ODF3L17985259 1.048015 1.1042227 1.168297 1.11 ZFAND67985266 1.2341272 1.2666059 1.195359 1.237985577 1.127577 1.0449512 1.0859976 1.09 ZSCAN27986092 1.1753209 1.2619061 1.0093756 1.15 FURIN7986442 1.0110474 1.2182997 1.6815044 1.307987439 1.0421199 1.1289489 1.137474 1.10 GPR1767987454 1.1417826 1.0999888 1.1277804 1.12 BMF7987466 1.0325743 1.1813614 1.1756399 1.137987554 1.1673186 1.2118264 1.0806401 1.15 DNAJC17

173  

7987772 1.131289 1.086165 1.0134532 1.08 EHD47988344 1.4107089 1.1495728 1 1.197988605 1.2267666 1.0405142 1.1007311 1.12 COPS27988767 1.2261326 1.1154406 1.1879791 1.18 CYP19A17988970 1.1497496 1.1412175 1.1024679 1.13 KIAA13707989132 1.1146766 1.1231774 1.0022594 1.08 RFXDC27989243 1.1848475 1.3885108 1.0430361 1.217989387 1.0235163 1.1058377 1.0896668 1.07 VPS13C7989628 1.0783031 1.1652937 1.1153659 1.12 CSNK1G17990080 1.0855659 1.1390898 1.007214 1.08 LARP67990391 1.1897768 1.1844732 1.1232929 1.17 CYP1A17990736 1.1062205 1.0785295 1.0869116 1.09 ADAMTS77991332 1.0101154 1.1088485 1.1400498 1.09 MESP17991512 1.2363735 1.0002819 1.1832633 1.14 C15orf517991540 1.085924 1.2476517 1.4647379 1.277991598 1.3920518 1.0886657 1.0132167 1.16 SNRPA17992010 1.0860816 1.2000749 1.4015839 1.23 RHBDL17992269 1.0117226 1.1604125 1.0945789 1.097992439 1.1792384 1.2640398 1.0726395 1.17 GFER7992795 1.1520286 1.3738303 1.0463477 1.19 THOC67992861 1.112365 1.0961595 1.174602 1.137992987 1.1692795 1.1235157 1.0373291 1.11 HMOX27993114 1.0809804 1.0895361 1.1123701 1.09 C16orf687993310 1.0996104 1.1430331 1.0326347 1.09 MKL27993737 1.0832559 1.1292201 1.2053181 1.14 ACSM2A|ACSM2B7993798 1.1459872 1.0068792 1.1207037 1.097993821 1.1516284 1.012576 1.1332433 1.10 FLJ417667994582 1.02038 1.1979686 1.1432679 1.12 SULT1A3|GIYD1|GIYD27994781 1.0222586 1.1966896 1.1289283 1.12 SULT1A3|GIYD1|GIYD27994804 1.1244626 1.0482734 1.1149449 1.10 MYLPF7994824 1.2689855 1.266793 1.0655808 1.207994926 1.2178738 1.1528318 1.0649657 1.15 SNORA307995258 1.0618587 1.2270639 1.119456 1.14 ZNF2677995310 1.0873858 1.0350366 1.1259755 1.08 MGC348007995477 1.3889308 1.2058476 1.095362 1.237995712 1.0662756 1.2237407 1.0878661 1.13 CAPNS27995895 1.1074691 1.0690786 1.1036417 1.09 HERPUD17996185 1.0644273 1.1119875 1.108667 1.10 MMP157996593 1.0860633 1.1640873 1.009804 1.09 CTCF7996608 1.0863581 1.1592932 1.0789808 1.11 RLTPR7996759 1.3815032 1.1655991 1.1755847 1.247996837 1.092714 1.0253667 1.1531295 1.09 CDH17996947 1.0351788 1.1888369 1.1153455 1.11 CYB5B7997336 1.0015212 1.142313 1.1092359 1.08 KIAA15767997396 1.1956921 1.0283272 1.1252043 1.12 ATMIN7997626 1.0137795 1.1514218 1.1088341 1.09 C16orf447997700 1.1996664 1.178701 1.0882423 1.167997746 1.048049 1.1584375 1.1705872 1.13 JPH37997962 1.1052253 1.134366 1.0507773 1.10 DPEP17998759 1.1218729 1.1241769 1.2996548 1.18 LOC283871

174  

7999253 1.1995828 1.1639338 1.1589242 1.17 PPL7999387 1.1006489 1.2138785 1.0006566 1.11 EMP27999427 1.4258915 1.0068566 1.2340657 1.22 TNP27999435 1.2868649 1.0909429 1.0098978 1.13 PRM27999752 1.002207 1.0943358 1.1343687 1.087999965 1.0174118 1.1235164 1.1832852 1.11 ACSM2B|ACSM2A8000407 1.0893899 1.1842877 1.1664674 1.158000411 1.163313 1.0741888 1.1427313 1.138000465 1.2093744 1.282371 1.0333343 1.188000467 1.1141713 1.0798538 1.0841471 1.09 GSG1L8000746 1.1087912 1.0956571 1.0253481 1.088000957 1.0944146 1.124809 1.0086572 1.08 LOC2839328000974 1.0978557 1.0160508 1.1209348 1.08 ZNF6688001030 1.070668 1.3272351 1.2469769 1.21 PYCARD8001552 1.1898173 1.1506544 1.2058214 1.18 CIAPIN18001800 1.3694799 1.2314364 1.0838622 1.23 CDH118002181 1.1280044 1.1114668 1.1424183 1.13 DPEP38002209 1.0488133 1.0910089 1.1467783 1.108002262 1.095937 1.0961037 1.079372 1.098002266 1.0887408 1.1591797 1.0508189 1.10 CTF88002312 1.1312715 1.093 1.053097 1.09 NOB18002344 1.0808529 1.0929419 1.1625277 1.11 EXOSC68002992 1.0847833 1.1017444 1.114893 1.10 C16orf468003332 1.3093789 1.5064822 1.1704128 1.33 MVD8003444 1.0311412 1.2269135 1.0863422 1.11 LOC1464298003605 1.1772624 1.5924035 1.1016129 1.298003656 1.041855 1.2235769 1.3967426 1.22 SERPINF28003812 1.1337674 1.1440598 1.4369242 1.24 OR3A38003824 1.1893297 1.1343563 1.1907091 1.17 CTNS8004167 1.0845137 1.0399116 1.0970066 1.07 FAM64A8004219 1.0243803 1.1837707 1.1942095 1.138005089 1.1931292 1.1968703 1.0642089 1.15 COX108005743 1.0322092 1.1908324 1.1875083 1.14 FLJ360008005839 1.1996229 1.8263687 1.1805004 1.40 TMEM978006323 1.1130961 1.0405288 1.1000903 1.088006367 1.1356021 1.0156969 1.1704766 1.11 RHBDL38006423 1.006006 1.1000518 1.2387348 1.11 SPACA38006590 1.1136106 1.0010692 1.2378942 1.128006779 1.181573 1.053165 1.1849493 1.14 ARHGAP238006786 1.0556784 1.2208792 1.124136 1.138006836 1.0827494 1.036712 1.1177361 1.08 FLJ438268007071 1.2016506 1.1071901 1.0730101 1.13 CDC68007148 1.1042011 1.1127062 1.3389119 1.19 GAST8007388 1.0688294 1.0922204 1.2640142 1.14 CNTD18007548 1.0356264 1.1140163 1.3520757 1.17 C17orf888007828 1.1870351 1.035157 1.2893823 1.17 MAPT8007902 1.100092 1.0929043 1.0254744 1.078008711 1.0514582 1.1205696 1.1228697 1.10 OR4D18009727 1.1611476 1.1709166 1.1948096 1.18 ICT18009796 1.047944 1.1041256 1.0857812 1.08 KIAA0195

