chapter_2 - Detectinggeneticalterations clinicalcontext...

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Unformatted text preview: Detectinggeneticalterations clinicalcontext Demonstrating the presence of a genetic defect in a patient with a presumed genetic condition is of major importance. It allows to accurately diagnose a particular condition, which offers several advantages for the patient andhisorherrelatives: estimatingtherecurrenceriskfortheparents offeringthepossibilitytoperformprenatalorpreimplantationdiagnosis performingpresymptomaticgenetictestingtounaffectedfamilymembers evaluatingtheexpected(future)diseasemanifestations The implications towards treating the condition remain insufficient. While for a number of inborn errors of metabolism,enzymereplacementtherapyisavailable,thecurrentpotentialofgenetherapyisextremelylimited. tissuesforgeneticanalysis MostmolecularanalysesareperformedonPCRamplifiedgeneticmaterialisolatedfromvarioustissues. AwidevarietyofsamplescanbeusedasasourceforgenomicDNA: blood: prelevated in tubes containing EDTA as anticoagulation agent remains the predominant source for routinegenetictesting buccalcells:noninvasiveprelevation,yieldsalimitedamountofDNA(suitableformutationdetection,usually notformutationscanning) chorionic villi and amniocytes: requires an invasive biopsy, suitable source for prenatal testing early in the pregnancy(1112weeksforchorionicvillisamplingand1516weeksforamniocentesis) embryonicbiopsy:DNAsourceinthecontextofpreimplantationdiagnosis cancercells:tumourbiopsiesorbonemarrowsamples(note:DNAextractedfromparaffinembeddedtissueis degradedandissuitedforlimitedanalysesonly,suchasmutationdetectionormicrosatellitephenotypingsee below) fibroblastculturesandEBVtransformedcelllines:vitalcells,importantsourceforRNAanalysesandfunctional studies Guthriecard:eachnewbornisscreenedforalimitednumberofmetabolicdiseasesfortheseanalysesadried bloodspotonacardisused;DNAcanbeextractedfromthedriedbloodforfurtheranalyses hair,sperm:forensicapplications Althoughlesscommonlyused,RNAisaninvaluablesourceforgenetictesting.Amajoradvantageresidesinthefact that RNA has a reduced complexity as compared to DNA. Sequencing RNA (usually cDNA) often requires fewer fragmentstobeinvestigated,asintronsarenolongerretainedinthesetranscripts.Inaddition,thefunctionaleffectof apresumedsplicingmutationcanbeevaluatedeasily.Also,aberrantsplicingasaresultofadeepintronicmutation can be detected. Disadvantages of working with RNA are related to its rapid degradation. RNA has to be extracted fromfreshsamples(bloodorcellcultures),manipulationsneedtobestrictlyRNasefree.Inaddition,mutantRNAis often instable as it is selectively degraded by NMD (see above). As a consequence, if no stabilising agents (such as puromycin) are added, only the wild type transcript will be available for analyses and pathogenic mutations can be missed. Finally,aprerequisite for performing RNA analyses is that the geneofinterest isexpressedinthe available tissue. conventionalcytogenetics:karyotyping In spite of the introduction of highly specialised technologies in the diagnostic laboratory, karyotyping currently remainsanextremelyimportanttechnique.Theprocedureforkaryotypingincludesashorttermcultureafterwhich metaphasespreadsareobtainedandstained.Dependingonthestainingprocedure,G,QorRbandedchromosomes canbevisualisedmicroscopically. Allowing an evaluation of the entire genome represents one of the major advantages of conventional cytogenetics. Compaction of chromatin at the metaphase stage, however, limits its sensitivity to detecting rearrangements comprising minimally several megabases. In addition, the procedure is quite time consuming and laborious and requires the presence of dividing cells in order to obtain chromosomes. Furthermore, in complex chromosomal rearrangements it is often difficult or even impossible to provide an accurate description of the karyotypic aberrations,whilebalancedrearrangements,resultingintheexchangeofsimilarorverysmallchromosomalregions (i.e.crypticaberrations)bydefinitionremainunnoticedinaconventionalcytogeneticevaluation. Indicationsforkaryotypinginconstitutionalgeneticsinclude: unexplainedmentalretardation congenitalmalformations,dysmorphicfeatures