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1、Medical Genetics18 疾病的产前诊断 Prenatal Diagnosis of DiseaseMedical GeneticsGeneticdiseasesandcongenitalmalformationsoccurinapproximately2to5%ofalllivebirths,accountforupto30%ofpaediatricadmissionstohospital,andareanimportantcauseofdeathundertheageof15years.Medical GeneticsFurthermore,thepsychologicalst
2、ressonfamilieswithchildrenwithseriousgeneticdisordersisincalculable.Untilgenetherapybecomesapracticalreality,theonlyoptionavailableforthecontrolofgeneticdiseaseisprenataldiagnosis.Medical Genetics1. Indications for prenatal diagnosisTheuseofprenataldiagnosisisdeterminedbybalancingtheriskofthebirthof
3、anabnormalchildagainsttheriskofaninvestigativeprocedure.Medical GeneticsThegeneralindicationsofprenataldiagnosisincludematernalageandtheresultsofnoninvasiveserumbiochemicalscreening.Medical GeneticsSpecificindicationsincludeapositivefamilyhistoryandthebirthofapreviouschildaffectedbyaparticulargeneti
4、cdisease.Medical Genetics2. Methods for obtaining fetal tissues for prenatal diagnosis2. Methods for obtaining fetal tissues for prenatal diagnosisToperformprenataldiagnosis,fetal-derivedtissuesmustfirstbeobtained.Allofthecommonlyusedmethodsthatyieldfetaltissuesareinvasive.Medical GeneticsA. Amnioce
5、ntesisAmniocentesisisthewithdrawalofamnioticfluidfromtheamnioticsacsurroundingthefetus.Forovertwodecadesthishasbeentheprimarytechniqueutilisedforthediagnosisoffetalgeneticdisorders.Medical GeneticsTraditionallyamniocentesishasbeenperformedaround15to16weeksgestation.Atthistimetheuterusiseasilyaccessi
6、bletoatransabdominalapproach,andasufficientvolumeofamnioticfluid(approximately200ml)existstopermit20to30mltobewithdrawnsafely.Medical GeneticsAmniocentesisisnormallyperformedasanoutpatientfacility.Anultrasoundexaminationisnormallydoneimmediatelybeforetheproceduretoevaluatefetalnumberandviability,per
7、formfetalbiometricmeasurements,establishplacentallocation,andestimateamnioticfluidvolume.Medical GeneticsAmniocentesis performed concurrently with ultrasound scanning.Medical GeneticsSafety of amniocentesisAnyprocedurethatinvolvespassingadeviceintoanorgan,especiallythepregnantuterus,carrieswithitris
8、ks;amniocentesisisnoexception.Medical GeneticsAmniocentesisinvolvespotentialdangertobothmotherandfetus.Seriousmaternalrisksarequitelowbutincludeamnionitiswhichcanleadtofetalloss,haemorrhageorinjurytoanintra-abdominalviscusandleakageofamnioticfluid.Medical GeneticsFetalrisksincludespontaneousabortion
9、,needlepunctureinjuries,placentalabruption,chorioamnionitis,pretermlabour,andamnioticbandformation.Medical GeneticsSeveralcontrolledstudieshavebeendonetoevaluatetherisksofamniocentesis.Thedataindicatethattheriskofpregnancylossattributabletoamniocentesismaybeashighas0.5%.Medical GeneticsIngeneral,ris
10、kswilldependon(1)theexperienceoftheobstetricianperformingtheprocedure;(2)clinicalcharacteristicsofaparticularcase(e.g.,presenceorabsenceofbiochemicalmarkersoffetalabnormality);(3)thequalityofthehigh-resolutionultrasoundutilised.Medical GeneticsEarly amniocentesisWithdevelopmentofhigherresolutionultr
11、asoundequipment,somecentreshavebegunofferingamniocentesisbefore15weeksgestation,usuallybetween10and14weeks.Themajorityofprocedureshavebeenperformedduringthe13thand14thweeksofgestation.Medical GeneticsThereisevidencethatearlyamniocentesisisassociatedwithahigherfetallossrateandamorefrequentoccurrenceo
