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KaryotypingforChromosomalAbnormalities
By:ClareO'Connor,Ph.D.(BiologyDepartment,BostonCollege)2008NatureEducation
Citation:O'Connor,C.(2008)Karyotypingforchromosomalabnormalities.Nature
Education1(1):27

Eachchromosomepairviewedinakaryotypeappearstohaveitsowndistinct"barcode"of
bands.Whatchangesdoscientistslookforinakaryotypewhendiagnosingdiseasesand
disorders?

Karyotypingistheprocessofpairingandorderingallthechromosomesofanorganism,thusprovidinga
genomewidesnapshotofanindividual'schromosomes.Karyotypesarepreparedusingstandardizedstaining
proceduresthatrevealcharacteristicstructuralfeaturesforeachchromosome.Clinicalcytogeneticistsanalyze
humankaryotypestodetectgrossgeneticchangesanomaliesinvolvingseveralmegabasesormoreofDNA.
Karyotypescanrevealchangesinchromosomenumberassociatedwithaneuploidconditions,suchastrisomy
21(Downsyndrome).Carefulanalysisofkaryotypescanalsorevealmoresubtlestructuralchanges,suchas
chromosomaldeletions,duplications,translocations,orinversions.Infact,asmedicalgeneticsbecomes
increasinglyintegratedwithclinicalmedicine,karyotypesarebecomingasourceofdiagnosticinformationfor
specificbirthdefects,geneticdisorders,andevencancers.

PreparingKaryotypesfromMitoticCells
Karyotypesarepreparedfrommitoticcellsthathavebeenarrestedinthemetaphaseorprometaphaseportionof
thecellcycle,whenchromosomesassumetheirmostcondensedconformations.Avarietyoftissuetypescanbe
usedasasourceofthesecells.Forcancerdiagnoses,typicalspecimensincludetumorbiopsiesorbonemarrow
samples.Forotherdiagnoses,karyotypesareoftengeneratedfromperipheralbloodspecimensoraskinbiopsy.
Forprenataldiagnosis,amnioticfluidorchorionicvillusspecimensareusedasthesourceofcells.

Theprocessofgeneratingakaryotypebeginswiththeshorttermcultureofcellsderivedfromaspecimen.After
aperiodofcellgrowthandmultiplication,dividingcellsarearrestedinmetaphasebyadditionofcolchicine,which
poisonsthemitoticspindle.Thecellsarenexttreatedwithahypotonicsolutionthatcausestheirnucleitoswell
andthecellstoburst.Thenucleiarethentreatedwithachemicalfixative,droppedonaglassslide,andtreated
withvariousstainsthatrevealstructuralfeaturesofthechromosomes.

BandingPatternsRevealtheStructuralDetailsofChromosomes
Withoutanytreatment,structuraldetailsofchromosomesaredifficulttodetectunderalightmicroscope.Thus,to
makeanalysismoreeffectiveandefficient,cytologistshavedevelopedstainsthatbindwithDNAandgenerate
characteristicbandingpatternsfordifferentchromosomes.Priortothedevelopmentofthesebanding
techniques,distinguishingchromosomesfromoneanotherprovedverydifficult,andchromosomesweresimply
groupedaccordingtotheirsizeandtheplacementoftheircentromeres.

Thischangedin1970,whenTorbjornCasperssonandhiscolleaguesdescribedthefirstbandingtechnique,
knownasQbanding.Qbandinginvolvesuseofthefluorescentdyequinacrine,whichalkylatesDNAandis
subjecttoquenchingovertime.Casperssonetal.demonstratedthatquinacrineproducedcharacteristicand
reproduciblebandingpatternsforindividualchromosomes.Sincethen,researchershavedevelopedavarietyof
otherchromosomebandingtechniquesthathavelargelysupplantedQbandinginclinicalcytogenetics.Today,
mostkaryotypesarestainedwithGiemsadye,whichoffersbetterresolutionofindividualbands,producesa
morestablepreparation,andcanbeanalyzedwithordinarybrightfieldmicroscopy.

