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NIPT

Non-Invasive Prenatal Testing


Ben Caljon, NGS platform (UZ Brussel, VUB, ULB)

Overview
Summary/introduction on invasive testing
History of prenatal diagnosis
Current first trimester screening strategy
Types of Invasive Prenatal Diagnosis (IPD)
Assessing IPD results

Non-invasive testing
Detection of cfDNA
NIPT methodologies
NIPT technique (Lo et al)
Claimed accuracy
Indications and limitations

NIPT - Non-invasive prenatal testing

09-05-2014

Invasive testing
Summary/introduction

History (1)
Prenatal diagnosis
1970 to early 1980 : advanced maternal age
(+35y)
<1/3 of Down syndrome pregnancies diagnosed
2% of IPD had chromosomal abnormalities

Data from England and Wales in the period of 1990-1998

Morris JK, Mutton DE, Alberman E. Revised estimates


of the maternal age specific live birth prevalence of
Down's syndrome. J Med Screen. 2002;9(1):2-6
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History (2)
Late 1980, early 1990 : introduction of
second-trimester maternal serum markers
Ca. 2/3 of Down syndrome pregnancies diagnosed
4% of IPD had chromosomal abnormalities
Marker

Full name

Origin

Tr 21

AFP

alphafetoprotein

Yolk sac/fetal
liver

(MoM : 0,73)

HCG

beta-human
chorionic
gonadotropin

Trophoblast

(MoM : 2)

UE3

unconjugated
estriol

Feto-placental
unit

(MoM : 0,73)

Marker levels vary with gestation week

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History (3)
Complex data analysis
Necessary to use MoM (multiple of median) values
Need for accurate population parameters
Need for likelihood ratio distribution
Need for correct curve fitting for medians of the markers
Need for correction for other factors such as maternal weight
Software prediction tools available

MoM =

Patient value
Median value normal pregnancies

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History (4)
Late 1990, early 2000 : combined first-trimester
testing
Ca. 90% of Down syndrome pregnancies diagnosed
6% of IPD had chromosomal abnormalities
Marker

Full name

Origin

Tr 21

HCG

beta-human chorionic
gonadotropin

Trophoblast

(MoM : 1,8)

PAPP-A

Pregnancy-associated
plasma protein A

trophoblast and decidualized endometrial


stroma cells

(MoM : 0,4)

Ultrasound measurement
NT

Nuchal translucency
thickness

NIPT - Non-invasive prenatal testing

(MoM : 2-2,5)

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History (5)

NIPT - Non-invasive prenatal testing

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History (6)
Normal

Disadvantage : inter-operator differences

Trisomy 21

Advantage : useful in twins, elevated in other trisomies


Fluid filled space (cardiac failure, structural malformation,
delayed/abnormal development of lymphatic system)

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History (7)
Factors influencing 1st trimester testing :

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Current first trimester screening


Curtosy Dr. Anniek Vorsselmans :

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Prenatal testing Invasive (1)


Chorion Villus Sampling
(CVS)
10-13 weeks after gestation
Transabdominal or
Transcervical (depending on
access to placenta/skills
physician)
Risk :
Vaginal bleeding/spotting
Chorioamnionitis
Fetal loss: 1/200

Pro :
Results are available earlier
in pregnancy
Less parental anxiety
Earlier/safer methods for
pregnancy termination

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Prenatal testing Invasive (2)


Amniocentesis
>14 weeks after gestation
(earlier possible but higher risk)
Risk
Transient vaginal spotting:
1-2%
Amniotic fluid leakage: 12%
Chorio-amnionitis: < 0.1%
Needle injuries to the fetus
are rare when
ultrasonographic guidance
is used.
Fetal loss: 1/200
(overestimate)

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NIPT - Non-invasive prenatal testing

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Prenatal testing Invasive (3)


Cordocentesis
>17 weeks after gestation
(mostly > 20 weeks)
Only if CVS or AC not possible
Risk (similar to AC)
Transient vaginal spotting:
1-2%
Amniotic fluid leakage: 12%
Chorio-amnionitis: < 0.1%
Needle injuries to the fetus
are rare when
ultrasonographic guidance
is used.
Fetal loss: 1/200

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Assessing invasive results (1)