12、fcertaincongenitalabnormalities.Medical GeneticsB. Chorionic villus sampling Chorionicvillussampling(CVS) istheonlytestedmethodforfirst-trimesterfetalgeneticdiagnosisthatiscurrentlyinclinicaluseandisusuallyperformedbetween9and11weeks.Medical GeneticsTheprocedureinvolvesthepassingofasamplinginstrumen
13、tintothechorion(developingplacenta).Agoodprocedureyieldsfrom10to25mgoftissuewhichisadequateforcytogenetic,enzymaticorDNAanalysis.Medical GeneticsThemainadvantageofCVSoveramniocentesisistheapplicabilityofCVSearlieringestation.Thisresultsinconsiderablyreducedsocial,emotionalandpsychologicalstressforth
14、ecouple.Medical GeneticsSafety of CVSMaternalcomplicationsincludebleedingandinfection.FetallossfollowingCVShasbeenreportedtobearound2%.TherearealsoreportsoflimbreductiondefectsininfantsborntomotherswhohavehadCVSbetween56and66daysofgestation.Medical GeneticsC. Fetal blood sampling (FBS)Fetalbloodcanb
15、esafelyanddirectlysampledfromapproximately18weeksgestationonwards.FBScanbeusedforbothdiagnosticandtherapeuticpurposes.Medical Genetics Indications for fetal blood samplingDiagnosticRapid karyotypingFetal anomaly on ultrasoundLate attending patients who require fetal karyotypingAlloimmunisationRhesus
16、Platelet antigensFetal infectionToxoplasmosisCytomegalovirus infectionGeneticHaemoglobinopathiesMetabolic disorders and enzyme deficienciesFetal well beingSevere intrauterine growth retardationTherapeuticTransfusionRed cell alloimmunisationTransplantationStem cellsMedical GeneticsFBSiscontraindicate
17、difthemotherissufferingfrominfectionsthatcanbetransmittedtothefetusbytheprocedure.ExamplesincludehumanimmunodeficiencyvirusandhepatitisBvirusinfection.Medical GeneticsSafetyofFBSMaternalcomplicationsfromFBSareuncommonbutincludeamnionitis,infection,rhesussensitisationandtransplacentalhaemorrhage.Medi
18、cal GeneticsFetallossratesfollowingFBShavebeenreportedtobeapproximately1%inseverallargeseries.Thepresenceofstructuralabnormalitiesorseveregrowthretardationofthefetusisassociatedwithamuchincreasedfetallossrate.Medical GeneticsOtherfetalcomplicationsincludeinfection,prematureruptureofmembranes,haemorr
19、hage,severebradycardiaandumbilicalcordthrombosis.Medical GeneticsD. Fetal biopsyAlthoughadvancesinmolecularandbiochemicalgeneticshavemadethediagnosisofmanyMendeliandisorderspossiblebyanalysisofamnioticfluidcellsorchorionicvilli,someconditionsstillrequiredirectanalysisoftissuesinwhichthedisorderisman
20、ifested.Tissueswhichhavebeensuccessfullybiopsiedincludefetalskin,liverandmuscle.Medical GeneticsSafetyoffetalbiopsyDuetotherelativelysmallnumbersperformedindifferentcentres,noprecisefiguresforthesafetyoffetalbiopsyisavailable.Medical Genetics3. Analytical methodsFollowingtheacquisitionoffetaltissues
21、,thesematerialsarethensubjectedtoanalysisusingavarietyoftechniques.Medical GeneticsA. Cell culture and conventional cytogenetics ThesearethemostcommonlyusedmethodsforthediagnosisofchromosomalaneuploidiessuchasDownsyndrome.Medical GeneticsB. Molecular cytogenetics using FISHFISHinvolvesthehybridizati
22、onofDNAprobesrepresentingaspecificchromosomeorchromosomalregiontotargetDNAsuchasmetaphasechromosomesorinterphasenuclei,wheretheprobebindstohomologoussequencesinthecell.Medical GeneticsUsingFISH,severalgroupshavedemonstratedthattrisomiessuchastrisomy21andtrisomy18canbedetectedinunculturedinterphasenu