175  

8009951 1.0943238 1.1527853 1.0367186 1.09 ITGB48010512 1.0197479 1.1625643 1.0972586 1.09 KIAA13038010550 1.1127979 1.1419733 1.0158436 1.09 CHMP68010780 1.0210466 1.1943929 1.1506366 1.12 FLJ357678010963 1.0291283 1.0850339 1.3442773 1.158011320 1.241717 1.0955245 1.1100123 1.15 OR1D5|OR1D48011375 1.0474495 1.1840708 1.0983831 1.11 TRPV18012079 1.124576 1.1046549 1.038746 1.09 DVL28012528 1.0289361 1.1140468 1.1560159 1.10 LOC3883338013035 1.0829638 1.0050365 1.2267103 1.10 ZNF6248013135 1.1092952 1.3578025 1.0151284 1.16 SREBF18013319 1.2802929 1.3376375 1.3635235 1.33 GRAP8013473 1.043633 1.2505599 1.0908455 1.13 LOC3392408014035 1.006125 1.2857502 1.1005998 1.138014487 1.2498661 1.4064792 1.1593804 1.27 ACACA8014787 1.1199657 1.0600078 1.1281208 1.10 LOC1001319338015511 1.1029085 1.199477 1.0060179 1.10 DHX588015642 1.2235154 1.1619585 1.0923183 1.16 PSMC3IP8015735 1.1893553 1.0870671 1.0214889 1.108015737 1.1043023 1.1071491 1.2865762 1.178015806 1.1039271 1.0006282 1.1479646 1.08 ETV48016094 1.0247376 1.198096 1.2717092 1.16 GJC18016239 1.1103065 1.1368725 1.0243989 1.09 PLEKHM18016463 1.0607456 1.2082378 1.0924212 1.12 HOXB68016473 1.0922648 1.1810955 1.1847837 1.15 HOXB88016519 1.1186882 1.0624428 1.1135101 1.108016615 1.0436248 1.1360942 1.1520369 1.11 SAMD148017096 1.1301181 1.1502517 1.2292368 1.178017253 1.0226841 1.0836982 1.4480606 1.18 LOC7296218017476 1.1296633 1.2560225 1.0370692 1.14 CSH2|CSH1|GH18019280 1.0399817 1.2170421 1.1059258 1.12 PCYT28019347 1.0839396 1.095706 1.0279282 1.078019392 1.5260879 1.4667207 1.0959201 1.36 FASN8019711 1.0231706 1.1080948 1.1088847 1.08 SCGB1C1|hCG_17413448019754 1.0455555 1.1280317 1.2422535 1.14 LOC3482628019778 1.1103601 1.2853881 1.1253959 1.17 PCYT28020226 1.1863377 1.050289 1.3032066 1.18 SLMO18020795 1.094254 1.0483708 1.294714 1.15 TTR8020847 1.0267242 1.120407 1.1183462 1.09 DTNA8020878 1.1741852 1.0350024 1.1097856 1.11 MAPRE28021129 1.2592072 1.0747237 1.1220658 1.15 C18orf23|RNF1658021243 1.0818232 1.2520355 1.1809367 1.178021286 1.1411254 1.141305 1.1350343 1.14 C18orf548021482 1.0956249 1.0971795 1.0196384 1.078021914 1.0636635 1.0911989 1.146274 1.10 ADNP28022412 1.1131451 1.1215338 1.1953112 1.14 MC2R8022488 1.0961356 1.0162147 1.1937114 1.10 ABHD38022531 1.0441654 1.1991677 1.2249871 1.16 NPC18022801 1.150295 1.2398093 1.131391 1.178023646 1.0969764 1.0155355 1.1099946 1.07 BCL2

176  

8023672 1.1556892 1.1132587 1.0901805 1.12 VPS4B8023710 1.088721 1.0689185 1.112376 1.09 CDH198024358 1.1561661 1.1478014 1.0544721 1.12 CSNK1G28024637 1.0345742 1.0935128 1.0924107 1.07 FZR18025183 1.0135192 1.198838 1.0943351 1.10 MCOLN18025285 1.1476917 1.0255924 1.1771255 1.12 C19orf598025828 1.342854 1.9312289 1.4032624 1.56 LDLR8026024 1.0718602 1.171986 1.1050807 1.12 ASNA18026294 1.2343777 1.0090717 1.2273779 1.16 LOC1132308026383 1.1044307 1.0832781 1.0194547 1.078026533 1.0324129 1.1247005 1.1223755 1.09 HSH2D8027006 1.1277384 1.087998 1.0673132 1.09 SSBP48027018 1.0507933 1.089239 1.1005405 1.08 C19orf508027268 1.1017265 1.0956006 1.067694 1.09 ZNF668027297 1.2429205 1.1064311 1.050386 1.13 ZNF7388027368 1.0881437 1.0816481 1.1237062 1.10 ZNF2548028613 1.0221069 1.2882127 1.1100236 1.14 IL298029289 1.0601476 1.0913533 1.2017281 1.12 TEX1018029726 1.0596019 1.1251943 1.1883341 1.128029978 1.0973939 1.1716045 1.155868 1.14 CRX8030092 1.1213454 1.1434053 1.0661032 1.11 SEC18030360 1.0969989 1.2803886 1.020122 1.13 SNORD32A|RPL13A8030416 1.1490396 1.1047487 1.0032324 1.09 SCAF18030641 1.0926497 1.2245147 1.0509089 1.12 POLD18030753 1.1023731 1.0852035 1.0846374 1.09 KLK38031122 1.1010189 1.2503743 1.0276161 1.13 CNOT38031475 1.2750847 1.1506517 1.0362085 1.15 TMEM1908031550 1.113002 1.0083191 1.1990746 1.11 EPN18031686 1.1528356 1.1079568 1.1188302 1.13 USP298032212 1.0423386 1.1248897 1.5171665 1.238032249 1.0215588 1.2098322 1.1780297 1.14 ADAMTSL58032265 1.0865175 1.1059024 1.0423824 1.08 MEX3D8032530 1.027805 1.1515329 1.0959002 1.09 SGTA8032996 1.2373424 1.1093712 1.0713977 1.14 P1178033043 1.0367426 1.1414834 1.1871414 1.12 FUT68034565 1.1639488 1.1730564 1.0133333 1.12 DNASE28034722 1.1947049 1.12514 1.1961514 1.17 RFX18034974 1.0388033 1.2565236 1.1935713 1.16 ABHD98035040 1.1099932 1.2761459 1.023432 1.14 WIZ8035737 1.0305477 1.116048 1.099045 1.08 ATP13A18035808 1.1343454 1.2239212 1.0771935 1.15 ZNF1008035884 1.0671002 1.091376 1.2106774 1.128036025 1.1145389 1.1227331 1.0890173 1.11 LOC3885328036055 1.0108515 1.108182 1.1061075 1.08 LGI48036318 1.1038665 1.1310426 1.2296996 1.15 ZNF5668036389 1.1304739 1.1949619 1.1592311 1.16 ZNF585B8036707 1.1006631 1.1334991 1.1710659 1.14 LRFN18037032 1.1217613 1.0707406 1.1420189 1.11 B3GNT88037309 1.1122673 1.2900051 1.0348873 1.15 PHLDB38037363 1.1151527 1.0059854 1.213049 1.11 CADM4

