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7/23/2019 NIPT - BeSHG 2014
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NIPTNon-Invasive Prenatal Testing
Ben Caljon, NGS platform (UZ Brussel, VUB, UB!
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NIPT - Non-invasive prenatal testing2 09-05-2014
"vervie#
Summar$%intro&u'tion on invasive testing istor$ of prenatal &iagnosis
Current first trimester s'reening strateg$ T$pes of Invasive Prenatal )iagnosis (IP)!
*ssessing IP) results
Non-invasive testing )ete'tion of 'f)N*
NIPT met+o&ologies
NIPT te'+niue (o et al! Claime& a''ura'$
In&i'ations an& limitations
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Invasive testing
Summar$%intro&u'tion
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istor$ (!
Prenatal &iagnosis ./0 to earl$ .10 2 a&van'e& maternal age
(345$! 6%4 of )o#n s$n&rome pregnan'ies &iagnose&
78 of IP) +a& '+romosomal a9normalities
NIPT - Non-invasive prenatal testing4 09-05-2014
:orris ;<, :utton )=, *l9erman => ?evise& estimatesof t+e maternal age spe'ifi' live 9irt+ prevalen'e of
)o#n@s s$n&rome> ; :e& S'reen> 7007A.(!27-
)ata from =nglan& an& ales in t+e perio& of ..0-..1
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istor$ (7!
ate .10, earl$ ..0 2 intro&u'tion ofse'on&-trimester maternal serum marDers
Ca> 7%4 of )o#n s$n&rome pregnan'ies &iagnose& E8 of IP) +a& '+romosomal a9normalities
NIPT - Non-invasive prenatal testing5 09-05-2014
Marker Full name Origin Tr 21
*FP alp+a-fetoprotein
olD sa'%fetalliver
H(:o: 2 0,/4!
CG 9eta-+uman'+orioni'gona&otropin
Trop+o9last (:o: 2 7!
U=4 un'onjugate&estriol
Feto-pla'entalunit
H(:o: 2 0,/4!
:arDer levels var$ #it+ gestation #eeD
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istor$ (4!
CompleJ &ata anal$sis Ne'essar$ to use :o: (multiple of me&ian! values Nee& for a''urate population parameters Nee& for liDeli+oo& ratio &istri9ution Nee& for 'orre't 'urve fitting for me&ians of t+e marDers Nee& for 'orre'tion for ot+er fa'tors su'+ as maternal #eig+t Soft#are pre&i'tion tools availa9le
NIPT - Non-invasive prenatal testing6 09-05-2014
s pregnancienormalvalue Median
valuePatient MoM =
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istor$ (E!
ate ..0, earl$ 7000 2 'om9ine& first-trimestertesting
Ca> .08 of )o#n s$n&rome pregnan'ies &iagnose& 8 of IP) +a& '+romosomal a9normalities
NIPT - Non-invasive prenatal testing7 09-05-2014
Marker Full name Origin Tr 21
CG 9eta-+uman '+orioni'
gona&otropin
Trop+o9last
(:o: 2 ,1!
P*PP-* Pregnan'$-asso'iate&plasma protein *
trop+o9last an& &e'i-&ualiKe& en&ometrialstroma 'ells
H(:o: 2 0,E!
Ultrasoun& measurement
NT Nu'+al translu'en'$t+i'Dness
(:o: 2 7-7,5!
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istor$ (5!
NIPT - Non-invasive prenatal testing8 09-05-2014
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istor$ (!
Normal
NIPT - Non-invasive prenatal testing9 09-05-2014
Trisom$ 7
)isa&vantage 2 inter-operator &ifferen'es *&vantage 2 useful in t#ins, elevate& in ot+er trisomies
Flui& fille& spa'e ('ar&ia' failure, stru'tural malformation,&ela$e&%a9normal &evelopment of l$mp+ati' s$stem!
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istor$ (/!
Fa'tors influen'ing st trimester testing 2
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Current first trimester s'reening
Curtos$ )r> *nnieD Vorsselmans 2
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Prenatal testing L Invasive (!
