QF QF - - PCR in substitution of direct method for PCR in substitution of direct method for the analysis of the chorionic the analysis of the chorionic villi villi in prenatal in prenatal diagnosis: limits, advantages and valuations diagnosis: limits, advantages and valuations from an experience of 44727 prenatal from an experience of 44727 prenatal diagnoses on CVS diagnoses on CVS Francesca R. Grati, Francesca R. Grati, Ph.D Ph.D . . Research Research & & Development Development , cytogenetics, , cytogenetics, molecular molecular cytogenetics and cytogenetics and molecular molecular biology biology TOMA, Advanced Biomedical Assays, S.p.A. TOMA, Advanced Biomedical Assays, S.p.A. [email protected][email protected]
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QFQF--PCR in substitution of direct method for PCR in substitution of direct method for
the analysis of the chorionic the analysis of the chorionic villivilli in prenatal in prenatal
diagnosis: limits, advantages and valuations diagnosis: limits, advantages and valuations
from an experience of 44727 prenatal from an experience of 44727 prenatal
diagnoses on CVSdiagnoses on CVS
Francesca R. Grati, Francesca R. Grati, Ph.DPh.D..
ResearchResearch & & DevelopmentDevelopment, cytogenetics, , cytogenetics, molecularmolecular cytogenetics and cytogenetics and molecularmolecular biologybiology
�� Gold standard of Gold standard of fetalfetal karyotyping on karyotyping on chorionicchorionic villi (CVS)villi (CVS)
ItIt requiresrequires: :
�� a a complexcomplex cytogeneticcytogenetic lab lab organizationorganization
�� standardizationstandardization of of cytogeneticcytogenetic protocolsprotocols
�� expert expert operatorsoperators in in cellcell colturescoltures & & karyotypekaryotype analysisanalysis
SimoniSimoni, , TerzoliTerzoli e Rossella, 1990; e Rossella, 1990; SimoniSimoni e Rossella, 1986; e Rossella, 1986; VejerslevVejerslev and and MikkelsenMikkelsen, , 19891989
HahnemannHahnemann and and VejerslevVejerslev, 1997, 1997
PREMESSAPREMESSABACKGROUNDBACKGROUND
�� ThisThis combinedcombined approachapproach isis the the mostmost reliablereliable methodmethod and and withwith the best the best
��ItIt waswas notnot untiluntil 2001 2001 thatthat the first report of the first report of clinicalclinical applicationapplication of of thisthis
test test appearedappeared ((CiriglianoCirigliano etet al, 2001)al, 2001)
��Best Best practicepractice guidelinesguidelines havehave beenbeen developeddeveloped byby a a collaborationcollaboration
betweenbetween the the associationassociation of of ClinicalClinical CytogeneticsCytogenetics (ACC) (ACC)
(http://www.cytogenetics.org.uk/) and the (http://www.cytogenetics.org.uk/) and the ClinicalClinical MolecularMolecular GeneticsGenetics
Society (CMGS) (Society (CMGS) (http://www.cmgs.orghttp://www.cmgs.org), and a 2012 ), and a 2012 versionversion hashas beenbeen
ratifiedratified byby bothboth councilscouncils
�� CECE--IVD commercial IVD commercial kitskits are are nownow availableavailable fromfrom a a numbernumber of of differentdifferent companiescompanies
�� In In manymany labslabs, , assaysassays are are designeddesigned toto test test forfor bothboth sex sex chromosomechromosome
aneuploidyaneuploidy and the and the viableviable trisomiestrisomies, and , and thusthus, , autosomeautosome and sex and sex
chromosomechromosome markersmarkers are are multiplexedmultiplexed togethertogether..
�� In the majority of UK NHS In the majority of UK NHS laboratorieslaboratories, a , a
separate sex separate sex chromosomechromosome assayassay isis appliedapplied onlyonly in in
a a minorityminority of of casescases referredreferred withwith anan indicationindication of of
TypeType of CPM or TFMof CPM or TFMNo UPD/No of No UPD/No of
studiedstudied casescasesChromosomeChromosome
AbnormalitiesAbnormalities
TOTAL CASES INVOLVING
IMPRINTED CHR230
UPD CASES 5
FREQUENCY (%) 2.17
UPD CONFIRMATIONS
[2] False negative [2] False negative casescases due due toto MCCMCC
�� 15.025 15.025 monitoredmonitored diagnosesdiagnoses on CVS : 135 on CVS : 135 casescases withwith MCC in male sex LTC MCC in male sex LTC
(1.8%)(1.8%)
N Incidence (%)
Total Monitored cases
(Jan2009 - May2011)15.025 //
Total cases w MCC
(XX cell line in a male sample)135x2 1.80%
Cases with Dir XY and LTC w MCC
(2-90% XX cell line) 128x2 1.7% (~1:60)
Cases with Dir XY and LTC w MCC
(100% XX); 5*x2 0.06% (~1:1600)
Only LTC w MCC (100%); 2**x2 0.026% (~1:3800)
��In 7/135 the In 7/135 the karyotypekaryotype fromfrom LTC LTC waswas femalefemale
ID sample Dir (# XY metaphases)LTC (# XX
metaphases)
QF-PCR on
native DNA
1* 16 20 Disomico,XY
2* 15 20 Disomico,XY
3* 16 12 Disomico,XY