23、cleiasthreepositivehybridisationsignalsratherthanthenormaltwo.Medical GeneticsThemainadvantageisspeed:thusresultsareavailablein24to48hourscomparedwiththe10to14daysmoretypicalofstandardculture-basedcytogeneticanalysis.Thistypeoftechnologycanbeappliedtofetalmaterialsobtainedfollowingamniocentesis,CVSo
24、rfetalbloodsampling.Medical GeneticsC. DNA-based techniquesThemainadvantageofDNA-basedtechniquesisthatanynucleatedfetalcellcanbeused.Techniqueswhichareusedincludethepolymerasechainreaction(PCR)andSouthernblotting.Medical GeneticsPCR-basedtechniquesallowarapiddiagnosistobemadeinseveralhours.Thesemeth
25、odscanbeusedfordirectmutationdetectionorlinkageanalysis.Thelattertypeofanalysisisneededwhentheexactmutationorgenecausingthediseaseisnotknown.Medical GeneticsMedical GeneticsGeneticdiagnosisisthencarriedoutbyanalysingDNAsequenceswithinthegeneitselforDNAlocicloselylinkedtoit.Ananalysabledifferenceorpo
26、lymorphismmustexistbetweenthedisease-carryingalleleandthenormalalleletodistinguishthem.Medical GeneticsMedical GeneticsMedical GeneticsD. Metabolic analysis of fetal tissuesFetaltissuesorfluidscanbesubjectedtoanalysistodetectthecharacteristicmetabolicorcellulardefectsofaninheritedmetabolicdisease.Me
27、dical GeneticsForthistypeofanalysistobecarriedout,thespecificenzymeormetaboliteofinterestmustbeexpressedinthefetaltissuessampled,andtherangeofnormalvaluesaswellastheassaysensitivityandreproducibilitymustbeestablishedwithinthetissueofinterest.Medical GeneticsAlthoughanincreasingnumberofinheritedmetab
28、olicdiseasesareamenabletodirectDNA-baseddiagnosis,enzyme-basedtechniquesarestillusefulinsituationswherethedisease-causinggenehasnotbeenidentifiedorwheretheprecisemutationisnotknown.Medical GeneticsE. Microarray Analysis Muchoftheexcitementtodaycentersongeneexpressionprofilingthatusesatechnologycalle
29、dmicroarrays.Medical GeneticsADNAmicroarrayisathin-sizedchipthathasbeenspottedatfixedlocationswiththousandsofsingle-strandedDNAfragmentscorrespondingtovariousgenesofinterest.Medical GeneticsAsinglemicroarraymaycontain10,000ormorespots,eachcontainingpiecesofDNAfromadifferentgene.Fluorescent-labeledpr
30、obeDNAfragmentsareaddedtoaskifthereareanyplacesonthemicroarraywheretheprobestrandscanmatchandbind.Completepatternsofgeneactivitycanbecapturedwiththistechnology.Medical GeneticsMedical Genetics4. New methods for prenatal diagnosis4. New methods for prenatal diagnosisA. Preimplantation diagnosisPreimp
31、lantationdiagnosisistheperformanceofprenatalgeneticanalysisonembryosoroocytespriortoimplantation.Medical GeneticsThistechnologyhastheadvantagethatitallowsprenataldiagnosistobecarriedoutmuchearlierthanexistingmethodssuchasamniocentesisandCVS.Medical GeneticsFurthermore,coupleswhoareatexceptionallyhig
32、hgeneticriskandthosewhohavehadpreviousterminationsforgeneticindicationsmayfindpreimplantationdiagnosisamoreacceptableformofprenataltesting.Medical GeneticsMedical GeneticsInthefuture,preimplantationdiagnosismayalsobeusedinconjunctionwithgenetherapy.Atpresent,giventheexpenseoftheprocedureandthesmalln
