Medical Genetics18 疾病的产前诊断 Prenatal Diagnosis of DiseaseMedical GeneticsGeneticdiseasesandcongenitalmalformationsoccurinapproximately2to5%ofalllivebirths,accountforupto30%ofpaediatricadmissionstohospital,andareanimportantcauseofdeathundertheageof15years.Medical GeneticsFurthermore,thepsychologicalstressonfamilieswithchildrenwithseriousgeneticdisordersisincalculable.Untilgenetherapybecomesapracticalreality,theonlyoptionavailableforthecontrolofgeneticdiseaseisprenataldiagnosis.Medical Genetics1. Indications for prenatal diagnosisTheuseofprenataldiagnosisisdeterminedbybalancingtheriskofthebirthofanabnormalchildagainsttheriskofaninvestigativeprocedure.Medical GeneticsThegeneralindicationsofprenataldiagnosisincludematernalageandtheresultsofnoninvasiveserumbiochemicalscreening.Medical GeneticsSpecificindicationsincludeapositivefamilyhistoryandthebirthofapreviouschildaffectedbyaparticulargeneticdisease.Medical Genetics2. Methods for obtaining fetal tissues for prenatal diagnosis2. Methods for obtaining fetal tissues for prenatal diagnosisToperformprenataldiagnosis,fetal-derivedtissuesmustfirstbeobtained.Allofthecommonlyusedmethodsthatyieldfetaltissuesareinvasive.Medical GeneticsA. AmniocentesisAmniocentesisisthewithdrawalofamnioticfluidfromtheamnioticsacsurroundingthefetus.Forovertwodecadesthishasbeentheprimarytechniqueutilisedforthediagnosisoffetalgeneticdisorders.Medical GeneticsTraditionallyamniocentesishasbeenperformedaround15to16weeksgestation.Atthistimetheuterusiseasilyaccessibletoatransabdominalapproach,andasufficientvolumeofamnioticfluid(approximately200ml)existstopermit20to30mltobewithdrawnsafely.Medical GeneticsAmniocentesisisnormallyperformedasanoutpatientfacility.Anultrasoundexaminationisnormallydoneimmediatelybeforetheproceduretoevaluatefetalnumberandviability,performfetalbiometricmeasurements,establishplacentallocation,andestimateamnioticfluidvolume.Medical GeneticsAmniocentesis performed concurrently with ultrasound scanning.Medical GeneticsSafety of amniocentesisAnyprocedurethatinvolvespassingadeviceintoanorgan,especiallythepregnantuterus,carrieswithitrisks;amniocentesisisnoexception.Medical GeneticsAmniocentesisinvolvespotentialdangertobothmotherandfetus.Seriousmaternalrisksarequitelowbutincludeamnionitiswhichcanleadtofetalloss,haemorrhageorinjurytoanintra-abdominalviscusandleakageofamnioticfluid.Medical GeneticsFetalrisksincludespontaneousabortion,needlepunctureinjuries,placentalabruption,chorioamnionitis,pretermlabour,andamnioticbandformation.Medical GeneticsSeveralcontrolledstudieshavebeendonetoevaluatetherisksofamniocentesis.Thedataindicatethattheriskofpregnancylossattributabletoamniocentesismaybeashighas0.5%.Medical GeneticsIngeneral,riskswilldependon(1)theexperienceoftheobstetricianperformingtheprocedure;(2)clinicalcharacteristicsofaparticularcase(e.g.,presenceorabsenceofbiochemicalmarkersoffetalabnormality);(3)thequalityofthehigh-resolutionultrasoundutilised.Medical GeneticsEarly amniocentesisWithdevelopmentofhigherresolutionultrasoundequipment,somecentreshavebegunofferingamniocentesisbefore15weeksgestation,usuallybetween10and14weeks.Themajorityofprocedureshavebeenperformedduringthe13thand14thweeksofgestation.Medical