C+orion Villus Sampling(CVS! 0-4 #eeDs after gestation
Transa9&ominal orTrans'ervi'al (&epen&ing ona''ess to pla'enta%sDillsp+$si'ian!
?isD 2
Vaginal 9lee&ing%spotting C+orioamnionitis Fetal loss2 %700
Pro 2 ?esults are availa9le earlier
in pregnan'$ ess parental anJiet$ =arlier%safer met+o&s for
pregnan'$ termination
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Prenatal testing L Invasive (7!
*mnio'entesis ME #eeDs after gestation
(earlier possi9le 9ut +ig+er risD!
?isD Transient vaginal spotting2
-78 *mnioti' flui& leaDage2 -
78
C+orio-amnionitis2 6 0>8 Nee&le injuries to t+e fetus
are rare #+enultrasonograp+i' gui&an'eis use&>
Fetal loss2 %700(overestimate!
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Prenatal testing L Invasive (4!
Cor&o'entesis M/ #eeDs after gestation
(mostl$ M 70 #eeDs!
"nl$ if CVS or *C not possi9le ?isD (similar to *C!
Transient vaginal spotting2-78
*mnioti' flui& leaDage2 -
78 C+orio-amnionitis2 6 0>8 Nee&le injuries to t+e fetus
are rare #+enultrasonograp+i' gui&an'eis use&>
Fetal loss2 %700
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*ssessing invasive results (!
Preparation of samples CVS 2 mi'ros'opi' separation
of maternal tissue from villi &isse'tion of &e'i&ua
Centrifugation of 'or& 9loo&(serum%plasma for9io'+emistr$, BC for)N*%9io'+emi'al!
C+e'D for maternal 'ell'ontamination (:C! Via fragment anal$sis
(Po#erPleJ S!
)epen&ent on anal$sis 2 put
*C%CVS 'ells in 'ulture ?isD for overgro#t+ #it+
maternal 'ells
NIPT - Non-invasive prenatal testing15 09-05-2014
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*ssessing invasive results (7!
=Jample :aternal Cell Contamination
NIPT - Non-invasive prenatal testing16 09-05-2014
Prenatal sample
Mother
Father
For 9io'+emistr$ anal$sis, :C&ete'tion is more &iffi'ult 2 'omparisonin enK$mati' a'tivities &e'i&ua%villi• O-glu'uroni&ase• O-+eJosamini&ase
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*ssessing invasive results (4!
Bio'+emistr$ anal$sis "nl$ possi9le if enK$me is eJpresse& in t+e
anal$Ke& tissue (*C%CVS%'or& 9loo&! Po#er 2 one assa$ for man$ mutations
:easure enK$mati' a'tivities via artifi'ialsu9strates (fluorimetri' &osage over time!
C$togeneti's CVS 2
)ire't%<T
T
*C
NIPT - Non-invasive prenatal testing17 09-05-2014
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*ssessing invasive results (E!
<ar$ot$pe
M5 :9 &up%&el
NIPT - Non-invasive prenatal testing18 09-05-2014
FIS
*C lo#er error rates t+an CVS 2• Confine& Pla'ental :osai'ism (CP:!• :C• Ban&ing resolution
F-PC? :P* aCG (3 SNP
arra$!
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*ssessing invasive results (5!
:ole'ular )N* anal$sis
i&e range of mole'ular test%te'+niues 'an
9e applie& if )N* is availa9le ?estri'tion &igestion
Sout+ern 9lotting
Fragment anal$sis Seuen'ing
Q
NIPT - Non-invasive prenatal testing19 09-05-2014
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Non-invasive testing
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)ete'tion of 'f)N*
)ete'tion of 'ell-free fetal )N*in maternal plasma in ../
S+e&&ing of trop+o9last 'ells mi'roparti'les of fragmente& )N* maternal 9loo&stream s+ort+alf life (7 + 'learan'e! Pro 2 multigravi& pregnan'$
:e&ian prevalen'e of 48 (real timePC?! to 087 (&igital PC?! in st
an& 7n& trimester ?elia9le &ete'tion from -7
#eeDs on In'reasing 8 &uring 7 an& 4r&
trimester
NIPT - Non-invasive prenatal testing21 09-05-2014
> o :), Cor9etta N, C+am9erlain PF, ?ai V, Sargent I, et al> (../! Presen'e of fetal )N* inmaternal plasma an& serum> an'et 4502 E15LE1/>
7> un F:F, C+iu ?<, C+an <C*, eung T, au T<, o :)> 7001 :i'roflui&i's &igital PC? reveals a+ig+er t+an eJpe'te& fra'tion of fetal )N* in maternal plasma> Clin> C+em> 5E, EL/7>
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Intro&u'tion - :e&ia
NIPT - Non-invasive prenatal testing22 09-05-2014
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing23 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
• Examination of differentiallyexpressed genes (trophoblastvs. Maternal RNA)
• Currently beinginvestigated.