miscarriage,morsinuteroorneonataldeath pregnancyadvancedmaternalage reducedfertility segregationofabalancedtranslocationinthefamily disturbedpubertaldevelopment Inthecontextofmalignantproliferationskaryotypingisfrequentlyperformedin: molecularcytogeneticanalyses:FISH,mFISH,CGHandarrayCGH FluorescentinsituhybridizationisessentiallybasedontheintrinsiccapabilityofsinglestrandedDNAprobestoanneal tocomplementarysequences,presentinthegenomeunderinvestigation.TheDNAprobesusedinFISHarelabelled with a reporter molecule that fluoresces when excited at a particular wavelength. This fluorescent signal can be visualizedusingafluorescencemicroscopeequippedwithadaptedfilters. FISHisextremelyusefulforanalyzinggenomicrearrangementsinmetaphasespreadsaswellasinterphasenuclei.In addition, clones covering the entire genome (i.e. BAC and PAC clones used in the human genome project) are hematologicalneoplasms(leukemia,lymphoma,...) solidtumours available, allowing an accurate detection of specific chromosomal regions and genes. FISH overcomes some of the limitationsofconventionalkaryotyping.Firstofall,thepresenceofdividingcellsisnolongerrequiredasinterphase nuclei can serve as extremely useful targets for FISH approaches. In addition, cryptic aberrations can readily be diagnosedusingappropriateprobecombinations.Finally,giventhespecificityoftheannealingprocess,FISHishighly genespecific:whereaskaryotypingcansuggesttheinvolvementofaspecificgene,FISHcanunequivocallyconfirmor refuteinvolvementofageneoraspecificregioninaparticulargenomicaberration. The high specificity of FISH using gene or chromosome region specific probes, however, also represents a major limitationandprecludesitsuseintheanalysisofcomplexchromosomalrearrangements.Inordertoovercomethese restrictions,severalinnovativetechniques,includingmultiplexFISH(MFISH)spectralkaryotyping(SKY)andCombined Binary Ratio Labelling (COBRA), were developed. These techniques allow to assign separate colours to all individual humanchromosomesandthereforemadeanalysingcomplexchromosomalaberrationsafeasibleobjective. Comparative genome hybridisation (CGH) is a variant of FISH: instead of hybridizing probes to metaphase spreads fromthepatient,thetestgenome(e.g.constitutionalDNAofapatientorDNAfrommalignantcells)andareference genome are differentially labelled with fluorophores and hybridized onto normal control metaphase spreads. With dedicated software, a fluorescence ratio is calculated along the axis of each of the chromosomes. This ratio is a measurement for the relative copy number status of each region in the DNA of the patient or tumour sample compared to the reference DNA. This technique made it possible to detect chromosomal gains and losses in DNA sampleswherenokaryotypecouldbeobtained(e.g.whennovitalcellsareavailable). In 1997 a variant of CGH was developed: instead of hybridizing the test and reference DNA to normal metaphase spreads,amicroarrayofDNAprobesimmobilizedonaglassslideisused.Thefluorescenceratioofeachspotonthe arrayisagainameasurementfortherelativecopynumberstatusofthelocusofthatparticularprobeinthetestDNA comparedtothereferenceDNA.Despiteitsrelativelyearlyintroduction,ittookseveralyearsbeforethisarrayCGH methodology became more widely used. Array CGH is now widely applied due to the availability of large sets of validated clones (e.g. BAC sets developed for the Human Genome Project) and the availability of commercial platforms, increasing steadily in density and resolution. Apart from BAC clone tiling path arrays which attain a resolution up to 75 kb, short oligonucleotide sequences are increasingly being used allowing further increase of resolution. For smaller genomes or specific sequences, arrays have been developed which can even detect single basepair alterations. This new technique called `arraybased sequencing` makes it possible to resequence whole stretchesofDNAfor1500/Mb. moleculargeneticanalyses Threeelementarytechniquesareavailableformoleculargenetictesting: mutationdetection:evaluationofthepresenceorabsenceofalimitednumberofmutations,suitedonlyfor diseasewitharestrictedallelicheterogeneity(e.g.:mucoviscidosis,hemochromatosis,achondroplasia...). mutation scanning: screening a