177  

8037444 1.1404817 1.1333302 1.0846306 1.12 ZNF2358037513 1.0016457 1.1998651 1.1394354 1.11 EXOC3L28037537 1.132253 1.2893683 1.0663266 1.16 ERCC28037594 1.1484252 1.1225996 1.0559969 1.11 RTN28037872 1.0363613 1.091552 1.1110451 1.08 BBC38037959 1.1015583 1.0117561 1.1456702 1.09 BSPH18038139 1.0949053 1.1953843 1.1232353 1.14 LOC1261478038213 1.0035601 1.1302202 1.104783 1.08 HSD17B148038309 1.1308845 1.2382035 1.0388597 1.14 KCNA78038861 1.0080763 1.2064408 1.1206759 1.11 SIGLEC68038949 1.1100028 1.0883803 1.0168601 1.07 LOC2843718039054 1.112999 1.1247158 1.0682883 1.10 ZNF3478039062 1.0534581 1.2284671 1.1171508 1.13 ZNF6658039070 1.142879 1.0263453 1.0875217 1.09 ZNF6778039484 1 1.1864887 1.0863243 1.09 IL118039771 1.0088001 1.1140265 1.1560957 1.09 ZNF1328039937 1.1295047 1.5711663 1.05858 1.25 GLTPD18040018 1.0855099 1.1221952 1.0110089 1.07 TTC158040077 1.1542975 1.2321513 1.157515 1.18 FLJ410468040247 1.1002251 1.2290207 1.1047484 1.148040334 1.0220647 1.1960003 1.4564226 1.228040340 1.3005749 1.2337475 1.2187976 1.25 LPIN18040490 1.0156469 1.1791928 1.1015697 1.10 KLHL298040690 1.2535655 1.3191674 1.1322863 1.24 LOC3397788041553 1.0976213 1.31082 1.0003332 1.14 GEMIN68041568 1.1762931 1.0331163 1.1027021 1.108041570 1.0248408 1.1375709 1.1730393 1.11 TMEM1788041913 1.0997006 1.0136445 1.1250601 1.08 CCDC1288042109 1.1179589 1.484534 1.0827075 1.238042283 1.1357613 1.2061709 1.0283625 1.12 HSPC1598042291 1.0960356 1.119539 1.0009179 1.07 AFTPH8042859 1.1315758 1.1109452 1.0331717 1.09 ZNHIT4|WBP18042925 1.0081285 1.1003175 1.0906909 1.07 SEMA4F8042993 1.0168675 1.126982 1.0827183 1.08 CTNNA28043155 1.2174774 1.0572513 1.1033552 1.138043381 1.0889463 1.1176873 1.1514727 1.12 SMYD18043666 1.1672145 1.1239223 1.063958 1.12 CNNM38043861 1.0895234 1.1593146 1.0300586 1.09 EIF5B8043902 1.1886759 1.0432957 1.0948567 1.11 PDCL38044698 1.0093678 1.0879905 1.1151546 1.078044745 1.1210052 1.1475796 1.0170625 1.10 DDX188045291 1.118862 1.0385957 1.0863498 1.08 TUBA3D|TUBA3E|TUBA3C8045309 1.2214955 1.0734627 1.1152003 1.14 CCDC74A|CCDC74B8045339 1.1033952 1.0515451 1.1184154 1.098045398 1.0073398 1.0879704 1.0879517 1.06 RAB3GAP18045664 1.2966574 1.0488575 1.140409 1.16 LOC1305768045846 1.1076678 1.211239 1.0775093 1.138046588 1.112647 1.2116097 1.1271175 1.158046746 1.0297823 1.1811045 1.125388 1.11 PPP1R1C8046848 1.1410908 1.1064249 1.0727328 1.11 ZC3H15

178  

8047125 1.1709634 1.2413892 1.0409181 1.158047702 1.073869 1.1250708 1.1406028 1.11 ICOS8047709 1.1260886 1.1332166 1.0359287 1.10 PARD3B8047865 1.0234149 1.2048035 1.1312759 1.12 PIP5K38047965 1.1697171 1.1214617 1.1816348 1.16 KIAA18438048249 1.0850217 1.264646 1.1255829 1.16 GPBAR18048707 1.0918534 1.1125114 1.030358 1.08 CCDC1408049297 1.067084 1.1029832 1.1025652 1.09 SCARNA58049574 1.0171082 1.1155137 1.1413985 1.09 UBE2F8050190 1.0062757 1.1033624 1.092167 1.07 ADAM178050238 1.0096729 1.3419073 1.1066549 1.158050423 1.1664112 1.3680679 1.2281935 1.25 MYCNOS8050869 1.0368019 1.1180809 1.1286008 1.098051028 1.1733855 1.1356539 1.6870941 1.338051119 1.1335284 1.1746734 1.016246 1.11 PPM1G8051275 1.1168337 1.2598221 1.0694344 1.15 CAPN138051573 1.135777 1.0954293 1.031939 1.09 CDC42EP38051622 1.0942366 1.0432395 1.1174754 1.08 SFRS78052233 1.1260399 1.1629074 1.0693333 1.12 C2orf638052581 1.1199516 1.1583039 1.1684129 1.158052902 1.2715251 1.0886272 1.0388349 1.13 FIGLA8052932 1.1997668 1.014188 1.1005005 1.108052940 1.1032085 1.1099741 1.1336812 1.12 PAIP2B8052947 1.1271831 1.0678142 1.094717 1.10 CYP26B18053288 1.2063495 1.1299982 1.0958154 1.14 C2orf38053349 1.0942814 1.2931936 1.1237864 1.17 LRRTM18054329 1.010041 1.1065339 1.1565193 1.09 RNF1498054344 1.0864116 1.0311092 1.0844786 1.07 FLJ429868054467 1.1713191 1.1276996 1.0094216 1.10 SEPT108054866 1.0865089 1.0417964 1.094415 1.07 FLJ148168055173 1.1143352 1.1167365 1.112515 1.11 CCDC74A|CCDC74B8055261 1.139507 1.0840005 1.0080233 1.08 RAB6C|RAB6A|LOC1507868055314 1.0853919 1.1059898 1.2114271 1.13 LYPD18056968 1.2236205 1.0058295 1.172049 1.13 LOC3752958057394 1.1193746 1.034479 1.0885063 1.08 SESTD18057480 1.0305 1.1719116 1.1271939 1.11 NEUROD18057959 1.1910133 1.0351857 1.0883445 1.10 PGAP18058203 1.0884304 1.0544676 1.1163502 1.09 ALS2CR128058295 1.0471013 1.113925 1.0889208 1.08 ALS28058348 1.1621474 1.2068162 1.0971066 1.168058458 1.0146344 1.1046023 1.3692735 1.16 LOC6459788059275 1.0914459 1.0932302 1.0192562 1.078059277 1.1198806 1.0619369 1.1881032 1.128059350 1.0939044 1.1000866 1.0825136 1.09 AP1S38059475 1.0328757 1.1228226 1.1184309 1.098059712 1.3682551 1.2878159 1.0861412 1.25 SNORD828060020 1.2291523 1.12031 1.0969434 1.15 FLJ438798060344 1.015644 1.114084 1.2066394 1.11 TRIB38060501 1.0879031 1.0058022 1.1641188 1.09 SNORA518060940 1.0840851 1.0896542 1.1466302 1.11 C20orf103

