hedgehog signaling modulates cholesterol homeostasis … · ii hedgehog signaling modulates...
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HEDGEHOGSIGNALINGMODULATESCHOLESTEROLHOMEOSTASISINCHONDROCYTESANDINOSTEOARTHRITIS
by
ShabanaAmandaAli
AthesissubmittedinconformitywiththerequirementsforthedegreeofDoctorofPhilosophy
InstituteofMedicalScienceUniversityofToronto
©CopyrightbyShabanaAmandaAli2014
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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
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developedmoresevereOAthancontrollittermates.TheyexpressedtypicalOA
markers,suchastypeXcollagen,indicatingchondrocytehypertrophyinthearticular
cartilage.GeneticmanipulationofHhsignalinginthesemicesuggeststhatHhsignaling
ismodulatingthephenotypebyregulatingsterolhomeostasis.Hhreductionandstatin
treatmentbothloweredcholesterolproductionandrescuedthephenotype,reducing
theseverityofOA.HereweidentifiednovelHhtargetgenesinchondrocyteswhich
regulateintracellularcholesterollevels,andfoundthatcholesteroldysregulationinthe
chondrocytespredisposestoOA.Thesedatasuggestthatpharmacologiccorrectionof
intra‐articularsterolimbalancecanbeusedasatreatmentforosteoarthritis.
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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.
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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.
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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.
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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
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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
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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
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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.
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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.
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ListofTablesTable1.ComparingRNAobtainedfromdifferentextractionmethods.Table2.ComparinggeneexpressionresultsusingRNAobtainedfromdifferentextractionmethods.Table3.Microarrayanalysis:top20genechanges.Table4.IngenuityPathwayAnalysisofmicroarrayresults.Table5.ICRSScoringofGli2+/‐,InsigDKO,andGli2+/‐;InsigDKOcartilage.Table6.ICRSScoringofCol2a1‐Gli2,InsigDKO,andCol2a1‐Gli2;InsigDKOcartilage.Table7.ICRSScoringofcartilageaftersurgically‐inducedOA.
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ChapterOne:
Introduction
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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).
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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
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burden,affecting250millionpeopleglobally,andalmosttwiceasmanywomenasmen
(Vosetal.2012).
Figure1.Representativetotalkneearthroplastysampleshowingfeaturesof
osteoarthritis.Typicalfeaturesofosteoarthritisincludecartilagefibrillation,cartilage
erosion,andosteophyte(bonespur)formation.Progressionofthisdiseasetypically
correlateswithdegenerationofthemenisci.Themedialsidetendstobemoreseverely
affected.
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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.
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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.
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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.
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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.
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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).
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Figure2.Schematicrepresentationofarticularchondrocytesandgrowthplate
chondrocytes.Thearticularcartilageisdividedintoseveralzoneswhichcanbe
identifiedbyarticularchondrocyteshapeandtypeIIcollagenfibreorientation.The
growthplatechondrocytesundergoacontrolledprocessofdifferentiationfromresting,
toproliferating,topre‐hypertrophic,tohypertrophic.Thisenableselongationofthe
longbones.
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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.
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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.
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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.
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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).
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Figure3.SchematicrepresentationoftheHedgehog(Hh)signalingpathway.In
theabsenceofHhligand,thetransmembranereceptorPtchrepressesasecond
transmembranereceptorSmo,resultinginpathwayinhibition.InthepresenceofHh
ligand,repressionofSmobyPtchisrelieved,andtheGlitranscriptionfactorsbecome
active,resultinginpathwayactivationandupregulationoftargetgenes.PM=plasma
membrane.
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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.
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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
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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.
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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
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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.
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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.
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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.
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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
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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.
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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
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distallytoregulatetherateofchondrocytedifferentiation(Vortkampetal.1996).
Modulationofsterollevelhasbeenshowntocausealteredgrowthplatephenotypes,
whichcouldpotentiallyarisefromimpairedHhligandtrafficking.Impairedgrowthof
thelongbones,asdescribedtoresultfromcholesteroldepletion(Gofflotetal.2003;S.
WuandDeLuca2004),hasalsobeenshowntoresultfromdecreasingHhsignaling
(Kobayashietal.2005).WhiletheseoverlappingphenotypesinSLOSandthegrowth
platehighlighttheinterdependencybetweenHhsignalingandsterolhomeostasis,the
exactregulatoryrelationshipremainsunclear.
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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‐
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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
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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.
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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.
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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
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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.
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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).
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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.
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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.
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HypothesisHhsignalingregulatescholesterolbiosynthesisinchondrocytes,andmodulating
cholesterolhomeostasiscanimpacttheseverityofOA.
Objectives1. IdentifyHhsignalingtargetgenesinchondrocytes.
2. DeterminewhethermodulationofHhsignalingtargetgenescanimpactchondrocyte
homeostasis.