• Proof-of-principle wassuccessful
• Clinical trial disappointing
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing24 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
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NIPT L )igital PC?
*9solute uantitation of non-pol$morp+i' '+r (referen'e!an& '+r7 marDer>
)ifferen'e in uantitation
allo#s for T7 &ete'tion *&vantage 2
=ase of use C+eap
)isa&vantage 2
"nl$ limite& amount of marDersto 9e assesse& in eJperiment Fetal fra'tion &etermines
'ompleJit$ (num9er ofR#ells%'ounts! nee&e& forsuffi'ient statisti'al po#er> For78, 7701 'ounts are nee&e&,#+i'+ is 9e$on& 'urrent'ommer'ial te'+nolog$
NIPT - Non-invasive prenatal testing25 09-05-2014
Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK,Leung TY, Zee BC, Cantor CR, Chiu RW. Digital PCR for themolecular detection of fetal chromosomal aneuploidy. Proc
Natl Acad Sci U S A. 2007 Aug 7;104(32):13116-21.
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing26 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
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NIPT L PC?
)ifferent arra$ of met+o&s,mostl$ 9ase& on &ifferentiall$met+$late& regions ():?s! :et+$lation )epen&ent
Immuno-Pre'ipitation (:e)IP-PC?!
:et+$lation Spe'ifi' PC? (:SP!
*&vantage 2 C+eap on 'onsuma9les
)isa&vantage 2 Sele'tion%optimiKation of test
is te&ious (sele'tion of ):?s! :ultiple ):?s nee&e& to
o9tain suffi'ient statisti'al
po#er Not +ig+l$ multipleJa9le a9orious
NIPT - Non-invasive prenatal testing27 09-05-2014
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing28 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
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NIPT L SNP 9ase& approa'+es (!
"ffere& 9$ 'ompan$ Natera Commer'ial name 2 Panorama N*TUS te'+nolog$ NeJt generation *neuploi&$ Testing
Using SNPs Using NGS, 000 SNPs are &etermine& #it+in t+e 'f)N* for
'+romosomes 4,1,7, an& PC? amplifi'ation of pol$morp+i' regions Seuen'ing of ampli'ons using NGS &evi'e
Base& on t+e SNP genot$ping (9ot+ plasma as 9uff$ 'oat!,
t+e N*TUS algorit+m performs a Rper '+romosome C an&esta9lis+es fetal '+romosomal anomalies using a Ba$esian-9ase& maJimum liDeli+oo& statisti'al met+o&
NIPT - Non-invasive prenatal testing29 09-05-2014
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NIPT L SNP 9ase& approa'+es (7!
*&vantages ig+ a''ura'$ (also for seJ
'+romosomes!
*9ilit$ to &ete't pol$ploi&$ )isa&vantage
Not usea9le for &onor-egg'ell pregnan'ies
"nl$ small 'o+ort stu&$pu9lis+e& (E5 samples!
In proof-of-prin'iple 2 7>8of samples +a& a Rno-'all&ue to poor )N* ualit$ (notpassing C filter!
Currentl$ in patent-'onfli't#it+ Seuenom
No information on ot+er'+romosomes
NIPT - Non-invasive prenatal testing30 09-05-2014
Zimmermann B, Hill M, Gemelos G, Demko Z, Banjevic M, Baner J, Ryan A, Sigurjonsson S, Chopra N, Dodd M, Levy B,Rabinowitz M. Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targetedsequencing of polymorphic loci. Prenat Diagn. 2012 Dec;32(13):1233-41.