Preparation of samples
CVS : microscopic separation
of maternal tissue from villi =
dissection of decidua
Centrifugation of cord blood
(serum/plasma for
biochemistry, WBC for
DNA/biochemical)

Check for maternal cell


contamination (MC)
Via fragment analysis
(PowerPlex 16HS)

Dependent on analysis : put


AC/CVS cells in culture
Risk for overgrowth with
maternal cells

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Assessing invasive results (2)


Example Maternal Cell Contamination
Prenatal sample

Mother

For biochemistry analysis, MC


detection is more difficult : comparison
in enzymatic activities decidua/villi
-glucuronidase
-hexosaminidase

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NIPT - Non-invasive prenatal testing

Father
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Assessing invasive results (3)


Biochemistry analysis
Only possible if enzyme is expressed in the
analyzed tissue (AC/CVS/cord blood)
Power : one assay for many mutations

Measure enzymatic activities via artificial


substrates (fluorimetric dosage over time)

Cytogenetics
CVS :
Direct/KT
LT

AC

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Assessing invasive results (4)


Karyotype

FISH

>5 Mb dup/del

AC lower error rates than CVS :


Confined Placental Mosaicism (CPM)
MC
Banding resolution

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NIPT - Non-invasive prenatal testing

QF-PCR
MLPA
aCGH (+ SNP
array)
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Assessing invasive results (5)


Molecular DNA analysis
Wide range of molecular test/techniques can
be applied if DNA is available
Restriction digestion
Southern blotting
Fragment analysis
Sequencing

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Non-invasive testing

Detection of cfDNA
Detection of cell-free fetal DNA
in maternal plasma in 19971
Shedding of trophoblast cells
microparticles of fragmented DNA
maternal bloodstream short
half life (2 h clearance)
Pro : multigravid pregnancy

Median prevalence of 3% (real time


PCR) to 10%2 (digital PCR) in 1st
and 2nd trimester
Reliable detection from 11-12
weeks on
Increasing % during 2 and 3rd
trimester

1.
2.

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Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, et al. (1997) Presence of fetal DNA in
maternal plasma and serum. Lancet 350: 485487.
Lun FMF, Chiu RWK, Chan KCA, Leung TY, Lau TK, Lo YMD. 2008 Microfluidics digital PCR reveals a
higher than expected fraction of fetal DNA in maternal plasma. Clin. Chem. 54, 16641672.

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Introduction - Media

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NIPT - Non-invasive prenatal testing

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NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS
(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT - Non-invasive prenatal testing

RNA based
approaches

qPCR
Examination of differentially
expressed genes (trophoblast
vs. Maternal RNA)
Currently being
investigated.
Proof-of-principle was
successful
Clinical trial disappointing
09-05-2014

NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS

RNA based
approaches

qPCR

(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT Digital PCR


Absolute quantitation of nonpolymorphic chr1 (reference)
and chr21 marker.
Difference in quantitation
allows for T21 detection
Advantage :
Ease of use
Cheap

Disadvantage :
Only limited amount of markers
to be assessed in 1 experiment
Fetal fraction determines
complexity (number of
wells/counts) needed for
sufficient statistical power. For
2%, 220816 counts are needed,
which is beyond current
commercial technology
Lo YM, Lun FM, Chan KC, Tsui NB, Chong KC, Lau TK,
Leung TY, Zee BC, Cantor CR, Chiu RW. Digital PCR for the
molecular detection of fetal chromosomal aneuploidy. Proc
Natl Acad Sci U S A. 2007 Aug 7;104(32):13116-21.
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NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS

RNA based
approaches

qPCR

(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT - Non-invasive prenatal testing

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NIPT qPCR
Different array of methods,
mostly based on differentially
methylated regions (DMRs)
Methylation Dependent
Immuno-Precipitation (MeDIPPCR)
Methylation Specific PCR (MSP)

Advantage :
Cheap on consumables

Disadvantage :
Selection/optimization of test
is tedious (selection of DMRs)
Multiple DMRs needed to
obtain sufficient statistical
power
Not highly multiplexable
Laborious

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NIPT - Non-invasive prenatal testing

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NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS

RNA based
approaches

qPCR

(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT SNP based approaches (1)