179  

8060997 1.1012875 1.0691595 1.0847528 1.09 SPTLC38061073 1.1521202 1.033899 1.1530662 1.118061082 1.0414917 1.0997874 1.0952533 1.08 OTOR8061562 1.1270437 1.371754 1.0547127 1.188061772 1.1140752 1.1123098 1.0031422 1.08 MAPRE18062034 1.0123056 1.1108696 1.0953751 1.07 TP53INP28062251 1.2576936 1.1678226 1.0021715 1.14 EPB41L18062395 1.2002261 1.2184396 1.037624 1.15 NNAT8062404 1.135014 1.1217039 1.1953064 1.158062539 1.0212471 1.2342712 1.1742077 1.14 SLC32A18062576 1.1551306 1.3200786 1.1851801 1.22 DHX358063380 1.1745375 1.4031214 1.1093928 1.238064098 1.6033744 2.116185 1.076793 1.608064203 1.372524 1.2163411 1.0273006 1.21 C20orf1358064208 1.0832462 1.1532807 1.101936 1.11 DNAJC58064904 1.0382837 1.1172911 1.1067946 1.09 FERMT18065084 1.1607689 1.094352 1.2964474 1.18 LOC6132668065120 1.1970372 1.2106566 1.0758492 1.16 LOC1001316428065416 1.0996456 1.0684042 1.2246082 1.13 CST28065421 1.105321 1.0262113 1.1512756 1.09 CST58065668 1.3222821 1.0968618 1.0237637 1.15 CDK5RAP18065752 1.1070796 1.0944349 1.0386262 1.088065756 1.1156029 1.4172776 1.1648909 1.238065868 1.0857489 1.1283491 1.0045909 1.07 EIF68066482 1.0643868 1.113456 1.2204403 1.13 WFDC58067087 1.0878898 1.0982342 1.3012619 1.16 SALL48067546 1.0952411 1.1349968 1.2036597 1.14 LOC1001357778067798 1.0356162 1.0900875 1.1397492 1.09 SOX188067818 1.1533815 1.250562 1.1654543 1.19 NPBWR28068200 1.083691 1.0558423 1.1111436 1.08 C21orf1198068952 1.1247782 1.134591 1.0444733 1.10 AGPAT38069085 1.1040555 1.1546607 1.0059222 1.09 TRPM28069470 1.1211905 1.1510926 1.0290399 1.10 FLJ450558069505 1.0241995 1.2080194 1.200622 1.14 C21orf158069764 1.0947967 1.0162036 1.1071374 1.078069868 1.2053615 1.1780374 1.2567815 1.21 KRTAP7‐18070295 1.2947075 1.0891966 1.0799944 1.158070689 1.1884036 1.3389566 1.021784 1.18 HSF2BP8070716 1.1459842 1.1244931 1.1530684 1.14 C21orf328070777 1.05599 1.0951904 1.096032 1.08 KRTAP10‐28070961 1.1622511 1.3456504 1.0130118 1.17 LSS8071196 1.0285329 1.1128931 1.0904436 1.08 TSSK28071532 1.1072937 1.1031947 1.0157355 1.08 RIMBP3|RIMBP3B8072304 1.0353328 1.1104218 1.2180107 1.12 HORMAD28072454 1.0355922 1.0928086 1.0835334 1.07 RNF1858072482 1.1206452 1.3031067 1.3505179 1.268072494 1.1465476 1.088265 1.067767 1.10 SFI18072678 1.060807 1.1958834 1.1087348 1.12 HMOX18072817 1.0296036 1.3502027 1.1260772 1.17 CDC42EP18072894 1.0502821 1.1948193 1.1617411 1.14 TRIOBP

180  

8072924 1.1786925 1.1181917 1.0637169 1.128073522 1.3821472 1.2623159 1.1185522 1.25 SREBF28073605 1.067412 1.0979903 1.2558036 1.14 BIK8073631 1.011309 1.1952214 1.126076 1.118073633 1.3042239 1.3415449 1.07715 1.24 PNPLA38074335 1.161173 1.0233363 1.2035892 1.13 PRODH8074769 1.0485368 1.1116483 1.1457751 1.10 RIMBP3B8074897 1.0035793 1.1316619 1.1104409 1.08 LOC3888828075164 1.1207159 1.2166504 1.0036118 1.11 CHEK28075217 1.2406746 1.1246005 1.0622226 1.14 AP1B18075695 1.1291765 1.108224 1.0531102 1.10 APOL38076300 1.0409745 1.1611803 1.2756956 1.168077779 1.0604779 1.2618691 1.1101829 1.148077879 1.0093129 1.1707269 1.0968992 1.09 SYN28077942 1.1739038 1.0586137 1.1473857 1.138078448 1.1046932 1.1408131 1.085699 1.118078688 1.0041285 1.4246714 1.1846654 1.208078918 1.139042 1.0443114 1.2673261 1.15 SNORA62|RPSA8079392 1.1149122 1.0621967 1.093917 1.09 CCR2|FLJ783028079401 1.1760712 1.0099475 1.2082905 1.13 CCR5|LOC7277978079407 1.1910795 1.1463323 1.127524 1.15 CCRL2|LOC7278118079415 1.0463772 1.1503224 1.1702793 1.12 RTP38080676 1.0623767 1.1652055 1.0970656 1.11 PDE128080683 1.0524477 1.2931359 1.3070289 1.228080855 1.10761 1.2061267 1.0740469 1.13 SYNPR8081676 1.0431571 1.2572551 1.1175374 1.14 GTPBP88081758 1.0426064 1.1470599 1.1918478 1.13 GRAMD1C8081820 1.0916423 1.349632 1.2146478 1.228082254 1.1193899 1.0157654 1.387496 1.17 LOC1001255568082368 1.0587201 1.1356084 1.1381934 1.11 PODXL28082431 1.0926493 1.0371193 1.1514267 1.09 RAB7A8082583 1.3166375 1.1310732 1.0166894 1.15 UNQ61258082767 1.0137396 1.2310498 1.1265763 1.12 TMEM1088082965 1.1457464 1.0566317 1.1443876 1.12 MRAS8083599 1.062492 1.0926054 1.1026024 1.09 C3orf558083656 1.0814718 1.11551 1.0839983 1.09 MFSD18084219 1.1930149 1.0136374 1.0891447 1.10 KLHL248084323 1.0866141 1.1163129 1.0222502 1.08 DVL38084648 1.1018784 1.0638505 1.1023946 1.09 AHSG8084929 1.1677003 1.0857611 1.0269703 1.09 OSTalpha8084955 1.1056045 1.3706174 1.0796925 1.19 PIGX8085114 1.173778 1.790764 1.3402411 1.438085195 1.0221235 1.0875745 1.1205 1.08 LHFPL48086352 1.1327134 1.1000757 1.0693913 1.10 ULK48086462 1.1142468 1.0197085 1.1575338 1.10 C3orf398086698 1.1149176 1.1143717 1.0651959 1.10 CCDC128086876 1.1161406 1.0864607 1.2975092 1.178087611 1.0802108 1.1485919 1.1062574 1.11 HYAL18087624 1.1683686 1.0012949 1.0874135 1.09 HYAL28087640 1.0071754 1.2355593 1.1221403 1.12 RASSF1