GeneticsThereisevidencethatearlyamniocentesisisassociatedwithahigherfetallossrateandamorefrequentoccurrenceofcertaincongenitalabnormalities.Medical GeneticsB. Chorionic villus sampling Chorionicvillussampling(CVS) istheonlytestedmethodforfirst-trimesterfetalgeneticdiagnosisthatiscurrentlyinclinicaluseandisusuallyperformedbetween9and11weeks.Medical GeneticsTheprocedureinvolvesthepassingofasamplinginstrumentintothechorion(developingplacenta).Agoodprocedureyieldsfrom10to25mgoftissuewhichisadequateforcytogenetic,enzymaticorDNAanalysis.Medical GeneticsThemainadvantageofCVSoveramniocentesisistheapplicabilityofCVSearlieringestation.Thisresultsinconsiderablyreducedsocial,emotionalandpsychologicalstressforthecouple.Medical GeneticsSafety of CVSMaternalcomplicationsincludebleedingandinfection.FetallossfollowingCVShasbeenreportedtobearound2%.TherearealsoreportsoflimbreductiondefectsininfantsborntomotherswhohavehadCVSbetween56and66daysofgestation.Medical GeneticsC. Fetal blood sampling (FBS)Fetalbloodcanbesafelyanddirectlysampledfromapproximately18weeksgestationonwards.FBScanbeusedforbothdiagnosticandtherapeuticpurposes.Medical Genetics Indications for fetal blood samplingDiagnosticRapid karyotypingFetal anomaly on ultrasoundLate attending patients who require fetal karyotypingAlloimmunisationRhesusPlatelet antigensFetal infectionToxoplasmosisCytomegalovirus infectionGeneticHaemoglobinopathiesMetabolic disorders and enzyme deficienciesFetal well beingSevere intrauterine growth retardationTherapeuticTransfusionRed cell alloimmunisationTransplantationStem cellsMedical GeneticsFBSiscontraindicatedifthemotherissufferingfrominfectionsthatcanbetransmittedtothefetusbytheprocedure.ExamplesincludehumanimmunodeficiencyvirusandhepatitisBvirusinfection.Medical GeneticsSafetyofFBSMaternalcomplicationsfromFBSareuncommonbutincludeamnionitis,infection,rhesussensitisationandtransplacentalhaemorrhage.Medical GeneticsFetallossratesfollowingFBShavebeenreportedtobeapproximately1%inseverallargeseries.Thepresenceofstructuralabnormalitiesorseveregrowthretardationofthefetusisassociatedwithamuchincreasedfetallossrate.Medical GeneticsOtherfetalcomplicationsincludeinfection,prematureruptureofmembranes,haemorrhage,severebradycardiaandumbilicalcordthrombosis.Medical GeneticsD. Fetal biopsyAlthoughadvancesinmolecularandbiochemicalgeneticshavemadethediagnosisofmanyMendeliandisorderspossiblebyanalysisofamnioticfluidcellsorchorionicvilli,someconditionsstillrequiredirectanalysisoftissuesinwhichthedisorderismanifested.Tissueswhichhavebeensuccessfullybiopsiedincludefetalskin,liverandmuscle.Medical GeneticsSafetyoffetalbiopsyDuetotherelativelysmallnumbersperformedindifferentcentres,noprecisefiguresforthesafetyoffetalbiopsyisavailable.Medical Genetics3. Analytical methodsFollowingtheacquisitionoffetaltissues,thesematerialsarethensubjectedtoanalysisusingavarietyoftechniques.Medical GeneticsA. Cell culture and conventional cytogenetics ThesearethemostcommonlyusedmethodsforthediagnosisofchromosomalaneuploidiessuchasDownsyndrome.Medical GeneticsB. Molecular cytogenetics using FISHFISHinvolvesthehybridizationofDNAprobesrepresentingaspecificchromosomeorchromosomalregiontotargetDNAsuchasmetaphasechromosomesorinterphasenuclei,wheretheprobebindstohomologoussequencesinthecell.Medical