Offered by company Natera
Commercial name : Panorama
NATUS technology = Next generation Aneuploidy Testing
Using SNPs
Using NGS, 11000 SNPs are determined within the cfDNA for
chromosomes 13,18,21, X and Y
PCR amplification of polymorphic regions
Sequencing of amplicons using NGS device

Based on the SNP genotyping (both plasma as buffy coat),


the NATUS algorithm performs a per chromosome QC and
establishes fetal chromosomal anomalies using a Bayesianbased maximum likelihood statistical method

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NIPT SNP based approaches (2)


Advantages
High accuracy (also for sex
chromosomes)
Ability to detect polyploidy

Disadvantage
Not useable for donor-egg
cell pregnancies
Only small cohort study
published (145 samples)
In proof-of-principle : 12.6%
of samples had a no-call
due to poor DNA quality (not
passing QC filter)
Currently in patent-conflict
with Sequenom
No information on other
chromosomes
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 targeted
sequencing of polymorphic loci. Prenat Diagn. 2012 Dec;32(13):1233-41.

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NIPT - Non-invasive prenatal testing

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NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS

RNA based
approaches

qPCR

(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT tMPS (1)


Offered by company
Ariosa Diagnostics
Commercial name :
Harmony Prenatal Test
Scope : T13, T18, T21, X
& Y (optional)
Technology : DANSR
(Digital Analysis of
Selected Regions) assay
and FORTE (fetal-fraction
optimized risk of trisomy
evaluation)
Sparks AB, Struble CA, Wang ET, Song K, Oliphant A.
Noninvasive prenatal detection and selective analysis
of cell-free DNA obtained from maternal blood:
evaluation for trisomy 21 and trisomy 18. Am J Obstet
Gynecol. 2012 Apr;206(4):319.e1-9.

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NIPT tMPS (2)


DANSR : probes for both polymorphic
(determine fetal fraction) as non-polymorphic
regions (chromosome proportion
determination)
FORTE algoritm : calculates an individualized
odd score for trisomy, based on fetal fraction,
chromosome proportion data and maternal age

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NIPT tMPS (3)


Advantages
Easy fetal fraction determination, using probes for polymorphic regions
No need for reference data (FORTE normalizes using intra-run data) : less
process drift
Cheaper, because of higher multiplexibility

Disadvantages :
False negative results have been obtained for 1/232 T21, 2/105 T13 and 2/10
T13
No information on other chromosomes/limited normalization capabilities

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NIPT - Non-invasive prenatal testing

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NIPT - Methodologies
NIPT
cfDNA based

cfRNA based

Clinical utility
s-MPS

Digital PCR

(Shotgun Massive
parallel Sequencing)

t-MPS

RNA based
approaches

qPCR

(Targeted Massive
parallel Sequencing)

SNP based
approaches

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NIPT sMPS (1)


Offered by companies Sequenom and
Verinata
Commercial names : MaterniT21 Plus and
Verifi resp.
Technology : see next slides
Scope
Sequenom : T21, T18, T13, X/Y aneuploidy,
T16,T22 (and specific microdeletions)
Verinata : T21, T18, T13, X/Y aneuploidy

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NIPT sMPS (2)


Verinata (website verinata)
N

Sensitivit 95%
y
CI

Specificity 95% CI

21

500

>99.9%
(90/90)

96100.0

99.8%
98.7-100.0
(409/410)

18

501

97.4%
(37/38)

86.299.9

99.6%
98.5-100.0
(461/463)

13

501

87.5%
(14/16)

61.798.5

>99.9%
99.2-100.0
(485/485)

MX

508

95.0%
(19/20)

75.199.9

99.0%
97.6-99.7
(483/488)

XX

XY

Sensitivity

95% CI Specificity 95% CI Accura 95% CI


cy

508

97.6%
(243/249)

94.899.1

99.2%
(257/259
)

97.299.9

98.4%

96.999.3

508

99.1%
(227/229)

96.999.9

98.9%
(276/279
)

96.999.8

99.0%

97.799.7

Sequenom (website verinata)

Results of published clinical


trials will be discussed later
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NIPT sMPS (3)


