interpretation of altered methylation patterns in

©2005NaturePublishingGroupPERSPECTIVEStheyaremoresusceptibletomutation6.However,clustersofCpGsitesareocca-sionallypresentinthegenome,andtheseclustersaredesignatedasCpGislands(CGIs;FIG.1).AlthoughCGIsweretradi-tionallyconsideredtobelocatedin5′regionsofgenesandtobeconsistentlykeptunmethylated,theyareactuallylocatedatvariouspositionsthroughoutgenes7,suchasinexonsandintrons,orfurtherdown-stream.SomeCGIsaremethylatedundernormalconditions8,9.ChallengesinanalysingCGImethylationincludedistin-guishingthemfromrepetitiveDNAsequences,whichareusuallyheavilymethylated,andtoidentifythosethatlieinpromoterregionsthatregulategeneexpres-sion.StrictcriteriaforestablishingasequencetobeatrueCGIhavethereforebeenproposed7.CGIsthatlieingenepromoterregionsareespeciallyimportantforanalysis,astheirdownstreamgenesareconsistentlysilencedwhentheseCGIsaremethylated5,10.Incan-cercells,tumour-suppressorgenes,suchasCDKN2A,RB,VHLandBRCA1,areinacti-vatedbymethylationoftheirpromoterCGIs.Inadditiontoincreasedmethylationofcertaingenes,genome-widehypomethy-lationhasalsobeendetectedincancercells11.Thisisassociatedwithdemethyla-tionofpromoterCGIsthatarenormallymethylatedandthereforesilencedinnon-cancercells12.Alteredmethylationlevels,especiallyhypomethylation,cancausegenomicinstabilityand,consequently,tumourformation13–15.Inresponsetothefindingthatmanyimportanttumour-suppressorgenesaresilencedbymethylationofCGIsintheirpro-moterregions,genome-widescreeningtech-niquesfordifferencesinDNAmethylationweredevelopedinthelate1990s.Mostdevel-opersofthesetechniquesexpectedthatifitwerepossibletoidentifyaCGIthatwasaber-rantlymethylatedinatumour,atumour-suppressorgenewouldbepresentatthissite.Thankstocompletionofthehumangenomesequence,onceadifferentiallymethylatedsiteisdetected,itispossibletoanalysethegenomestructurearoundthesitetodeterminewhetherornotitislocatedwithinapromoterCGI.WhenmethylationoftheCGIisappropriatelyvalidated,expressionofthedownstreamgeneisalmostalwaysfoundtoberepressed.Asinitiallyexpected,noveltumour-suppressorgeneshavebeenidentifiedatsitesofalteredmethylation16–18.Atthesametime,ithasbecomerecognizedthatmethyla-tionincancercellsfrequentlyoccursinCGIsoutsidepromoterregions,whichdonotrepressgenetranscription,andalsoinpro-moterCGIsofgenesthatcannotberegardedastumour-suppressorgenes.Eveninnormalcells,methylationofspecificCGIsfrequentlyoccurs.Therefore,toidentifynoveltumour-suppressorgenessilencedincancercellsbyCGImethylationitisnecessarytocarefullyselecttheparticularCGIstobeincludedintheanalysis.Differencesinmethylationpatternshavealsoemergedasmarkers.Forexample,dif-ferencesbetweentumourandnormalcellscanbeusedtodetectthepresenceoftumourcellsinbiopsyspecimensortoidentifytumour-derivedDNAinbloodsamples19–22.Differencesinmethylationpatternsamongtumourscanbeassociatedwithpatientout-comeorotherclinicalresponses,andcanbeusedasmarkerstoclassifytumours.Fortheseuses,methylationdoesnotnecessarilyhavetoinducegenesilencing,butsimplybespecifictotumourcellsorhaveapatternthatisassociatedwithclinicallyimportantinformation.Abstract|Epigeneticalterations,suchasabnormalDNA-methylationpatterns,areassociatedwithmanyhumantumourtypes.Newtechniqueshavebeendevelopedtoperformgenome-widescreeningforalterationsinDNA-methylationpatterns,notonlytoidentifytumour-suppressorgenes,butalsotofindpatternsthatcanbeusedindiagnosisandprognosis.However,interpretationofdifferentialmethylationhasprovendifficultbecausethesignificanceofmethylationalterationsdependsonthegenomicregion,andfunctionsofCpGislandsatspecificsiteshavenotbeenfullyclarified.Whattechniquescanbeusedtoidentifynewtumoursuppressorsanddiagnosticmarkers?Epigeneticmodificationsaredefinedasheri-tableinformationotherthannucleotidesequences.TheseincludeDNAmethylationandhistonemodifications,whicharenowknowntoregulateawiderangeofphysiologi-calandpathologicalprocesses1.DNAmethy-lationalterschromosomestructure,excludingDNA-bindingproteinssuchasCTCF(whichregulatesgenetranscription2),anddefinesregionsfortranscriptionalregulation3,4.DNAmethylationcanalsopromotethebindingofproteinssuchasMECP2,MBD1,MBD2,MBD3andMBD4,whichinducehistonemodification5.DNAmethylationtypicallyoccursatCpGsites,whichare5′-CG-3′dinucleotidesequencesthatoccuratarelativelylowfre-quencyinthegenome.ThisisbecausemethylatedCpGsitesarelostovertime,asNATUREREVIEWS|CANCERVOLUME5|MARCH2005|223DetectionandinterpretationofalteredmethylationpatternsincancercellsToshikazuUshijimaINNOVATION©2005NaturePublishingGroup224|MARCH2005|VOLUME5www.nature.com/reviews/cancerPERSPECTIVESnecessary,asmethylationpatternsarespecifictocelltypes9,23.Toisolatemarkersfortumourclassification,itishelpfultohavegoodtumoursampleswithrelevantpatientbackgroundinformationavailable.Tosearchformethyla-tiondifferencesthataresharedbyalargenumberofsamples,poolingofsamplesiseffective.Topreparesamplesforanalysis,itisimportanttohaveahomogeneouspopulationofcells,aseachscreeningmethodhasalimita-tioninthedegreeofdifferencesthatitcandetect.Useofcelllinesisconvenientintermsofanalysis,butthefindingsmustthenbevali-datedin