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing31 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
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NIPT L t:PS (!
"ffere& 9$ 'ompan$*riosa )iagnosti's Commer'ial name 2
armon$ Prenatal Test S'ope 2 T4, T1, T7,
(optional! Te'+nolog$ 2 )*NS?
()igital *nal$sis of
Sele'te& ?egions! assa$an& F"?T= (fetal-fra'tionoptimiKe& risD of trisom$evaluation!
NIPT - Non-invasive prenatal testing32 09-05-2014
Sparks AB, Struble CA, Wang ET, Song K, Oliphant A.Noninvasive prenatal detection and selective analysisof cell-free DNA obtained from maternal blood:evaluation for trisomy 21 and trisomy 18. Am J ObstetGynecol. 2012 Apr;206(4):319.e1-9.
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NIPT L t:PS (7!
)*NS? 2 pro9es for 9ot+ pol$morp+i'(&etermine fetal fra'tion! as non-pol$morp+i'regions ('+romosome proportion
&etermination! F"?T= algoritm 2 'al'ulates an in&ivi&ualiKe&
o&& s'ore for trisom$, 9ase& on fetal fra'tion,'+romosome proportion &ata an& maternal age
NIPT - Non-invasive prenatal testing33 09-05-2014
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NIPT L t:PS (4!
*&vantages =as$ fetal fra'tion &etermination, using pro9es for pol$morp+i' regions No nee& for referen'e &ata (F"?T= normaliKes using intra-run &ata! 2 less
pro'ess &rift C+eaper, 9e'ause of +ig+er multipleJi9ilit$
)isa&vantages 2 False negative results +ave 9een o9taine& for %747 T7, 7%05 T4 an& 7%0
T4 No information on ot+er '+romosomes%limite& normaliKation 'apa9ilities
NIPT - Non-invasive prenatal testing34 09-05-2014
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NIPT - :et+o&ologies
NIPT - Non-invasive prenatal testing35 09-05-2014
NIPT
s-MPS(Shotgun Massive
parallel Sequencing)
Digital PCR
qPCRt-MPS(Targeted Massive
parallel Sequencing)
RNA basedapproaches
cfDNA based cfRNA based
SNP basedapproaches
Clinical utility
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NIPT L s:PS (!
"ffere& 9$ 'ompanies Seuenom an&
Verinata Commer'ial names 2 :aterniT7 Plus an&
Verifi resp>
Te'+nolog$ 2 see neJt sli&es S'ope
Seuenom 2 T7, T1, T4, % aneuploi&$,
T,T77 (an& Rspe'ifi' mi'ro&eletions! Verinata 2 T7, T1, T4, % aneuploi&$
NIPT - Non-invasive prenatal testing36 09-05-2014
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NIPT L s:PS (7!
Verinata (#e9site verinata!
NIPT - Non-invasive prenatal testing37 09-05-2014
N Sensitivit$
.58CI
Spe'ifi'it$ .58 CI
7 500M..>.8(.0%.0!
.-00>0
..>18(E0.%E0!
.1>/-00>0
1 50./>E8(4/%41!
1>7-..>.
..>8(E%E4!
.1>5-00>0
4 501/>58(E%!
>/-.1>5
M..>.8(E15%E15!
..>7-00>0
: 501 .5>08(.%70!
/5>-..>.
..>08(E14%E11!
./>-..>/
N Sensitivit$ .58 CI Spe'ifi'it$ .58 CI *''ura'$
.58 CI
501./>8(7E4%7E.!
.E>1-
..>
..>78(75/%75.!
./>7-
..>..1>E8
.>.-
..>4
501..>8(77/%77.!
.>.-
..>.
.1>.8(7/%7/.!
.>.-
..>1..>08
./>/-
..>/
Seuenom (#e9site verinata!
?esults of pu9lis+e& 'lini'altrials #ill 9e &is'usse& later
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NIPT L s:PS (4!
+$ s:PS 2 False positive%negative rates &e'rease over time (mainl$ 9$ improve&
9ioinformati's algorit+ms! No test is perfe't (t:PS, s:PS nor SNP 9ase&!