gene with a number of complementary techniques for a large number of possible(knownandunknown)mutations linkage:evaluatingthesegregationofasetoftightlylinkedmarkersinaffectedandunaffectedrelativesina pedigree mutationscanning The goal of mutation scanning is to identify a pathogenic mutation in a gene for a disease that is characterized by extensive allelic heterogeneity. Molecular genetic analyses in hereditary breast and ovarian cancer comprise an illustrative example of mutation scanning. In BRCA1 and BRCA2 several hundreds of different mutations have been reported. These mutations include splice site mutations, missense and nonsense mutations and deletions and insertions.Asaconsequence,ifwewishtoexploreahereditarycontributiontobreastandovariancancerinafamily, differenttechniques,thatenabledetectingthesedifferentmutations,havetobeused. Themostimportantmethodsusedinmutationscanningare: DNAsequencing Directsequencingremainsthegoldenstandardformutationscanning.Sequencingcanbeusedasascanningmethod, in addition, a large number of aberrant results obtained by other scanning methods need to be confirmed by sequencing in order to explore their nature. For instance: if the formation of heteroduplexes is documented with DGGE, sequencing will have to determine the nature of the difference between the two alleles. Currently, dideoxyterminationsequencing(Sangermethod)currentlyisthemostwidelyusedsequencingmethodintheclinical laboratory.Thehighcostcurrentlyremainsthemajordisadvantageofsequencing.Probably,however,sequencingwill moreandmorebecomethemethodofchoiceassequencingtechnologiesbecomemoreandmoreperformantandas pricesareexpectedtodropprogressively. detectingheteroduplexes DGGEanddHPLC(andcurrentlyalsohighresolutionmeltinganalysisHRM)arehighlysensitivetoolstodetectthe presence of small nucleotide changes and depend on the detection of heteroduplexes. Heterozygous PCR products (containing wild type and mutant amplicons) form heteroduplexes after denaturation and slow annealing. These heteroduplexeshavedifferentphysicalandchemicalcharacteristicsthatcanbedetectedinappropriateconditions. DGGEisbasedonthepropertythatpartiallydenaturedDNAmoleculesmigrateslowlyinagelelectrophoresis.When aPCRproductisplacedinanelectrophoreticfield,inagradientofadenaturingagent,itwillmigrateuntilitwillstart to denature. Denaturation is essentially dependent on the nucleotide content of the amplicons and therefore differentallelesandheteroduplexeswillshowadifferentmigrationpattern,visualisedasabandshift.DGGEishighly sensitive,cheapandcanbemultiplexed.Disadvantagesincludethefactthatonlyrelativelysmall(200500bp)canbe directsequencing denaturinggelelectrophoresis(DGGE) denaturinghighperformanceliquidchromatography(dHPLC) proteintruncationtest(PTT) Southernblot multiplexligationdependentprobeamplification(MLPA) quantitativePCR FISH analysed,thatitisagelbasedtechniqueandthatitrequiresconfirmationbysequencing. dHPLCisbasedonthesameprinciplesasDGGEbutusesadaptedchromatographycolumnstoseparatehomoand heteroduplexes.ItisequallysensitiveasDGGE,therelativelyexpensivecolumnsandinstrumentationformitsmajor disadvantages. NeitherDGGEnodHPLCprovideinformationonthenatureandpositionofthenucleotidealterationthatcausesthe formationofheteroduplexes:aberrantresultsalwaysneedtobeconfirmedbysequencing. proteintruncationtestPTT PTTisahighlysensitive,butlaboriousandradioactivetechniquetodetectthepresenceofanonsensemutationina PCRfragment.Inbrief,apeptideissynthesizedfromaPCRfragmentinvitro.Ifanonsensemutationispresentinthe amplicon, this will become evident by a truncation of the synthesized peptide, which can be detected by gelelectrophoresis.ThemainadvantagesofPTTresideinthefactthatlargeamplicons(upto1.5kb)canbescreened andthatthetechniquegivessome(althoughlimited)informationtothepositionofthemutation.Disadvantagesare: the laborious optimization, the fact that the procedure involves radioactive compounds, the inability to detect missensemutationsandthefactthattheprocedureislabourintensive.Inthepast,PTTwasthemethodofchoicefor screeningtumoursuppressorgenessuchasAPCandBRCA1andBRCA2.Currently,PTTanalysesaremoreandmore replacedbysequencingbasedapproaches.Note:ifPTTisappliedontheRNAlevel(cDNA)stabilisingagentsshouldbe