Why sMPS :
False positive/negative rates decrease over time (mainly by improved
bioinformatics algorithms)
No test is perfect (tMPS, sMPS nor SNP based)
-

Numerous factors responsible for test failure / altered DRs, but mainly due to
low % fetal DNA

Natera test has not been widely validated

Applicable to egg cell donor pregnancies


Lowest result failure (retest/new sample needed)

Generic test :
Expandable to other chromosomes
Possibility to pick up subchromosomal alterations (if sequencing costs
decreases)
Possibility to determine fetal SNVs (if sequencing costs decreases)
Possibility to determine polyploidy using SNVs (if sequencing costs decreases)

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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique Phlebotomy (1)


1. Phlebotomy

Maternal plasma + DNA isolation


Collection of maternal peripheral
blood (from 12 weeks gestation) in
10 ml EDTA tubes (Streck tubes)
with a proprietary stabilizing
agent
Inhibits gDNA release from
nucleated cells
Inhibits nuclease activity

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NIPT technique Phlebotomy (2)


gDNA release from nucleated (maternal)
cells over time (0-14 days)

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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique Plasma isolation (1)


2. Plasma isolation

Centrifuge blood @ 1600g for 10 at 4C (Lo, 2013) /


@300g for 20 at 22C (Streck)

Recentrifuge plasma portion @ 16000g for 10 at 4C


(Lo, 2013) / @ 5000g for 10 at 22C (Streck)

Ca. 55% of total blood

<1% of total blood

Removal of nucleated cells, as a


source of maternal DNA

Ca. 45% of total blood

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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique cfDNA extraction (1)


3. cfDNA extraction

Detection of cell-free fetal


DNA (cfDNA) in maternal
plasma in 19971
Shedding of trophoblast cells
microparticles of fragmented DNA
maternal bloodstream short half
life (2 h clearance)

Median prevalence of 3% (real


time PCR) to 10%2 (digital PCR)
in 1st and 2nd trimester
Increasing % during pregnancy
Reliable detection from 11-12
weeks on

1.
2.

45

Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, et al. (1997) Presence of fetal DNA in
maternal plasma and serum. Lancet 350: 485487.
Lun FMF, Chiu RWK, Chan KCA, Leung TY, Lau TK, Lo YMD. 2008 Microfluidics digital PCR reveals a
higher than expected fraction of fetal DNA in maternal plasma. Clin. Chem. 54, 16641672.

NIPT - Non-invasive prenatal testing

09-05-2014

Overview NIPT technique


1. Phlebotomy

5. Cluster generation

46

2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique Library prep. (1)


4. Library preparation

The cfDNA has to be modified for the


sequencing instrument (eg HiSeq) to be
able to read the DNA sequences of each
individual fragment. The following actions
are required :

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NIPT - Non-invasive prenatal testing

End repair
Adenylation (3)
Adapter ligation
Library purification (x2)
PCR amplification
Library purification
Library validation

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NIPT technique Library prep. (2)


Library validation
Average size cfDNA fragments = 150170 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), the
instrument can discriminate
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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique Cluster gen. (1)


5. Cluster generation

The pooled library sample has to be attached


to glass slide (flowcell), so every unique
fragment binds to a random but distinct zone
on this slide
Per zone, there will be 1 unique fragment that
will be amplified, so multiple copies of the
same fragment exist on that zone (=cluster
generation)
Amplification needed to overcome current detection
limitations (not possible to detect/genotype a
single DNA fragment)
Maternal cfDNA fragment

Fetal cfDNA fragment

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NIPT technique Cluster gen. (2)


Glass slide coated with DNA fragments
that recognize the adapters

Random binding of cfDNA fragments to probes on flowcell

Maternal cfDNA fragment

Fetal cfDNA fragment

mm
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NIPT technique Cluster gen. (3)

Amplification of each unique


fragment per cluster (clonal
amplification)

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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

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NIPT technique Sequencing (1)


6. Sequencing

Each cluster is interrogated for


its sequence

Create correct single stranded template for sequencing reaction

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NIPT - Non-invasive prenatal testing

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NIPT technique Sequencing (2)


Anneal sequencing primer
Sequencing By Synthesis (SBS)
Iteration of DNA polymerisation (1
base per cycle), laser scanning and
decapping