- Numerous fa'tors responsi9le for test failure % altere& )?s, 9ut mainl$ &ue to
lo# 8 fetal )N* Natera test +as not 9een #i&el$ vali&ate&
*ppli'a9le to egg 'ell &onor pregnan'ies o#est result failure (retest%ne# sample nee&e&!
Generi' test 2
=Jpan&a9le to ot+er '+romosomes Possi9ilit$ to pi'D up su9'+romosomal alterations (if seuen'ing 'osts
&e'reases! Possi9ilit$ to &etermine fetal SNVs (if seuen'ing 'osts &e'reases! Possi9ilit$ to &etermine pol$ploi&$ using SNVs (if seuen'ing 'osts &e'reases!
NIPT - Non-invasive prenatal testing38 09-05-2014
+
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"vervie# NIPT te'+niue
NIPT - Non-invasive prenatal testing39 09-05-2014
1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT + i P+l 9 (!
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NIPT te'+niue L P+le9otom$ (!
:aternal plasma 3 )N* isolation
Colle'tion of maternal perip+eral9loo& (from 7 #eeDs gestation! in0 ml =)T* tu9es (Stre'D tu9es!#it+ a proprietar$ sta9iliKingagent
In+i9its g)N* release fromnu'leate& 'ells
In+i9its nu'lease a'tivit$
NIPT - Non-invasive prenatal testing40 09-05-2014
1. Phlebotomy
NIPT + i P+l 9 (7!
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NIPT te'+niue L P+le9otom$ (7!
g)N* release from nu'leate& (maternal!'ells over time (0-E &a$s!
NIPT - Non-invasive prenatal testing41 09-05-2014
" i NIPT t + i
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"vervie# NIPT te'+niue
NIPT - Non-invasive prenatal testing42 09-05-2014
1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT t + i Pl i l ti (!
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NIPT te'+niue L Plasma isolation (!
Centrifuge 9loo& 00g for 0W at EXC (o, 704! %400g for 70W at 77XC (Stre'D!
?e'entrifuge plasma portion 000g for 0W at EXC(o, 704! % 5000g for 0W at 77XC (Stre'D!
NIPT - Non-invasive prenatal testing43 09-05-2014
2. Plasma isolation
Ca. 55% of total blood
<1% of total blood
Ca. 45% of total blood
Removal of nucleated cells, as asource of maternal DNA
"vervie# NIPT te'+niue
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"vervie# NIPT te'+niue
NIPT - Non-invasive prenatal testing44 09-05-2014
1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT te'+niue 'f)N* eJtra'tion (!
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NIPT te'+niue L 'f)N* eJtra'tion (!
)ete'tion of 'ell-free fetal)N* ('f)N*! in maternalplasma in ../
S+e&&ing of trop+o9last 'ells mi'roparti'les of fragmente& )N*
maternal 9loo&stream s+ort +alflife (7 + 'learan'e!
:e&ian prevalen'e of 48 (real
time PC?! to 087
(&igital PC?!in st an& 7n& trimester
In'reasing 8 &uring pregnan'$
?elia9le &ete'tion from -7#eeDs on
NIPT - Non-invasive prenatal testing45 09-05-2014
> o :), Cor9etta N, C+am9erlain PF, ?ai V, Sargent I, et al> (../! Presen'e of fetal )N* inmaternal plasma an& serum> an'et 4502 E15LE1/>
7> un F:F, C+iu ?<, C+an <C*, eung T, au T<, o :)> 7001 :i'roflui&i's &igital PC? reveals a+ig+er t+an eJpe'te& fra'tion of fetal )N* in maternal plasma> Clin> C+em> 5E, EL/7>
3. cfDNA extraction
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"vervie# NIPT te'+niue
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1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT te'+niue i9rar$ prep (!
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NIPT te'+niue L i9rar$ prep> (!
T+e 'f)N* +as to 9e mo&ifie& for t+eseuen'ing instrument (eg iSe! to 9ea9le to rea& t+e )N* seuen'es of ea'+in&ivi&ual fragment> T+e follo#ing a'tions
are reuire& 2
NIPT - Non-invasive prenatal testing47 09-05-2014
4. Library preparation
• End repair• Adenylation (3’)• Adapter ligation• Library purification (x2)• PCR amplification
• Library purification• Library validation
NIPT te'+niue i9rar$ prep (7!