33、umberofcentresequippedtoperformthisformofdiagnosis,preimplantationdiagnosisisunlikelytobecomeastandardprocedureintheforeseeablefuture.Medical GeneticsAccess to oocyte and embryonic cellsPreimplantationgeneticanalysiscanbecarriedoneitherembryoniccellsoroocytes.Inthelatersituation,diagnosisiscarriedou
34、tevenpriortofertilisation.Individualoocytesareaspiratedandtheirpolarbodybiopsied.Medical GeneticsForpreimplantationdiagnosiscarriedoutonembryoniccells,theembryomaybefertilisedinvitroandthenindividualblastomeresbiopsied.Alternatively,theembryomaybefertilisedinvivoandthentheembryosareobtainedbyuterine
35、lavagefollowedbybiopsyandgeneticanalysis.Medical GeneticsForheterozygouswomencarryingonemutantandonenormalalleleofadisease-causinggene,intheabsenceofchromosomalcrossing-over,theaspiratedpolarbodycontainingamutantallelewouldindicatethattheprimaryoocytepronucleusiscarryingthenormalallele.Medical Genet
36、icsInbothsituations,onlytheembryosconfirmednottopossessthefulldisease-causinggenotypearethenimplantedbackintotheuterus.Medical GeneticsMedical GeneticsDiagnostic methodsPreimplantationdiagnosismaybeachievedusingPCR,FISHorbymeasurementofembryonicsecretoryproductssuchascertainenzymes.Medical GeneticsT
37、histypeofanalysishasbeencarriedoutsuccessfullyforthedeterminationoffetalsexfortheavoidanceofsex-linkeddisorderssuchasDuchennemusculardystrophyandhaemophiliaA,andforthediagnosisofsinglegenedisorderssuchascysticfibrosis,alpha-1-antitrypsindeficiency,Tay-Sachsdisease,fragileXandsicklecellanaemia.Medica
38、l GeneticsWorldwide,preimplantationdiagnosisofembryoshasbeenattemptedonover1,200invitrofertilisationcyclesin1997,withclinicalpregnancyresultedin20%.Noincreaseintheoccurrenceofabnormalitieshasbeenobservedintheliveborns.Medical GeneticsB. Noninvasive prenatal diagnosis using fetal cells isolated from
39、maternal bloodCircumstantialevidencethatfetalnucleatedcellsexistinmaternalperipheralbloodcanbetracedbackto1969.Medical GeneticsHowever,convincingproofoftheexistenceofthesecellshavetoawaitthedevelopmentofmolecularbiologicaltechniques,especiallythePCR.Medical GeneticsUsingthePCR,investigatorsareableto
40、demonstratethepresenceofcellspossessingfetalgeneticmarkerscirculatingintheperipheralbloodofpregnantwomen.Theisolationofthesecellsofferthepossibilityofanoninvasiveandsafemethodforprenataldiagnosis.Medical GeneticsFetal nucleated cell types in maternal bloodThreepopulationsoffetalnucleatedcellsarecurr
41、entlyknowntobepresentinmaternalperipheralblood:fetallymphocytes,trophoblastsandfetalnucleatedredcells.Medical GeneticsAtpresent,theisolationoffetalnucleatedredcellsisreceivingthemostattentionfrominvestigatorsinthefieldduetotheavailabilityofrelativelyspecificmonoclonalantibodiesagainstthesecells.Medi
42、cal GeneticsIsolation of fetal cells and genetic analysisAcombinationofphysicalandimmunologicalmethodsareusedtoisolatefetalnucleatedcellsfrommaternalblood.Physicalmethodsincludedensitygradientcentrifugationandmicromanipulationtechniqueswhileimmunologicalmethodsincludetheuseofmonoclonalantibodies.Med
43、ical GeneticsGeneticanalysisoftheseisolatedfetalcellscanbeperformedusingPCRorFISH.Fetalcellsinmaternalbloodhavebeensuccessfullyusedonaresearchleveltodiagnosetrisomy21,trisomy18,beta-thalassaemiaandsicklecellanaemia.Actualclinicalusewillhavetoawaitfurthertechnologicaldevelopmenttoimproveitsreliabilityandclinicaltrialstoassessthesensitivityandspecificityofthisapproach.