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Themolecularcausesforstainingdifferencesalongthelengthofachromosomearecomplexandincludethe
basecompositionoftheDNAandlocaldifferencesinchromatinstructure.InGbanding,thevariantofGiemsa
stainingmostcommonlyusedinNorthAmerica,metaphasechromosomesarefirsttreatedbrieflywithtrypsin,an
enzymethatdegradesproteins,beforethechromosomesarestainedwithGiemsa.Trypsinpartiallydigests
someofthechromosomalproteins,therebyrelaxingthechromatinstructureandallowingtheGiemsadye
accesstotheDNA.

Ingeneral,heterochromaticregions,whichtendtobeATrichDNAandrelativelygenepoor,stainmoredarklyin
Gbanding.Incontrast,lesscondensedchromatinwhichtendstobeGCrichandmoretranscriptionallyactive
incorporateslessGiemsastain,andtheseregionsappearaslightbandsinGbanding.Mostimportantly,G
bandingproducesreproduciblepatternsforeachchromosome,andthesepatternsaresharedbetweenthe
individualsofaspecies.AnexampleofGiemsastainedhumanchromosomes,astheywouldappearundera
microscope,isshowninFigure1a.Typically,Giemsastainingproducesbetween400and800bandsdistributed
amongthe23pairsofhumanchromosomes.MeasuredinDNAterms,aGbandrepresentsseveralmillionto10
millionbasepairsofDNA,astretchlongenoughtocontainhundredsofgenes.

Figure1:Chromosomebandingrevealedbydifferentstainingtechniques.
Differentchromosomalstainingtechniquesrevealvariationsinchromosomestructure.Cytogeneticistsusethesepatternstorecognizethe
differencesbetweenchromosomesandenablethemtolinkdifferentdiseasephenotypestochromosomalabnormalities.Giemsabanding(a),
Qbanding(b),Rbanding(c)andCbanding(d)areshown.
2001NaturePublishingGroupRowley,J.Chromosometranslocations.NatureReviewsCancer1,246Stamatoullas,A.etal.
ConventionalcytogeneticsofnodularlymphocytepredominantHodgkin'slymphoma.Leukemia21,2065Vega,H.etal.Robertssyndrome
iscausedbymutationsinESCO2,ahumanhomologofyeastECO1thatisessentialfortheestablishmentofsisterchromatidcohesion.
NatureGenetics35,469(2001).Allrightsreserved.

FigureDetail

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Gbandingisnottheonlytechniqueusedtostainchromosomes,however.Rbanding,whichisusedinpartsof
Europe,alsoinvolvesGiemsastain,buttheproceduregeneratesthereversepatternfromGbanding.InR
banding(Figure1c),thechromosomesareheatedbeforeGiemsastainisapplied.Theheattreatmentisthought
topreferentiallymelttheDNAhelixintheATrichregionsthatusuallybindGiemsastainmoststrongly,leaving
onlythecomparativelyGCrichregionstotakeupthestain.Rbandingisoftenusedtoprovidecriticaldetails
aboutgenerichregionsthatarelocatednearthetelomeres.

YetanothermethodisCbanding(Figure1d),whichcanbeusedtospecificallystainconstitutive
heterochromatin,orgeneticallyinactiveDNA,butitisrarelyusedfordiagnosticpurposesthesedays.Cbanding
isaspecializedGiemsatechniquethatprimarilystainschromosomesatthecentromeres,whichhavelarge
amountsofATrichsatelliteDNA.

Thefirstmethodtobeusedtoidentifyall46humanchromosomeswasQbanding(Figure1b),whichisachieved
bystainingthechromosomeswithquinacrineandexaminingthemunderUVlight.Thismethodismostusefulfor
examiningchromosomaltranslocations,especiallyonesinvolvingtheYchromosome.Takentogether,these
bandingtechniquesofferclinicalcytogeneticistsanarsenalofstainingmethodsfordiagnosingchromosomal
abnormalitiesinpatients.

OrganizingChromosomesinKaryogramsforReview
Inordertomaximizethediagnosticinformationobtainedfromachromosomepreparation,imagesofthe
individualchromosomesarearrangedintoastandardizedformatknownasakaryotype,ormoreprecisely,a
karyogram(Figure1ac).Accordingtointernationalconventions,humanautosomes,ornonsexchromosomes,
arenumberedfrom1to22,indescendingorderbysize,withtheexceptionsofchromosomes21and22,the
formeractuallybeingthesmallestautosome.Thesexchromosomesaregenerallyplacedattheendofa
karyogram.