181  

8087746 1.3380895 1.0796232 1.1351863 1.188087830 1.1304798 1.0322174 1.2474369 1.14 RPL29|RPL29P48088106 1.1322964 1.2320483 1.0179752 1.13 TKT8088339 1.0807765 1.1483634 1.0853542 1.10 ARF48088384 1.1198887 1.134029 1.1049134 1.12 PDHB8088548 1.0891746 1.0561428 1.1148387 1.09 LOC4010708088560 1.0756171 1.0907398 1.0868533 1.08 ADAMTS98088739 1.1240164 1.08984 1.0071647 1.07 LMOD38088915 1.244291 1.1418823 1.3153348 1.238089185 1.0518382 1.1373851 1.1892256 1.13 IMPG28089261 1.1746129 1.0142635 1.1008526 1.10 CBLB8089372 1.0270263 1.1540025 1.1148137 1.10 KIAA15248090030 1.086557 1.1068846 1.0102016 1.07 HSPBAP18090349 1.0709288 1.1256685 1.1785047 1.138090639 1.1267011 1.1218644 1.0621141 1.10 PIK3R48090772 1.1096172 1.1863471 1.0246999 1.11 TOPBP18091032 1.2216873 1.0976093 1.0455153 1.12 FOXL28091118 1.0624642 1.1157618 1.0915153 1.098091511 1.0380384 1.1226084 1.0856854 1.08 P2RY148091546 1.0925367 1.2684324 1.0154489 1.13 LOC6458438091696 1.07076 1.2137485 1.0859147 1.128091941 1.163363 1.035801 1.1197432 1.11 PDCD108092187 1.0052066 1.0869352 1.108785 1.07 SPATA168092358 1.0216057 1.0890774 1.3083009 1.14 MCF2L28092552 1.1022503 1.1281611 1.120264 1.12 IGF2BP28092578 1.0024505 1.162364 1.2097371 1.12 ETV58092627 1.096805 1.1555724 1.0299456 1.09 TBCCD18093166 1.098469 1.0498685 1.2169946 1.12 PIGZ8093298 1.2313472 1.0112141 1.208703 1.15 CCR5|LOC7277978093304 1.2640184 1.1644728 1.0646574 1.16 CCRL2|LOC7278118093332 1.4434346 1.1100413 1.0831928 1.21 MGC263568094028 1.2191719 1.0076215 1.1901772 1.14 MGC109818094271 1.112554 1.1025108 1.0232978 1.08 MED288095043 1.1092124 1.0972053 1.1730796 1.13 RASL11B8095535 1.0796311 1.2166375 1.6135966 1.30 ENAM8095562 1.1259034 1.4725097 1.01102 1.208095806 1.111116 1.0660285 1.1184926 1.10 ART38096744 1.2097026 1.2136682 1.055665 1.16 CYP2U18096905 1.0509305 1.1191146 1.2114524 1.13 C4orf168097080 1.0627059 1.1816308 1.3656329 1.20 SYNPO28097305 1.3179964 1.0714883 1.085143 1.168097521 1.1564451 1.3110752 1.0437499 1.17 SCOC8097813 1.0393463 1.17111 1.1088755 1.11 ARFIP18097938 1.0057414 1.1278822 1.1116755 1.08 NPY2R8098103 1.2633426 1.117578 1.1821023 1.19 FNIP28098163 1.1315991 1.0858716 1.1595863 1.138098195 1.2796769 1.4979651 1.3891957 1.39 SC4MOL8098246 1.0937886 1.0596247 1.1885862 1.11 ANXA108098307 1.1009861 1.1176859 1.0522985 1.09 GALNT178098439 1.0369273 1.1587243 1.1990623 1.13

182  

8098697 1.0422035 1.159538 1.1514883 1.12 TRIML18098902 1.2467214 1.0078471 1.3266749 1.198098904 1.0364634 1.1885725 1.114094 1.11 SLBP8099093 1.0000705 1.1748334 1.2199346 1.13 OTOP18099130 1.0985798 1.1651673 1.1660734 1.148099713 1.0274769 1.1424938 1.2204685 1.138099897 1.1094203 1.1026253 1.025056 1.08 UGDH8100360 1.087425 1.1283834 1.0169301 1.088100476 1.0193365 1.1923935 1.115482 1.118100941 1.1273556 1.1903751 1.4589944 1.268100990 1.0672321 1.2177129 1.211691 1.17 PPBPL28101065 1.0861491 1.2985483 1.1014524 1.16 PPEF28101945 1.0859692 1.1368676 1.1283267 1.12 H2AFZ8101957 1.0923392 1.1965808 1.0426807 1.11 EMCN8102127 1.1270326 1.0923514 1.006276 1.08 TACR38102350 1.1348528 1.0902828 1.0047051 1.088102695 1.0326033 1.1007409 1.3635288 1.178102800 1.1098788 1.0252416 1.1082915 1.08 SLC7A118102988 1.2251945 1.2743807 1.0597644 1.19 GYPE8103289 1.1344129 1.0945115 1.0350981 1.09 PLRG18103431 1.0082549 1.1053396 1.1180351 1.08 LOC2017258103542 1.0352918 1.0856032 1.1446664 1.098103847 1.1530807 1.0351614 1.2246268 1.148103951 1.0803565 1.445028 1.1406714 1.22 ACSL18105061 1.358969 1.1496751 1.0837351 1.20 FYB8105681 1.1498834 1.0063429 1.098101 1.08 ERBB2IP8106098 1.1452423 1.0476351 1.1141897 1.10 MAP1B8106280 1.2202277 1.5617967 1.3801378 1.39 HMGCR8106352 1.062583 1.2256234 1.093025 1.13 NBLA11895|LOC1001295668106512 1.1874758 1.0509329 1.1594855 1.138106602 1.0681391 1.1958035 1.1203319 1.13 ZFYVE168106727 1.0116898 1.1098315 1.1117427 1.08 FLJ41309|LOC922708107202 1.090789 1.1154649 1.0394652 1.088107208 1.0993909 1.0778117 1.1067924 1.09 FER8107353 1.0800687 1.2695737 1.0975372 1.15 ZRSR18107563 1.1601192 1.1742254 1.1195197 1.15 PRR168107769 1.1997886 1.1773881 1.025725 1.13 SLC12A28107850 1.0545132 1.2956613 1.1010921 1.15 CHSY38108180 1.2168953 1.161417 1.1113706 1.168108593 1.1227034 1.1118246 1.0625745 1.10 WDR558108713 1.6116 1.0085168 1.3863844 1.34 PCDHB88108979 1.1151649 1.0594062 1.2000531 1.128109350 1.1005517 1.2102486 1.0461987 1.12 SLC36A18109752 1.2340273 1.3939427 1.0959743 1.24 ODZ28109908 1.0152781 1.0830003 1.1265793 1.07 LOC2573588109990 1.1623844 1.199245 1.095869 1.15 LOC6449948110327 1.0882304 1.1181097 1.0494618 1.09 RGS148110562 1.0387175 1.1065565 1.117091 1.09 LTC4S|MAML18110872 1.098481 1.0046712 1.1118968 1.07 IRX28110980 1.0905156 1.0389407 1.0924009 1.07