3. AssesstheimpactofHhsignalingtargetgenemodulationontheseverityof
osteoarthritis.
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ChapterTwo:
Hhsignalingregulatesexpressionofcholesterolbiosyntheticgenesinchondrocytes
RNAextractionfromhumanarticularcartilagebychondrocyteisolation
(AliandAlman2012)
PublishedinAnalyticalBiochemistry
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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.
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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
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expressionofHhtargetgenesinchondrocytespromotesOApathogenesisbydisrupting
normalarticularchondrocytebehaviour.Toelucidatethebiologicalnetworksthat
couldberesponsibleforthisdisruption,functionalpathwayanalysisofmicroarraydata
wasconducted(Downey2006).RegulationoftargetgenesbyHhsignalingwasverified
usingindependenthumansamplesthatweretreatedwithaHhantagonistoraHh
agonist.Additionally,insilicoanalyseswereconductedtodeterminewhethertarget
genescontainedGliconsensusbindingsitesandwhethertargetgeneswere
dysregulatedinosteoarthriticcartilagewhencomparedtonormalcartilage.Theresults
oftheseexperimentsidentifyHhtargetgenesthatareexpressedinosteoarthritic
cartilage.
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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.
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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
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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).
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Figure5.RepresentativeelectropherogramscomparingRNAquality.RNA
extractedusingthetraditionalTRIzol®methodcoupledwithQiagencolumn
purification(a,RIN2.3)andRNAextractedusingthemodifiedprotocol(b,RIN8.2).
Peaksandbandslabeledas18Sand28SrepresentrRNA.RIN=RNAintegritynumber.
TodeterminetheeffectsofRNAqualityongeneexpressionassays,cartilage
fromarepresentativesamplewasdividedandRNAwasextractedusingthetraditional
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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.
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Table2.ComparinggeneexpressionresultsusingRNAobtainedfromdifferent
extractionmethods.(a)ComparingqualityandquantityofRNAisolatedfromthe
samecartilagesampleusingtwodifferentextractionmethods.(b)CTvaluesforgenes
typicallyassayedincartilageexperiments,COL2A1,COL10A1,andMMP13.Inthis
representativesample,thequalityofRNA[shownin(a)]impactsgeneexpression
resultsbyaltering∆CTvalues.
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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).
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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.
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Figure7.Microarrayanalysis:genefiltering.Eachcolourrepresentsadifferent
microarraydatasetafteridentifyinggenechangesinthetreated(Media+HhAntagonist)
groupoverthecontrolgroup(Media).Eachspikerepresentsadifferentgene,where
inwardspikesrepresentdecreasesingeneexpressionandoutwardspikesrepresent
increasesingeneexpression.Differentiallyexpressedgeneswerefilteredforthose
whichchangedinthesamedirectionacrossdatasets.
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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.
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Table3.Microarrayanalysis:top20genechanges.Thetop10upregulatedandtop
10downregulatedgenesinhumanOAchondrocyteswithHhinhibition,afterfiltering
forgeneswhichchangedinthesamedirectionacrossthreedatasets.
Table4.IngenuityPathwayAnalysisofmicroarrayresults.Microarrayresultswere
analyzedinanunbiasedmannerforbiologicalnetworks,signalingpathways,andother
functionalgenegroupsusingIngenuityPathwayAnalysis.Thetopresultsforeach
categoryareshown.
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IdentifyingHhtargets:cholesterolbiosyntheticgenes
Microarrayanalysesrevealedgenesinvolvedinthecholesterolhomeostatic
pathwaytobeamongthetopdysregulatedwithHhpathwayinhibition(Figure8).
Figure8.Heatmapofcholesterolhomeostaticgenes.ResultsfromAffymetrix
HumanGene1.0STmicroarrayofhumanosteoarthriticcartilagetreatedwithHh
antagonist.Eachofthreepairsofsamples(Controlvs.Treated)wereanalyzed
independentlyandfilteredforgeneswhichwereeitherupregulatedordownregulated
acrossallthreesamples.Greaterintensityofredrepresentsincreasedgeneexpression
withHhinhibition(Treated).
Becausecholesterolisvitalforcellularprocessesandbecauseitsdysregulationhas
beenimplicatedinOA,wesoughttoassesstheroleofHhsignalinginregulatingthe
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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).
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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.
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Figure11.Insilicoanalysesofcholesterolhomeostaticgenes.Mulaninquiries
(http://mulan.dcode.org)wereconductedtoexaminethe5kbpromoterregionof
cholesterolhomeostaticgenesidentifiedbymicroarrayanalyses(3of3arrays)forGli
consensussequence‐bindingsitesthatareconservedbetweenhumansandmice(Gli
site).Publiclyavailableexpressiondatacomparingosteoarthriticcartilagetonormal
cartilagewerefilteredforchangestocholesterolbiosyntheticgenes(OAvs.normal).