GeneticsUsingFISH,severalgroupshavedemonstratedthattrisomiessuchastrisomy21andtrisomy18canbedetectedinunculturedinterphasenucleiasthreepositivehybridisationsignalsratherthanthenormaltwo.Medical GeneticsThemainadvantageisspeed:thusresultsareavailablein24to48hourscomparedwiththe10to14daysmoretypicalofstandardculture-basedcytogeneticanalysis.Thistypeoftechnologycanbeappliedtofetalmaterialsobtainedfollowingamniocentesis,CVSorfetalbloodsampling.Medical GeneticsC. DNA-based techniquesThemainadvantageofDNA-basedtechniquesisthatanynucleatedfetalcellcanbeused.Techniqueswhichareusedincludethepolymerasechainreaction(PCR)andSouthernblotting.Medical GeneticsPCR-basedtechniquesallowarapiddiagnosistobemadeinseveralhours.Thesemethodscanbeusedfordirectmutationdetectionorlinkageanalysis.Thelattertypeofanalysisisneededwhentheexactmutationorgenecausingthediseaseisnotknown.Medical GeneticsMedical GeneticsGeneticdiagnosisisthencarriedoutbyanalysingDNAsequenceswithinthegeneitselforDNAlocicloselylinkedtoit.Ananalysabledifferenceorpolymorphismmustexistbetweenthedisease-carryingalleleandthenormalalleletodistinguishthem.Medical GeneticsMedical GeneticsMedical GeneticsD. Metabolic analysis of fetal tissuesFetaltissuesorfluidscanbesubjectedtoanalysistodetectthecharacteristicmetabolicorcellulardefectsofaninheritedmetabolicdisease.Medical GeneticsForthistypeofanalysistobecarriedout,thespecificenzymeormetaboliteofinterestmustbeexpressedinthefetaltissuessampled,andtherangeofnormalvaluesaswellastheassaysensitivityandreproducibilitymustbeestablishedwithinthetissueofinterest.Medical GeneticsAlthoughanincreasingnumberofinheritedmetabolicdiseasesareamenabletodirectDNA-baseddiagnosis,enzyme-basedtechniquesarestillusefulinsituationswherethedisease-causinggenehasnotbeenidentifiedorwheretheprecisemutationisnotknown.Medical GeneticsE. Microarray Analysis Muchoftheexcitementtodaycentersongeneexpressionprofilingthatusesatechnologycalledmicroarrays.Medical GeneticsADNAmicroarrayisathin-sizedchipthathasbeenspottedatfixedlocationswiththousandsofsingle-strandedDNAfragmentscorrespondingtovariousgenesofinterest.Medical GeneticsAsinglemicroarraymaycontain10,000ormorespots,eachcontainingpiecesofDNAfromadifferentgene.Fluorescent-labeledprobeDNAfragmentsareaddedtoaskifthereareanyplacesonthemicroarraywheretheprobestrandscanmatchandbind.Completepatternsofgeneactivitycanbecapturedwiththistechnology.Medical GeneticsMedical Genetics4. New methods for prenatal diagnosis4. New methods for prenatal diagnosisA. Preimplantation diagnosisPreimplantationdiagnosisistheperformanceofprenatalgeneticanalysisonembryosoroocytespriortoimplantation.Medical GeneticsThistechnologyhastheadvantagethatitallowsprenataldiagnosistobecarriedoutmuchearlierthanexistingmethodssuchasamniocentesisandCVS.Medical GeneticsFurthermore,coupleswhoareatexceptionallyhighgeneticriskandthosewhohavehadpreviousterminationsforgeneticindicationsmayfindpreimplantationdiagnosisamoreacceptableformofprenataltesting.Medical GeneticsMedical GeneticsInthefuture,preimplantationdiagnosismayalsobeusedinconjunctionwithgenetherapy.Atpresent,giventheexpenseoftheprocedureandthesmallnumberofcentresequippedtoperformthisformofdiagnosis,preimplantationdiagnosisisunlikelytobecomeastandardprocedureintheforeseeablefuture.Medical