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NIPT te'+niue L i9rar$ prep> (7!
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Library validation
Average size cfDNA fragments = 150-170 bp
Average size cfDNA fragments +adapters = ca. 300 bp
• Validated libraries are pooled in
equal ratios (equimolar).• Because of the unique adapter (1
adapter per cfDNA sample), theinstrument can discriminate
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"vervie# NIPT te'+niue
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1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT te'+niue L Cluster gen (!
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NIPT te'+niue Cluster gen> (!
T+e poole& li9rar$ sample +as to 9e atta'+e&to glass sli&e (flo#'ell!, so ever$ uniuefragment 9in&s to a ran&om 9ut &istin't Koneon t+is sli&e
Per Kone, t+ere #ill 9e uniue fragment t+at#ill 9e amplifie&, so multiple 'opies of t+esame fragment eJist on t+at Kone ('lustergeneration! *mplifi'ation nee&e& to over'ome 'urrent &ete'tion
limitations (not possi9le to &ete't%genot$pe a
single )N* fragment!
NIPT - Non-invasive prenatal testing50 09-05-2014
5. Cluster generation
Maternal cfDNA fragment
Fetal cfDNA fragment
NIPT te'+niue L Cluster gen (7!
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NIPT te'+niue Cluster gen> (7!
mm
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Glass slide coated with DNA fragmentsthat recognize the adapters
Random binding of cfDNA fragments to probes on flowcell
Maternal cfDNA fragment
Fetal cfDNA fragment
NIPT te'+niue L Cluster gen (4!
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NIPT te'+niue Cluster gen> (4!
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Amplification of each unique
fragment per cluster (clonalamplification)
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"vervie# NIPT te'+niue
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1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT te'+niue L Seuen'ing (!
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NIPT te'+niue Seuen'ing (!
=a'+ 'luster is interrogate& forits seuen'e
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6. Sequencing
Create correct single stranded template for sequencing reaction
NIPT te'+niue L Seuen'ing (7!
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NIPT te'+niue Seuen'ing (7!
*nneal seuen'ing primer Seuen'ing B$ S$nt+esis (SBS! Iteration of )N* pol$merisation (
9ase per '$'le!, laser s'anning an&
&e'apping
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NIPT - Non-invasive prenatal testing56 09-05-2014
1. Phlebotomy 2. Plasma isolation 3. cfDNA extraction 4. Library preparation
5. Cluster generation 6. Sequencing 7. Data-analysis 8. Reporting
NIPT te'+niue L )ata anal$sis (!
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$ ( !
?esolution of &ete'tion is &etermine& 9$Coverage (no> of rea&s! 4 :9 2 'a> 50J0 rea&s reuire&
7 :9 2 'a> .7J0
rea&s reuire& :9 2 'a> 410J0 rea&s reuire&
ig+er resolution +as +ig+ impa't on total'ost
?ange of 'urrentl$ implemente& no> of rea&sfor &ete'ting aneuploi&ies 2 :in 2 7J0 rea&s :aJ 2 75J0 rea&s
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Yu et al, 2013, Noninvasive Prenatal Molecular Karyotyping from Maternal Plasma
NIPT te'+niue L )ata anal$sis (!
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$ ( !
NIPT - Non-invasive prenatal testing58 09-05-2014
Raw sequencing data isaligned to the genome
Raw Data
GC correction
Counting Statistics
Maternal cfDNA fragment
Fetal cfDNA fragment
NIPT te'+niue L )ata anal$sis (7!
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$
Z-s'ore 'al'ulation
Step 2 normaliKe for amount of &ata
)ivi&e no fragments for '+r N #it+ total no offragments
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Sample 1 : 1x representation Sample 2 : 2x representation
NIPT te'+niue L )ata anal$sis (4!
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Z-s'ore 'al'ulation Step 7 2 &etermine t+e num9er of stan&ar&
&eviations from mean ('ontrol S)s s+oul& 9e
esta9lis+e& for 'omparison purpose!
