nipt - beshg 2014

70
NIPT Non-Invasive Prenatal Testing Ben Caljon, NGS platform (UZ Brussel, VUB, UB!

Upload: sarlitaindahpermatasari

Post on 17-Feb-2018

220 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 1/70

NIPTNon-Invasive Prenatal Testing

Ben Caljon, NGS platform (UZ Brussel, VUB, UB!

Page 2: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 2/70

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

Page 3: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 3/70

Invasive testing

Summar$%intro&u'tion

Page 4: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 4/70

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

Page 5: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 5/70

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

Page 6: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 6/70

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   =

Page 7: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 7/70

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!

Page 8: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 8/70

istor$ (5!

NIPT - Non-invasive prenatal testing8 09-05-2014

Page 9: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 9/70

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!

Page 10: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 10/70

istor$ (/!

Fa'tors influen'ing st trimester testing 2

NIPT - Non-invasive prenatal testing10 09-05-2014

Page 11: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 11/70

Current first trimester s'reening

Curtos$ )r> *nnieD Vorsselmans 2

NIPT - Non-invasive prenatal testing11 09-05-2014

Page 12: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 12/70

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

NIPT - Non-invasive prenatal testing12 09-05-2014

Page 13: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 13/70

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!

NIPT - Non-invasive prenatal testing13 09-05-2014

Page 14: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 14/70

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

NIPT - Non-invasive prenatal testing14 09-05-2014

Page 15: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 15/70

*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

Page 16: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 16/70

*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

Page 17: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 17/70

*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

Page 18: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 18/70

*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$!

Page 19: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 19/70

*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

Page 20: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 20/70

Non-invasive testing

Page 21: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 21/70

)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>

Page 22: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 22/70

Intro&u'tion - :e&ia

NIPT - Non-invasive prenatal testing22 09-05-2014

Page 23: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 23/70

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

Page 24: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 24/70

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

Page 25: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 25/70

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.

Page 26: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 26/70

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

Page 27: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 27/70

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

Page 28: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 28/70

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

Page 29: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 29/70

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

Page 30: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 30/70

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.

Page 31: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 31/70

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

Page 32: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 32/70

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.

Page 33: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 33/70

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

Page 34: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 34/70

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

Page 35: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 35/70

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

Page 36: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 36/70

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

Page 37: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 37/70

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

Page 38: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 38/70

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

+

Page 39: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 39/70

"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 (!

Page 40: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 40/70

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!

Page 41: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 41/70

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

Page 42: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 42/70

"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 (!

Page 43: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 43/70

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

Page 44: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 44/70

"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 (!

Page 45: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 45/70

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

"vervie# NIPT te'+niue

Page 46: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 46/70

"vervie# NIPT te'+niue

NIPT - Non-invasive prenatal testing46 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 i9rar$ prep (!

Page 47: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 47/70

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!

Page 48: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 48/70

NIPT te'+niue L i9rar$ prep> (7!

NIPT - Non-invasive prenatal testing48 09-05-2014

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 

"vervie# NIPT te'+niue

Page 49: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 49/70

"vervie# NIPT te'+niue

NIPT - Non-invasive prenatal testing49 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 Cluster gen (!

Page 50: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 50/70

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!

Page 51: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 51/70

NIPT te'+niue Cluster gen> (7!

mm

NIPT - Non-invasive prenatal testing51 09-05-2014

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!

Page 52: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 52/70

NIPT te'+niue Cluster gen> (4!

NIPT - Non-invasive prenatal testing52 09-05-2014

Amplification of each unique

fragment per cluster (clonalamplification)

"vervie# NIPT te'+niue

Page 53: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 53/70

"vervie# NIPT te'+niue

NIPT - Non-invasive prenatal testing53 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 Seuen'ing (!

Page 54: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 54/70

NIPT te'+niue Seuen'ing (!

=a'+ 'luster is interrogate& forits seuen'e

NIPT - Non-invasive prenatal testing54 09-05-2014

6. Sequencing

Create correct single stranded template for sequencing reaction

NIPT te'+niue L Seuen'ing (7!

Page 55: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 55/70

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

NIPT - Non-invasive prenatal testing55 09-05-2014

"vervie# NIPT te'+niue

Page 56: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 56/70

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 (!

Page 57: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 57/70

$ ( !

?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

NIPT - Non-invasive prenatal testing57 09-05-2014

Yu et al, 2013, Noninvasive Prenatal Molecular Karyotyping from Maternal Plasma

NIPT te'+niue L )ata anal$sis (!

Page 58: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 58/70

$ ( !

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!

Page 59: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 59/70

$

Z-s'ore 'al'ulation

Step 2 normaliKe for amount of &ata

)ivi&e no fragments for '+r N #it+ total no offragments

NIPT - Non-invasive prenatal testing59 09-05-2014

Sample 1 : 1x representation Sample 2 : 2x representation

NIPT te'+niue L )ata anal$sis (4!

Page 60: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 60/70

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!

NIPT - Non-invasive prenatal testing60 09-05-2014

Claime& a''ura'$ (!

Page 61: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 61/70

T+e sensitivit$%spe'ifi'it$ of t+e testvaries per '+romosome

Near 008 sensitivit$ an& spe'ifi'it$

NIPT - Non-invasive prenatal testing61 09-05-2014

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!

Page 62: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 62/70

T+e sensitivit$%spe'ifi'it$ of t+e testvaries per '+romosome #it+

9ioinformati's 'orre'tions

NIPT - Non-invasive prenatal testing62 09-05-2014

> 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

Page 63: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 63/70

#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

NIPT - Non-invasive prenatal testing63 09-05-2014

Claime& positive pre&i'tive value

Page 64: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 64/70

4' #ositive #reditive value, ..8sensitivit$ an& 0>78 false positives

If %50 risD .0>18 If %500 risD E.>/8

NIPT - Non-invasive prenatal testing64 09-05-2014

In&i'ations

Page 65: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 65/70

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

Page 66: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 66/70

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

NIPT - Non-invasive prenatal testing66 09-05-2014

Pra'ti'al informationimitations of NIPT

Page 67: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 67/70

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

09-05-2014NIPT - Non-invasive prenatal testing67

Pra'ti'al informationimitations of NIPT

Page 68: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 68/70

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&

09-05-2014NIPT - Non-invasive prenatal testing68

Pra'ti'al informationContra in&i'ations for NIPT

Page 69: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 69/70

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

Page 70: NIPT - BeSHG 2014

7/23/2019 NIPT - BeSHG 2014

http://slidepdf.com/reader/full/nipt-beshg-2014 70/70

NIPT - Non-invasive prenatal testing70 09-05-2014