GeneswithaGlisiteorwithdysregulatedexpressioninOAaredesignatedbyblack
boxesintherespectivecolumns.
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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.
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Figure12.SREBF2chromatinimmunoprecipitation.Chromatinfromprimary
humanOAchondrocyteswasprobedwithanti‐Gli1,IgG(Neg),andanti‐H3(Pos)
antibodies.PCRamplificationoftheSREBF2geneshowsenrichmentintheGli1fraction,
withprimersdesignedtoflanktheputativeGlibindingsite.Additionalcontrols
includedprimersdesignedadjacenttotheGli‐bindingsiteandprimersdesignedto
amplifyrandomlyselectedDNAonthesamechromosome.
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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
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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
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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
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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.
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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.
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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
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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
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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
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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.
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ChapterThree:
Hhsignalingmodulatescholesterolaccumulationinchondrocytes
CholesterolhomeostasismediatesHedgehogsignalinginosteoarthritis
ShabanaAmandaAli,MushriqAl‐Jazrawe,HeatherWhetstone,RaymondPoon,HenryMa,SarahFarr,MarkNaples,KhosrowAdeli,BenjaminAlman
Manuscriptunderpreparation
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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.
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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
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Figure13.Schematicofthecholesterolbiosyntheticpathway.Colouredboxes
signifyalteredgeneexpressionwithHhinhibition,asdeterminedbymicroarray
experiments.Redindicatesupregulationandgreenrepresentsdownregulation.Figure
adaptedfromGenMAPP2.0.
NobelPrizeinMedicinein1985fortheirworkoncholesterolhomeostasis,describea
mousewithafloxedInsig1geneandgermlinedisruptionoftheInsig2gene(Brownand
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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.
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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).
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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).
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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.
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Figure16.Visualizingtotallipidandsterolaccumulationinchondrocytes.
RepresentativeimagesofculturedprimarychondrocytesstainedwithOil‐Red‐O(red
colour)toshowtotalsterolandlipidaccumulationaccordingtogenotype.Scalebar,
100μm.
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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.
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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).
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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.
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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
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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.
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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.
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Figure21.GrosssizeofInsigDKOmice.(a)InsigDKOmicearevisiblysmallerthan
theirControllittermatesat52weeks.(b)InsigDKOmice(greybars)weighsignificantly
lessthantheirControllittermates(blackbars)at24weeksand52weeks.Errorbars
are95%confidenceintervals(n=5‐9;*P<0.05).
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Figure22.BonelengthinInsigDKOmice.(a)Thefemurand(b)thetibiaofInsigDKO
mice(greybars)aresignificantlyshorterthanthoseofControlmice(blackbars)at16
weeks,24weeks,and52weeks.Errorbarsare95%confidenceintervals(n=10‐32;*P
<0.05).
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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.
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Figure23.GrowthplateanalysesinInsigDKOmice.Thetibiaeof17.5dpcmicewere
stainedwithanantibodyagainsttypeXcollagen(Col10)toidentifythehypertrophic
zoneinthegrowthplate.InsigDKOmicehadashorterhypertrophiczonethanControl
littermates.Errorbaris95%confidenceinterval(n=3;*P<0.05).
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Figure24.SecondaryossificationcentredevelopmentinInsigDKOmice.InsigDKO
miceshowedadelayindevelopmentofthesecondaryossificationcentre,visibleby
radiographyat7daysinControlmice(redarrow).
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Figure25.SkeletalanalysesinInsigDKOmice.Totalskeletonswerestainedwith
AlcianbluetoidentifycartilageandAlizarenredtoidentifybone.Therewerenoovert
differencesbetweenInsigDKOandControlmiceat2days.
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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.
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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.
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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
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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.
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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
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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
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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.
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ChapterFour:
Cholesterolmodulationcanaltertheseverityofosteoarthritis
CholesterolhomeostasismediatesHedgehogsignalinginosteoarthritis
ShabanaAmandaAli,MushriqAl‐Jazrawe,HeatherWhetstone,RaymondPoon,HenryMa,SarahFarr,MarkNaples,KhosrowAdeli,BenjaminAlman
Manuscriptunderpreparation
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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.
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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
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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.
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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.
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Figure26.HistologicanalysesofInsigDKOcartilage.Representativehistological
sectionsshowinghaematoxylinandeosinstainingofthekneejointsin24‐week‐old
mice.Boxesdelineatemagnifiedregionsshownbelowtohighlightchangesin
chondrocytemorphologyanddistribution.InsigDKOmiceshowcharacteristicOA
changes,includingcartilageerosion.Scalebar,100μm.
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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).