183  

8111668 1.1349878 1.1425977 1.0859965 1.128111922 1.0538038 1.1525743 1.1088209 1.11 LOC6489878111941 1.4250731 1.7165734 1.262038 1.47 HMGCS18112014 1.1258032 1.0370429 1.1068466 1.098112337 1.0531186 1.2481421 1.1109082 1.14 SFRS12IP18112666 1.2467922 1.2033628 1.3176323 1.268112886 1.2623788 1.1329335 1.3279916 1.248112890 1.1336977 1.129739 1.01999 1.09 MED27|LOC4410898112894 1.1183105 1.1006234 1.0622013 1.098112994 1.1253554 1.2606223 1.0932415 1.168112996 1.0903474 1.0537603 1.0966951 1.088113120 1.0996453 1.0146316 1.1621674 1.098113344 1.0998285 1.1377695 1.0084978 1.088113491 1.3779963 1.3541964 1.3548331 1.36 STARD48113691 1.086562 1.1103528 1.0602566 1.09 DTWD28113914 1.0611106 1.1483223 1.1667798 1.13 FNIP1|RAPGEF68114207 1.0997052 1.1278207 1.0101378 1.088114625 1.0487309 1.2473701 1.6051635 1.30 DND18114647 1.0981504 1.098264 1.1449388 1.11 SLC25A28114658 1.132524 1.0943061 1.0678016 1.10 DIAPH18114787 1.1468227 1.113735 1.0373682 1.10 GNPDA18114918 1.1911771 1.2217528 1.2318499 1.218115144 1.0824567 1.1476694 1.3145565 1.18 ARSI8115166 1.040689 1.2585461 1.11497 1.148115455 1.2573137 1.104576 1.0736351 1.15 HAVCR18115679 1.3310843 1.2092618 1.1996447 1.258115681 1.1716567 1.1275505 1.0761662 1.13 PANK38115849 1.0153888 1.187256 1.185961 1.138115927 1.0281916 1.1903431 1.0939788 1.10 RNF448116502 1.1097827 1.1479262 1.0350091 1.108116571 1.1174418 1.0007075 1.3770894 1.178117079 1.4278411 1.1212153 1.0609981 1.20 LOC6447148117118 1.0966759 1.1001091 1.0727743 1.098117301 1.0517519 1.1838669 1.0905877 1.11 HIST1H2BA8117389 1.2761223 1.0722673 1.1558055 1.17 HIST1H2BE|HIST1H2BG8117531 1.0982335 1.2382228 1.1764798 1.178117653 1.0600208 1.1165568 1.2274873 1.138117900 1.0145338 1.2092681 1.0839106 1.10 DDR18118061 1.1019957 1.0777109 1.1147577 1.10 DPCR18118142 1.1294072 1.0544529 1.2123348 1.13 TNF8118235 1.1177952 1.1244166 1.0588737 1.10 C6orf21|LY6G6D8118655 1.0902076 1.1091813 1.0495194 1.08 B3GALT48118945 1.0081528 1.1200165 1.0910082 1.07 PPARD8119338 1.1160557 1.1959937 1.063687 1.13 GLP1R8119466 1.1144029 1.1686428 1.0180607 1.10 MDFI8119712 1.0626606 1.308009 1.1943839 1.19 SRF8119842 1.0459995 1.1777766 1.0956429 1.11 POLR1C8119951 1.0823082 1.0530636 1.100164 1.08 CAPN118120208 1.1089935 1.089553 1.1232083 1.118120269 1.1018332 1.0085509 1.2281367 1.11 FBXO9

184  

8120717 1.2461965 1.1134405 1.1070837 1.168121159 1.1346071 1.0684094 1.1058168 1.108121429 1.1429939 1.2051648 1.0138301 1.12 FIG48121936 1.0853385 1.0917534 1.1721994 1.12 C6orf588122240 1.0931258 1.1349849 1.0266112 1.088122554 1.1558781 1.0947547 1.0226614 1.09 RAB328122703 1.1244128 1.8360835 1.2831087 1.418123060 1.2967709 1.0687546 1.1604788 1.188123463 1.0863329 1.1762267 1.0999709 1.12 C6orf1208123760 1.0669852 1.2443653 1.2079962 1.17 RP3‐398D13.18123910 1.0067434 1.0937933 1.2388757 1.11 GCM28123949 1.1122205 1.0973488 1.0792947 1.108124055 1.0881318 1.0048698 1.2527914 1.128124430 1.0724534 1.3050238 1.1286482 1.17 HIST1H1D8124467 1.0915481 1.2284136 1.1877557 1.178125123 1.1193928 1.0120867 1.2569854 1.138125321 1.0766495 1.1435021 1.1522696 1.12 PRRT18125415 1.105344 1.1179084 1.0882872 1.10 C6orf108125426 1.1444012 1.0987494 1.0235386 1.09 BTNL28125752 1.1063268 1.1001848 1.0114374 1.07 CUTA8126760 1.1200553 1.0241125 1.2982603 1.15 RCAN28126770 1.2401724 1.1886314 1.0504571 1.16 CYP39A18126855 1.0534406 1.0997034 1.1302661 1.09 C6orf1388127145 1.0878912 1.2473116 1.1333523 1.16 ELOVL58127824 1.1008563 1.1100299 1.1133629 1.11 UBE2CBP8127854 1.1345043 1.2509531 1.0559585 1.15 ME18128034 1.0537056 1.1417592 1.0854051 1.09 C6orf1668128133 1.0916628 1.2349828 1.0931118 1.14 LYRM2|ZC3H11A8128447 1.0992764 1.2497706 1.0947865 1.15 MCHR28128737 1.1578338 1.2576853 1.0104086 1.14 MICAL18128888 1.1014436 1.085251 1.0590638 1.088129039 1.0239207 1.258759 1.1480939 1.14 LOC4422498129099 1.0938382 1.0643182 1.0943335 1.08 TSPYL18129309 1.1805983 1.0473307 1.133782 1.128129773 1.0912577 1.0901452 1.1183296 1.10 BCLAF18129783 1.2213676 1.0321215 1.1300102 1.13 MAP78129963 1.1100801 1.1043829 1.046939 1.09 ADAT28130013 1.1299796 1.0474256 1.1172918 1.10 EPM2A8130071 1.1348224 1.3036649 1.017729 1.15 C15orf29|LOC7291768131263 1.1120886 1.1187074 1.0206047 1.08 RNU13P28131519 1.1575855 1.115584 1.0321887 1.10 PHF148131583 1.1750431 1.2364291 1.0243549 1.15 BZW28131844 1.216325 1.1027594 1.2336179 1.18 GPNMB8131867 1.0847112 1.1424341 1.1795359 1.148131917 1.0924782 1.1092836 1.1579192 1.128131975 1.1089488 1.0925981 1.0933639 1.10 TAX1BP18132118 1.1180189 1.1455827 1.1367306 1.13 AQP18132840 1.1391896 1.1352553 1.1271812 1.13 DKFZp564N24728132851 1.1390684 1.0205723 1.2631643 1.14 VSTM2A8132897 1.1741191 1.2215388 1.083567 1.16 LANCL2