4* 22 50 Disomico,XY
5* 15 50 (1met 46,XY) Disomico,XY
6** // 26 Disomico,XX
7** // 20 Disomico,XX
�� MCC1MCC1-->5: QF>5: QF--PCR PCR appliedapplied on DNA on DNA fromfrom direct CVS direct CVS showedshowed a a normalnormal disomicdisomic
male pattern male pattern unmaskingunmasking, , asas the STC the STC methodmethod doesdoes, the MCC, the MCC
�� MCC6MCC6--7: STC 7: STC withoutwithout spontaneousspontaneous metaphasesmetaphases ((probablyprobably due due toto the the presencepresence of of
the decidua the decidua onlyonly) and the ) and the followingfollowing US US investigationsinvestigations showedshowed a male a male fetusfetus
�� The The retrospectiveretrospective MCC MCC comparingcomparing the the fetalfetal and and maternalmaternal allelesalleles on DNA on DNA fromfrom
direct CVS direct CVS showedshowed a a normalnormal disomicdisomic fenalefenale pattern pattern corrispondingcorrisponding toto the the maternalmaternal
componentcomponent indicatingindicating a a substantialsubstantial MCCMCC
1) The 1) The failurefailure of the STC of the STC combinedcombined withwith a a femalefemale karyotypekaryotype in LTC in LTC
couldcould bebe the the ““alarmalarm bellbell”” of the of the presencepresence of a massive MCC of a massive MCC thatthat shouldshould
bebe investigatedinvestigated byby a MCC a MCC esclusionesclusion test test
2) 2) WithWith a routine a routine approachapproach QFQF--PCR+LTC the MCC PCR+LTC the MCC
esclusionesclusion test test withwith the the comparisoncomparison of the of the fetalfetal and and
maternalmaternal allelesalleles isis notnot performedperformed. In case of . In case of disclosuredisclosure
of a of a femalefemale fetusfetus byby sonographicsonographic investigationsinvestigations, the MCC , the MCC
wouldwould havehave beenbeen undetectedundetected
P (false negative) due P (false negative) due toto MCC: ~1/7500 MCC: ~1/7500
3) 3) PreferencePreference of LTC: of LTC:
P (false P (false resultresult) due ) due toto MCC=MCC=
1/1100 (7x2/15025);1/1100 (7x2/15025);
4) MCC 4) MCC esclusionesclusion isis advisableadvisable withwith the the comparisoncomparison of of fetalfetal and and
maternalmaternal allelesalleles on DNA on DNA fromfrom culture media culture media
PoorPoor amountamount CVSCVS
[2] False negative [2] False negative casescases due due toto MCCMCC
3) 3) ConfirmationsConfirmations on AFon AF
�� EachEach abnormalabnormal resultresult byby QFQF--PCR (PCR (mainlymainly in in casescases withoutwithout US US
abnormalitiesabnormalities) ) albeitalbeit in in combinationcombination withwith anan abnormalabnormal non non mosaicmosaic
mesenchymemesenchyme hashas toto bebe confirmedconfirmed byby amniocentesisamniocentesis
�� WithoutWithout a a cytogeneticcytogenetic resultresult on on cytotrophoblastcytotrophoblast, QF, QF--PCR PCR cannotcannot toto
discriminate a discriminate a homogeneoushomogeneous trisomytrisomy fromfrom high high levellevel trisomytrisomy (>50%) (>50%)
(CPM (CPM typetype II & III and TFM II & III and TFM typetype V & VI) V & VI)
ThisThis conditioncondition happenshappens forfor cr13, 18, 21 in ~1/2600 cr13, 18, 21 in ~1/2600
�� BiologistBiologist//SupervisorSupervisor ((electrpherogramelectrpherogram interpretationinterpretation-->>reportingreporting): 3 working ): 3 working
weeksweeks
CytogeneticCytogenetic staff training staff training requiresrequires 55--fold time fold time comparedcompared
toto QFQF--PCR staff trainingPCR staff training
CONCLUSIONSCONCLUSIONS
4) 4) QFQF--PCR staff training requires much less time than PCR staff training requires much less time than
cytogenetic staff trainingcytogenetic staff training
3) 3) ConsideringConsidering a a horizontalhorizontal organizationorganization, , QFQF--PCR CEPCR CE--IVD (not IVD (not
homehome--brew) and STC has similar to the cost/sample and brew) and STC has similar to the cost/sample and
identical TATidentical TAT
1) QF1) QF--PCR+LTC PCR+LTC comparedcompared toto STC+LTCSTC+LTC in CVS in CVS analysisanalysis
associatedassociated toto a a cumulative cumulative riskrisk of false negative of false negative resultresult of of
~1/2000~1/2000 mainlymainly due due toto the TFM the TFM typetype IV, UPD IV, UPD conditionsconditions
associatedassociated toto imprinting imprinting syndromessyndromes in the in the fetusfetus and and toto
erroneouserroneous diagnosisdiagnosis consequentconsequent toto MCC. MCC.
��The The differentlydifferently labelledlabelled productsproducts are are separatedseparated byby gel gel electrophoresiselectrophoresis whichwhich resolvesresolves
the allele the allele productsproducts on the on the basisbasis ofof the allele the allele sizesize//numbernumber ofof repeatsrepeats