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Figure27.TypeXcollagenstainingofInsigDKOcartilage.Representative
histologicalsectionsshowingtypeXcollagenimmunohistochemistryinthearticular
cartilageofthefemurat24weeksforeachgenotypeshowninFigure26.InsigDKOmice
showincreasedchondrocytehypertrophyinthesuperficialzone.Scalebar,100μm.
Figure28.RadiographsofInsigDKOknees.Representativeradiographicimagesof
mousekneescorrespondingtothegenotypesshowninFigure26,showingalateral
viewat16weeksofage.InsigDKOmiceshowirregularityinthebonecontourand
increasedsubchondralsclerosis.
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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
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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.
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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.
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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).
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Figure31.Serumcholesterollevelfollowingstatintreatment.Systemiclevelsof
cholesterolintheserumofmicethatwereimplantedwithaslow‐releasePlaceboor
StatinpelletweremeasuredusingtheAmplexRedassay.Nostatisticallysignificant
differencesweredetectedafterthe8‐weektreatmentperiod.Errorbarsare95%
confidenceintervals(n=3).
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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.
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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.
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Figure33.Statintreatmentinmicewithsurgically‐inducedOA.Representative
histologicalsectionsshowinghaematoxylinandeosinstainingofthekneejointsin16‐
week‐oldmicethatwereimplantedwithaslow‐releasePlaceboorStatinpellet.Mice
weresubjecttoexcisionofthemedialmeniscus(Surgery)orshamoperations(Sham)
at8weeksofage.StatintreatmentreducesfeaturesofOA.Scalebar,100μm.
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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).
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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).
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Figure34.TypeXcollagenstainingofCol2a1‐Gli2,InsigDKO,andCol2a1‐
Gli2;InsigDKOcartilage.RepresentativehistologicalsectionsshowingtypeXcollagen
immunohistochemistryinthearticularcartilageofthefemurat16weeksforeach
genotype/treatmentgroupshowninFigure32.StatintreatmentreducestypeX
collagenstaininginthesuperficialzone.Scalebar,100μm.
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Figure35.TypeXcollagenstainingofcartilageaftersurgically‐inducedOA.
RepresentativehistologicalsectionsshowingtypeXcollagenimmunohistochemistryin
thearticularcartilageofthefemurat16weeksforeachgenotype/treatmentgroup
showninFigure33.StatintreatmentreducestypeXcollagenstaininginthesuperficial
zone.Scalebar,100μm.
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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.
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Figure36.Real‐timePCRofOAmarkersafterstatintreatment.Geneexpression
showingareductioninOAmarkersMMP13andADAMTS5inhumanosteoarthritic
cartilageexplantstreatedwithstatin(greybars).Expressioninthecontrolgroup(black
bars)wasarbitrarilydefinedas‘1’anddatafromthestatin‐treatedgroupgivenasthe
mean.Errorbarsare95%confidenceintervals(n=4;*P<0.05).
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Figure37.ADAMTS5reporterconstructassay.LuciferaseactivityfromtheADAMTS5
promoterconstructtransfectedintoprimaryhumanOAchondrocytes,treatedwith
controlorstatin.Measuredintriplicateandreportedasrelativelightunits(RLU).Error
barsareSEM.
Figure38.ADAMTS5promoteranalysis.SchematicoftheADAMTS5promoter
showingaSREBFconsensusbindingsite(redtick)thatisconservedbetweenmouse
andhuman(fromMULANanalysis,http://mulan.dcode.org).
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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
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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
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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.
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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.
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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.
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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.
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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
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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).
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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.
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ChapterFive:
Conclusionsandfutureresearch
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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.
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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.
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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
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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
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alsotranslatetoothertissuetypesandothercartilagepathologies.Regardingother
tissues,simplestainssuchasOil‐Red‐OcanbeusedtocrudelyassesswhetherHh
modulationalterslipidcontent.Regardingothercartilagepathologies,thecontribution
ofcholesteroldysregulationtochondrosarcoma(L.Hoetal.2009),whichalsoresults
fromactivationofHhsignaling,meritsfurtherinvestigation.Thiscanbeexploredusing
themicewithcartilage‐specificcholesterolaccumulationandHhmodulationdescribed
inChapterThree.
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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
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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
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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
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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.
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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
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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.
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139
Relevance
IdentifyingHhtargetgenesandnetworksimprovesourunderstandingof
cartilagebiologyandpathology,andproposespotentialmechanismsbywhichHh
activationcontributestoOApathogenesis.Withhumanandanimalsamples,weshow
thatHhsignalingregulatescholesterolhomeostasisandthatcholesterolmodulation
canalterOApathogenesis.Theseresultshighlighttheneedtofurtherexplorethe
complexinteractionsamongHhsignaling,cholesterolhomeostasis,andchondrocyte
biology.Theabilitytopharmacologicallycorrectsterolimbalancesinthecartilageto
preventOApathologyrepresentsapromisingtherapeuticintervention.
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140
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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