185  

8133018 1.0998638 1.1385399 1.0315083 1.09 ZNF7168133106 1.0961124 1.1659393 1.0406518 1.10 SNORA228133504 1.1306741 1.0362352 1.0846815 1.08 GTF2I8133860 1.0571015 1.2628796 1.123619 1.15 GNAI18134211 1.1107507 1.2066586 1.0527855 1.12 DKFZP564O05238134420 1.2928214 1.3182987 1.0322874 1.21 TAC18134550 1.2847217 1.0577432 1.2523807 1.208135514 1.0406098 1.1427038 1.1652428 1.12 IFRD18135544 1.0810394 1.1690712 1.1870645 1.15 FOXP28135734 1.0278134 1.1014451 1.1489328 1.09 C7orf588135922 1.1247734 1.1646588 1.051844 1.11 METTL2B8135969 1.0996795 1.2272773 1.2276428 1.18 CCDC1368136177 1.0057164 1.1160557 1.1873981 1.10 KIAA02658136181 1.1465778 1.112018 1.0075703 1.09 C7orf458136471 1.1087356 1.0379505 1.0832505 1.088136709 1.1194812 1.0214102 1.2165209 1.12 LOC934328136849 1.1088686 1.1205922 1.0638769 1.10 GSTK18137240 1.1169957 1.0674512 1.1516277 1.11 GIMAP78137517 1.2553642 1.3094624 1.232021 1.27 HTR5A8137526 1.4404664 1.6578734 1.4730247 1.52 INSIG18137833 1.1655428 1.1064253 1.1164203 1.13 SNX88138189 1.1341456 1.2267083 1.1122195 1.16 RPA38138527 1.190082 1.1214306 1.1195275 1.14 MGC870428138757 1.091138 1.0596775 1.1024911 1.08 HOXA108138922 1.1590073 1.0231063 1.0837289 1.09 KBTBD28138941 1.261633 1.2117302 1.133226 1.20 NT5C38138988 1.1472188 1.0710167 1.241623 1.15 DPY19L2P18139125 1.0752083 1.1607711 1.1070405 1.118139201 1.0141542 1.0841954 1.0879797 1.06 8139242 1.2266543 1.0536234 1.2966862 1.198139314 1.0477213 1.2403716 1.1955312 1.16 GCK8139458 1.1357976 1.0963519 1.0003008 1.08 LOC1001283648139697 1.1830213 1.1421118 1.0582925 1.138139780 1.2792262 1.184631 1.0897791 1.188140420 1.2636291 1.1881655 1.1385956 1.20 FDPSL2A8140443 1.2641546 1.2175925 1.1312666 1.20 FDPSL2A8140864 1.1610378 1.5999483 1.0626613 1.27 CYP51A18141076 1.0629809 1.1230657 1.1098076 1.10 PON28141140 1.0823333 1.1239539 1.0400367 1.08 DLX58141169 1.0813296 1.1053166 1.1755702 1.12 MGC720808141173 1.1597561 1.1373818 1.1190207 1.14 LOC4951|OCM8141317 1.1454536 1.1653842 1.0191869 1.11 CYP3A48141526 1.0811384 1.3324625 1.1056919 1.17 TSC22D48142538 1.087222 1.0115049 1.1107397 1.078142880 1.1199117 1.1034762 1.0433085 1.098142882 1.2430623 1.121002 1.0093288 1.128143385 1.0690697 1.2041085 1.0866547 1.128143482 1.1329223 1.0776333 1.2899702 1.17 TAS2R388143605 1.1130792 1.3609872 1.3286572 1.27 FAM115A8143714 1.1734108 1.2201434 1.0077955 1.13 ZNF777

186  

8143790 1.0657513 1.1108384 1.0942352 1.09 TMEM176B8144044 1.0942674 1.2373546 1.5165994 1.28 FLJ422918144405 1.0462593 1.1942734 1.1430331 1.13 DEFB107A|DEFB107B8144669 1.200071 1.432527 1.2145431 1.28 FDFT18144703 1.1446048 1.0871285 1.056406 1.108144810 1.116982 1.2208017 1.1979531 1.188144931 1.0566058 1.2122667 1.1473868 1.14 ATP6V1B28144995 1.1096909 1.0097656 1.2555625 1.13 FGF178145272 1.2131665 1.1013486 1.0646366 1.13 R3HCC18145660 1.1903507 1.095768 1.0505766 1.11 DCTN68145766 1.038845 1.1204685 1.3566672 1.17 MST1318145795 1.035105 1.2928765 1.1616582 1.168146649 1.1009473 1.0917014 1.0511045 1.08 MTFR18146906 1.1519033 1.324599 1.2853665 1.258147112 1.0694556 1.1934143 1.1056988 1.12 CA138147503 1.0373627 1.1714802 1.1794018 1.13 LAPTM4B8147724 1.1003222 1.0182929 1.1793674 1.10 ATP6V1C18148280 1.1962905 1.4785942 1.1612251 1.28 SQLE8148293 1.1620045 1.1548103 1.0442897 1.12 NSMCE28148325 1.1278055 1.1723021 1.0545069 1.128148783 1.1246827 1.0939466 1.160998 1.13 KIAA1833|LOC3777118148824 1.131059 1.1234322 1.0237825 1.09 HSF1|LOC6422558148982 1.1262897 1.370911 1.0435061 1.18 LOC3896078149104 1.0007354 1.2497393 1.1223966 1.12 DEFA68149230 1.0436689 1.1653006 1.1399734 1.12 DEFB107A|DEFB107B8149438 1.0737433 1.1049719 1.0954887 1.09 SGCZ8149918 1.0094689 1.2678624 1.2215368 1.17 CHRNA28150034 1.495197 1.4631358 2.0955126 1.688150197 1.0871303 1.0276362 1.0869039 1.07 DUSP268150217 1.1834346 1.0967858 1.2147366 1.16 hCG_16401718150757 1.1027207 1.0755843 1.0892757 1.09 RB1CC18150868 1.0098327 1.1944404 1.1704402 1.128151123 1.1908764 1.0870786 1.0427063 1.11 PTTG38151369 1.4045244 1.1299374 1.2180384 1.25 RPESP8151492 1.0846169 1.0358583 1.0831907 1.078151795 1.025104 1.094058 1.1386987 1.09 CDH178151917 1.0828693 1.2314999 1.0549304 1.12 MTERFD18152340 1.1488284 1.0392759 1.0903848 1.09 NUDCD18152617 1.1743839 1.1277674 1.1468053 1.15 HAS28152648 1.0036291 1.1065599 1.1796013 1.10 C8orf768153336 1.1064296 1.0030155 1.0925183 1.07 SLURP18153727 1.1019484 1.1238774 1.0066296 1.08 CPSF18153828 1.0900439 1.0115197 1.2051448 1.10 FOXH18154357 1.1772017 1.0942771 1.0517457 1.11 LOC3922858154365 1.0978593 1.2612057 1.0520822 1.148154476 1.11959 1.0183512 1.1087927 1.08 SH3GL28154979 1.1717652 1.0902108 1.0457231 1.108155460 1.1175282 1.2837061 1.0188383 1.14 CNTNAP3|CNTNAP3B8155497 1.0188043 1.0948122 1.2570777 1.12 FAM27A|LOC1001329488155540 1.2362591 1.1833643 1.015799 1.15 CNTNAP3B|LOC389725