Withinakaryogram,chromosomesarealignedalongahorizontalaxissharedbytheircentromeres.Individual
chromosomesarealwaysdepictedwiththeirshortparmspfor"petite,"theFrenchwordfor"small"atthe
top,andtheirlongqarmsqfor"queue"atthebottom.Centromereplacementcanalsobeusedtoidentifythe
grossmorphology,orshape,ofchromosomes.Forexample,metacentricchromosomes,suchaschromosomes
1,3,and16,havepandqarmsofnearlyequallengths.Submetacentricchromosomes,suchaschromosomes
2,6,and10,havecentromeresslightlydisplacedfromthecenter.Acrocentricchromosomes,suchas
chromosomes14,15,and21,havecentromereslocatedneartheirends.

Arrangingchromosomesintoakaryogramcansimplifytheidentificationofanyabnormalities.Notethatthe
bandingpatternsbetweenthetwochromosomecopies,orhomologues,ofanyautosomearenearlyidentical.
Somesubtledifferencesbetweenthehomologuesofagivenchromosomecanbeattributedtonaturalstructural
variabilityamongindividuals.Occasionally,technicalartifactsassociatedwiththeprocessingofchromosomeswill
alsogenerateapparentdifferencesbetweenthetwohomologues,buttheseartifactscanbeidentifiedby
analyzing1520metaphasespreadsfromoneindividual.Itishighlyunlikelythatthesametechnicalartifact
wouldoccurrepeatedlyinagivenspecimen.

UsingKaryogramstoDetectChromosomalAbnormalities
Today,Gbandedkaryogramsareroutinelyusedtodiagnoseawiderangeofchromosomalabnormalitiesin
individuals.Althoughtheresolutionofchromosomalchangesdetectablebykaryotypingistypicallyafew
megabases,thiscanbesufficienttodiagnosecertaincategoriesofabnormalities.Forexample,aneuploidy,
whichisoftencausedbytheabsenceoradditionofachromosome,issimpletodetectbykaryotypeanalysis.
Cytogeneticistscanalsofrequentlydetectmuchmoresubtledeletionsorinsertionsasdeviationsfromnormal
bandingpatterns.Likewise,translocationsareoftenreadilyapparentonkaryotypes.

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Whenregionalchangesinchromosomesareobservedonkaryotypes,researchersoftenareinterestedin
identifyingcandidategeneswithinthecriticalintervalwhosemisexpressionmaycausesymptomsinpatients.
ThissearchprocesshasbeengreatlyfacilitatedbythecompletionoftheHumanGenomeProject,whichhas
correlatedcytogeneticbandswithDNAsequenceinformation.Consequently,investigatorsarenowabletoapply
arangeofmolecularcytogenetictechniquestoachieveevenhigherresolutionofgenomicchanges.
Fluorescenceinsituhybridization(FISH)andcomparativegenomichybridization(CGH)areexamplesoftwo
approachesthatcanpotentiallyidentifyabnormalitiesatthelevelofindividualgenes.

Molecularcytogeneticsisadynamicdiscipline,andnewdiagnosticmethodscontinuetobedeveloped.Asthese
newtechnologiesareimplementedintheclinic,wecanexpectthatcytogeneticistswillbeabletomaketheleap
fromkaryotypetogenewithincreasingefficiency.

ReferencesandRecommendedReading

Caspersson,T.,Zech,L.,&Johansson,J.Differentialbandingofalkylatingfluorochromesinhumanchromosomes.ExperimentalCellResearch60,
315319(1970)doi:10.1016/00144827(70)905239

Gartler,S.M.Thechromosomenumberinhumans:Abriefhistory.NatureReviewsGenetics7,655660(2006)doi:10.1038/nrg1917( linkto
article)

Speicher,M.R.,Ballard,S.G.,&Ward,D.C.KaryotypinghumanchromosomesbycombinatorialmultifluorFISH.NatureGenetics12,368375
(1996)( linktoarticle)

Strachan,T.,&Read,A.P.HumanMolecularGenetics,2nded.(Wiley,NewYork,1999)

Tjio,J.H.,&Levan,A.Thechromosomenumberofman.Hereditas42,16(1956)

Trask,B.J.Humancytogenetics:46chromosomes,46yearsandcounting.NatureReviewsGenetics3,769778(2002)doi:10.1038/nrg905( link
toarticle)

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