GeneticsAccess to oocyte and embryonic cellsPreimplantationgeneticanalysiscanbecarriedoneitherembryoniccellsoroocytes.Inthelatersituation,diagnosisiscarriedoutevenpriortofertilisation.Individualoocytesareaspiratedandtheirpolarbodybiopsied.Medical GeneticsForpreimplantationdiagnosiscarriedoutonembryoniccells,theembryomaybefertilisedinvitroandthenindividualblastomeresbiopsied.Alternatively,theembryomaybefertilisedinvivoandthentheembryosareobtainedbyuterinelavagefollowedbybiopsyandgeneticanalysis.Medical GeneticsForheterozygouswomencarryingonemutantandonenormalalleleofadisease-causinggene,intheabsenceofchromosomalcrossing-over,theaspiratedpolarbodycontainingamutantallelewouldindicatethattheprimaryoocytepronucleusiscarryingthenormalallele.Medical GeneticsInbothsituations,onlytheembryosconfirmednottopossessthefulldisease-causinggenotypearethenimplantedbackintotheuterus.Medical GeneticsMedical GeneticsDiagnostic methodsPreimplantationdiagnosismaybeachievedusingPCR,FISHorbymeasurementofembryonicsecretoryproductssuchascertainenzymes.Medical GeneticsThistypeofanalysishasbeencarriedoutsuccessfullyforthedeterminationoffetalsexfortheavoidanceofsex-linkeddisorderssuchasDuchennemusculardystrophyandhaemophiliaA,andforthediagnosisofsinglegenedisorderssuchascysticfibrosis,alpha-1-antitrypsindeficiency,Tay-Sachsdisease,fragileXandsicklecellanaemia.Medical GeneticsWorldwide,preimplantationdiagnosisofembryoshasbeenattemptedonover1,200invitrofertilisationcyclesin1997,withclinicalpregnancyresultedin20%.Noincreaseintheoccurrenceofabnormalitieshasbeenobservedintheliveborns.Medical GeneticsB. Noninvasive prenatal diagnosis using fetal cells isolated from maternal bloodCircumstantialevidencethatfetalnucleatedcellsexistinmaternalperipheralbloodcanbetracedbackto1969.Medical GeneticsHowever,convincingproofoftheexistenceofthesecellshavetoawaitthedevelopmentofmolecularbiologicaltechniques,especiallythePCR.Medical GeneticsUsingthePCR,investigatorsareabletodemonstratethepresenceofcellspossessingfetalgeneticmarkerscirculatingintheperipheralbloodofpregnantwomen.Theisolationofthesecellsofferthepossibilityofanoninvasiveandsafemethodforprenataldiagnosis.Medical GeneticsFetal nucleated cell types in maternal bloodThreepopulationsoffetalnucleatedcellsarecurrentlyknowntobepresentinmaternalperipheralblood:fetallymphocytes,trophoblastsandfetalnucleatedredcells.Medical GeneticsAtpresent,theisolationoffetalnucleatedredcellsisreceivingthemostattentionfrominvestigatorsinthefieldduetotheavailabilityofrelativelyspecificmonoclonalantibodiesagainstthesecells.Medical GeneticsIsolation of fetal cells and genetic analysisAcombinationofphysicalandimmunologicalmethodsareusedtoisolatefetalnucleatedcellsfrommaternalblood.Physicalmethodsincludedensitygradientcentrifugationandmicromanipulationtechniqueswhileimmunologicalmethodsincludetheuseofmonoclonalantibodies.Medical GeneticsGeneticanalysisoftheseisolatedfetalcellscanbeperformedusingPCRorFISH.Fetalcellsinmaternalbloodhavebeensuccessfullyusedonaresearchleveltodiagnosetrisomy21,trisomy18,beta-thalassaemiaandsicklecellanaemia.Actualclinicalusewillhavetoawaitfurthertechnologicaldevelopmenttoimproveitsreliabilityandclinicaltrialstoassessthesensitivityandspecificityofthisapproach.