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Claime& a''ura'$ (!
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T+e sensitivit$%spe'ifi'it$ of t+e testvaries per '+romosome
Near 008 sensitivit$ an& spe'ifi'it$
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1. Devers PL, Cronister A, Ormond KE, Faio F, !rasington CK, Flodman P. "oninvasive #renatal testing$noninvasive#renatal diagnosis% t&e #osition o' t&e "ational (oiet) o' *eneti Counselors. + *enet Couns. 21-
+un22/-0%213
Claime& a''ura'$ (7!
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T+e sensitivit$%spe'ifi'it$ of t+e testvaries per '+romosome #it+
9ioinformati's 'orre'tions
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> C+en =Z, C+iu ?, Sun , *DoleDar ?, C+an <C, eung T, ;iang P, Z+eng , un F:, C+an , ;in , Go *T, au =T, To , eung C, Tang ?, *u-eung S<, am , <ung , Z+ang , van Vugt ;:, :ineDa#a ?, Tang :, ang ;, "u&ejans CB, au T<, Ni'olai&es <, o :> Noninvasive prenatal&iagnosis of fetal trisom$ 1 an& trisom$ 4 9$ maternal plasma )N* seuen'ing> PoS "ne> 70A(/!2e7/.>
Claime& positive pre&i'tive value
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#ositive #reditive value, or #reisionrate is t+e proportion of positive test
results t+at are true positives (su'+ as'orre't &iagnoses!>
refle'ts t+e pro9a9ilit$ t+at a positive
test refle'ts t+e un&erl$ing 'on&ition9eing teste& for> Its value &oes &epen&on t+e prevalen'e of t+e out'ome of
interest
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Claime& positive pre&i'tive value
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4' #ositive #reditive value, ..8sensitivit$ an& 0>78 false positives
If %50 risD .0>18 If %500 risD E.>/8
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In&i'ations
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RNIPT s+all repla'e amnio'entesis Gui&elines of C
See neJt sli&e for in&i'ations ?IZIV
No ?IZIV refun& for NIPT
Biolog$ NIPT #ill fail in 'ertain situations 2 t#ins,
translo'ations, su9'+romosomal a99erations,pla'ental mosai'ism,Q
NIPT - Non-invasive prenatal testing65 09-05-2014
In&i'ations
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Previous in&i'ations for invasive NIPT
*9normal TT (M%400! an& no ot+er US anomalies
*9normal US s'reening suggestive for T7
T7 in previous +istor$
?o9 translo'ation 4, 7
:an$ ot+er in&i'ations
NIPT optional, 9ut not prefera9le
No in&i'ation for invasiveQ9ut NIPT &esira9le YY Normal st trim an& ol&er age
o# risD st trim
Previous TT anomalies ICSI %IVF%PG) pregnan'ies
Qall pregnan'ies
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Pra'ti'al informationimitations of NIPT
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imitations of NIPT
Pregnan'$ &uration
NIPT is less relia9le if pregnan'$ 6 7#
NIPT is impossi9le 6 0 # :aternal #eig+t NIPT is less relia9le if +ig+ B:I
eig+t (9efore pregnan'$ ! an& lengt+ reueste&
"t+er fa'tors influen'ing out'ome
ig+ p+$si'al a'tivit$
Infe'tion Can'er
Q
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Pra'ti'al informationimitations of NIPT
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imitations of NIPT
Not &ete'ta9le
:osa'ism of '+romosome 7
)eletions or &upli'ations of '+romosome 7
"t+er '+romosomal anomalies
:ole'ular anomalies (monogeni' ea CF, Fragile Q!
:isinterpretations 'an o''ur if :aternal '+romosomal anomalies or variants
?eport on '+romosomal testing of mot+er nee&e&
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Pra'ti'al informationContra in&i'ations for NIPT
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Contra in&i'ations for NIPT
T#in or multiple pregnan'$
istor$ of (64mont+s!
maternal 9loo& transfusion stem'ell t+erap$
immunot+erap$
transplantation
US anomalies in foetus ( arra$!
09-05-2014NIPT - Non-invasive prenatal testing69
uestionsY
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