187  

8155598 1.1204321 1.0864356 1.1086205 1.118155600 1.1169566 1.0855013 1.1123775 1.108155877 1.0932345 1.1415263 1.2444632 1.168156043 1.4255711 1.1374127 1.1120727 1.23 PSAT18156082 1.1451217 1.1260239 1.0527076 1.118156228 1.0688051 1.2513236 1.1683512 1.16 CTSL18156371 1.0922865 1.074653 1.2985209 1.168157012 1.0623168 1.1991055 1.220366 1.16 OR13F18157189 1.0887934 1.1837585 1.0562266 1.118157191 1.0851548 1.0594902 1.1714817 1.118157270 1.0131303 1.1245633 1.1500993 1.10 SLC31A18157605 1.1711727 1.0038005 1.1191801 1.108157671 1.097877 1.1070179 1.1861688 1.13 OR1J48157798 1.1326007 1.1113757 1.018777 1.098157922 1.0048217 1.3315533 1.105234 1.15 LMX1B8158539 1.1149029 1.0944729 1.1405255 1.12 C9orf1068158686 1.1107867 1.1915709 1.0793692 1.13 FUBP3|LOC1583018159609 1.1124463 1.2225214 1.0130048 1.12 SSNA18159687 1.064707 1.1443086 1.1367707 1.12 MRPL418160020 1.0222918 1.31548 1.1643031 1.17 C9orf388160317 1.0323927 1.1096245 1.1743586 1.11 LOC3922888160478 1.1105914 1.103501 1.0314271 1.08 C9orf828160602 1.1767827 1.1346997 1.0107266 1.11 APTX8160898 1.0717897 1.2448257 1.2752609 1.20 C9orf144|LOC2593088161288 1.1058009 1.203981 1.0927559 1.13 CNTNAP3|CNTNAP3B8161353 1.0934535 1.153123 1.0206475 1.09 MGC218818161442 1.0167046 1.1378217 1.3235116 1.16 FAM27D18161460 1.2384274 1.1555728 1.0188305 1.14 CNTNAP3B8162132 1.3132415 1.12505 1.0015169 1.15 C9orf1538162466 1.0028349 1.1408199 1.1335175 1.09 FAM120AOS8162827 1.1041037 1.1476842 1.0497096 1.10 ALG28162934 1.1101322 1.1277272 1.1195179 1.12 OR13C58162940 1.1773858 1.5541046 1.2218634 1.32 ABCA18163147 1.4007379 1.1030204 1.0935711 1.208163348 1.1374443 1.1916264 1.0480826 1.13 C9orf848163424 1.0118339 1.2537329 1.0881228 1.128163509 1.0402774 1.1159592 1.0985936 1.08 ALAD8163825 1.1332594 1.0355532 1.0949364 1.09 TRAF18164062 1.1816832 1.034712 1.22171 1.15 LOC6132068164087 1.0397525 1.0915239 1.1548264 1.10 NR6A18164848 1.413336 1.0088072 1.2414905 1.22 LCN1L18164872 1.0187488 1.1398008 1.1040639 1.09 MED228164937 1.2778836 1.0630168 1.1228067 1.15 C9orf1668165295 1.1212502 1.0547701 2.0085402 1.39 LCN88165430 1.1394957 1.0988486 1.2136029 1.15 C9orf1408165552 1.1050174 1.2065797 1.0174502 1.11 NELF8165707 1.0996068 1.0145693 1.1622504 1.098166278 1.3462875 1.3991286 1.090374 1.28 SCML18166349 1.712062 1.089633 1.0301445 1.288166355 1.116527 1.0266292 1.2466373 1.13 CNKSR2

188  

8166440 1.087573 1.1628759 1.0766613 1.11 DDX538166525 1.1839311 1.0038052 1.1290929 1.11 POLA18166571 1.2587866 1.1510382 1.0584205 1.16 MAGEB68166948 1.215748 1.1078088 1.2184867 1.188167040 1.0611787 1.1831851 1.200182 1.15 RNU128167150 1.0625 1.1290246 1.0995166 1.108167161 1.2201802 1.2086483 1.3234901 1.258167305 1.1492254 1.881344 1.1490464 1.39 EBP8167603 1.161024 1.0968379 1.1741564 1.14 CLCN58167910 1.0995148 1.1201953 1.0608511 1.098168026 1.1084582 1.1610855 1.1355162 1.14 LOC1001301308168373 1.0541637 1.1458191 1.0991002 1.108169044 1.0147802 1.1717137 1.1385403 1.11 TCEAL38169158 1.0949765 1.0818429 1.11144 1.10 CXorf578169699 1.0873847 1.225836 1.1403404 1.158170135 1.0824031 1.1775193 1.0716027 1.11 GPR1128170260 1.0852451 1.1914855 1.0171967 1.10 MAGEC38170326 1.1273046 1.0001676 1.1015092 1.08 FMR18170590 1.2002372 1.3611622 1.101783 1.22 NSDHL8170671 1.0272422 1.096225 1.1576896 1.09 ATP2B38171041 1.0517544 1.1232461 1.1504529 1.11 VAMP78171161 1.232324 1.075585 1.1013486 1.14 ARSE8171418 1.0840124 1.0454328 1.1044213 1.08 PIGA8171537 1.0525298 1.115656 1.1074822 1.098172197 1.0576162 1.1209471 1.1844865 1.12 PPP1R2P98172266 1.2021059 1.3201519 1.1337903 1.228172538 1.0563872 1.110355 1.1853751 1.12 WDR458172776 1.0506797 1.1514826 1.3181976 1.178173349 1.1156648 1.0888233 1.147041 1.12 DGAT2L48173930 1.022486 1.1397591 1.096913 1.09 EPAG8174047 1.252137 1.1058059 1.1079828 1.16 TIMM8A8174193 1.1944609 1.0799495 1.2134553 1.168174207 1.0874211 1.1407509 1.09877 1.11 NXF38174226 1.6339853 1.3694111 1.1574363 1.398174304 1.0790232 1.1778498 1.201995 1.15 SERPINA78174568 1.1073577 1.1070614 1.5185852 1.24 LHFPL18174670 1.1166018 1.095806 1.0320758 1.08 RP13‐347D8.38175209 1.057184 1.1090962 1.1644659 1.11 TFDP38175269 1.0250143 1.2382797 1.1209238 1.13 FAM122B8175763 1.1456838 1.0340267 1.2429242 1.14 PNMA58176133 1.0985228 1.1873282 1.0221903 1.10 G6PD8176400 1.0390643 1.1455426 1.0823226 1.09 PCDH11Y|PCDH11X8176679 1.0459173 1.091029 1.1503589 1.108176962 1.0512643 1.1230724 1.1468902 1.11 VAMP78177212 1.046048 1.0933173 1.1566148 1.108177280 1.223822 1.1852745 1.078616 1.168177867 1.0209799 1.1932471 1.0828575 1.10 DDR18177983 1.1264933 1.0591383 1.210469 1.13 TNF8178063 1.1283132 1.1287776 1.0599024 1.11 C6orf21|LY6G6D8178754 1.078662 1.1413023 1.1123735 1.11 PRRT1

189  

8178795 1.1400728 1.1015059 1.0288314 1.09 BTNL28179184 1.0208236 1.1925173 1.0901499 1.10 DDR18179263 1.1225313 1.0565687 1.2115452 1.13 TNF8179950 1.0632282 1.1363646 1.1133192 1.10 PRRT18179996 1.135169 1.0851085 1.0311197 1.08 BTNL2