added,otherwisenonsensemutationsmightbemissedduetoNMD. Southernblot&MLPA Whilesmallnucleotidedeletionsandinsertionswillbedetectedbydirectsequencing,itwillmisslargerones(partial andtotalgenedeletionsandduplications). Becauseofthefactthatitistimeconsumingandlaborious,Southernblotisabandonedprogressivelyandisgradually replacedbyMLPA,ahighlysensitivePCRbasedtechniqueforthequantificationofgene(orratherofallexonsofthe geneunderinvestigation)copynumber. MLPA depends on the annealing of specifically designed probes. The probes contain a gene specific sequence, (ensuring unique hybridisation), a stuffer sequence (enabling discrimination of the different probe sets after amplification) and universal adaptors (facilitating simultaneous amplification of all probe sets). After hybridisation, ligation and amplification, fragment analysis allows quantifying the copy number of all investigated sequences, identifying both deletions and copy number gain. In a routine set up, probe sets are designed for each exon of the investigatedgene.MLPAischeap,fastandhighlysensitive.Inaddition,itissuitableformultiplexing(upto40targets can be analysed simultaneously i.e. up to 40 exons of a gene can be analysed in one experiment). The major disadvantageforMLPAisthelimitedavailabilityofcommerciallydevelopedprobesets. mutationdetection Mutationdetectioncomprisesthetechniquesthatareusedtodetectspecificsequencealterations.Evidentlyapriori knowledge of the nature of the disease causing mutations is required. Mutation detection is routinely used for investigating disease with a limited allelic heterogeneity and for evaluating the presence or absence of a known mutationinunaffectedrelativesofapatientinwhichapathogenicmutationwasdemonstratedbymutationscanning (e.g.presymptomatictestingforapathogenicBRCA1mutation). Methodsusedformutationdetectionare: restrictiondigestionofPCRamplifiedDNA In selected cases, a mutation creates an alteration in the susceptibility to enzymatic restriction (creating a new or abolishingapreviouslypresentrestrictionsite).ThesetypesofalterationscanbedetectedbyamplificationoftheDNA sequence flanking the mutation and analysis of the fragment length of the amplicon following enzymatic digestion: PCRRFLP(PCRandrestrictionfragmentlengthpolyporphism). Thistechniqueisusedinthediagnosticlaboratoryforthemolecularevaluationof: allelespecificoligonucleotidehybridisation(ASO) InanASOexperiment,PCRfragmentsarehybridisedtoanallelespecificoligonucleotideprobeonareversedotblot oraDNAchip.Thelineprobeassay(LiPA)testformucoviscidosisformsanillustrativeexampleofahighlyperformant implementationofthisprocedure. evaluatingthesizeofexpandedrepeats Tripletrepeatdiseasesarealwayscausedbythesametypeofmutation,locatedinawellknownregionofthedisease associatedgene.Forthesetypesofgeneticdefects,mutationdetectionisthereforefeasible.Characterisationofthe sizeoftherepeatiseitherperformedbyfragmentlengthanalysisofthePRCproductorbySouthernblot.Southern blotisparticularlyusefulfordetectingextremelylargeexpansions,asthesecannotbeamplifiedbyPCR. linkageanalysis In linkage analyses, the segregation of haplotypes in family members (affected and unaffected) is evaluated. If the causativegeneorlocusforacertainconditionisknown,linkageanalysiscanbeappliedtoidentifythehaplotypethat isassociatedwiththepresenceofaparticularphenotype. Inatypicalanalysis,highlypolymorphicmarkers,linkedtothegenelocusaregenotyped.Themarkersusedareusually microsatellites:shortoligonucleotiderepeats(di,triandtetranucleotiderepeats).Thefactthatthesemicrosatellites arehighlypolymorphic,usuallyenablesdeterminingtheinheritancepatternandreconstructingthehaplotypesthey determine. Markers that have a different length in different relatives are called informative. The availability of a sufficient number of affected and unaffected relatives is a prerequisite for performing linkage analysis. Therefore linkagedependsonanaccurateclinicalanalysisofthedifferentrelativesanalysed. hereditaryhemochromatosis achondroplasia restrictiondigestionofPCRamplifiedDNA hybridisationofPCRamplifiedDNAtoallelespecificoligonucleotidesonadotblotorgenechip evaluationofthesizeofanexpandedrepeat ...
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