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NIPT - Non-invasive prenatal testing

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Overview NIPT technique


1. Phlebotomy

5. Cluster generation

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2. Plasma isolation

6. Sequencing

NIPT - Non-invasive prenatal testing

3. cfDNA extraction

4. Library preparation

7. Data-analysis

8. Reporting

09-05-2014

NIPT technique Data analysis (1)


Resolution of detection is determined by
Coverage (no. of reads)
3 Mb : ca. 150x106 reads required
2 Mb : ca. 192x106 reads required
1 Mb : ca. 380x106 reads required

Higher resolution has high impact on total


cost
Range of currently implemented no. of reads
for detecting aneuploidies :
Min : 2x106 reads
Max : 25x106 reads

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

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NIPT technique Data analysis (1)


Raw Data
Maternal cfDNA fragment

Fetal cfDNA fragment

GC correction

Raw sequencing data is


aligned to the genome

Counting Statistics

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NIPT technique Data analysis (2)


Z-score calculation
Step 1 : normalize for amount of data
Divide no fragments for chr N with total no of
fragments
Sample 1 : 1x representation

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NIPT - Non-invasive prenatal testing

Sample 2 : 2x representation

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NIPT technique Data analysis (3)


Z-score calculation
Step 2 : determine the number of standard
deviations from mean (control SDs should be
established for comparison purpose)

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Claimed accuracy (1)


The sensitivity/specificity of the test
varies per chromosome
Near 100% sensitivity and specificity

1.

61

Devers PL, Cronister A, Ormond KE, Facio F, Brasington CK, Flodman P. Noninvasive prenatal testing/noninvasive
prenatal diagnosis: the position of the National Society of Genetic Counselors. J Genet Couns. 2013
Jun;22(3):291-5

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Claimed accuracy (2)


The sensitivity/specificity of the test
varies per chromosome & with
bioinformatics corrections

1.

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Chen EZ, Chiu RW, Sun H, Akolekar R, Chan KC, Leung TY, Jiang P, Zheng YW, Lun FM, Chan LY, Jin Y, Go AT, Lau ET, To WW, Leung WC, Tang RY, AuYeung SK, Lam H, Kung YY, Zhang X, van Vugt JM, Minekawa R, Tang MH, Wang J, Oudejans CB, Lau TK, Nicolaides KH, Lo YM. Noninvasive prenatal
diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PLoS One. 2011;6(7):e21791.

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Claimed positive predictive value


positive predictive value, or precision
rate is the proportion of positive test
results that are true positives (such as
correct diagnoses).
reflects the probability that a positive
test reflects the underlying condition
being tested for. Its value does depend
on the prevalence of the outcome of
interest

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Claimed positive predictive value


If positive predictive value, 99%
sensitivity and 0.2% false positives
If 1/50 risk
90.8%
If 1/500 risk
49.7%

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Indications
NIPT shall replace amniocentesis
Guidelines of HC
See next slide for indications

RIZIV
No RIZIV refund for NIPT

Biology
NIPT will fail in certain situations : twins,
translocations, subchromosomal abberations,
placental mosaicism,

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Indications
Previous indications for invasive

NIPT

Abnormal TT (>1/300) and no other US anomalies


Abnormal US screening suggestive for T21
T21 in previous history
Rob translocation 13, 21

Many other indications


NIPT optional, but not preferable

No indication for invasivebut NIPT desirable ??


Normal 1st trim and older age
Low risk 1st trim
Previous TT anomalies
ICSI /IVF/PGD pregnancies
all pregnancies

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Practical information
Limitations of NIPT
Pregnancy duration
NIPT is less reliable if pregnancy < 12w
NIPT is impossible < 10 w

Maternal weight
NIPT is less reliable if high BMI
Weight (before pregnancy ) and length requested

Other factors influencing outcome


High physical activity
Infection
Cancer

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Practical information
Limitations of NIPT
Not detectable
Mosacism of chromosome 21
Deletions or duplications of chromosome 21
Other chromosomal anomalies
Molecular anomalies (monogenic ea CF, Fragile X)

Misinterpretations can occur if


Maternal chromosomal anomalies or variants
Report on chromosomal testing of mother needed

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Practical information
Contra indications for NIPT
Twin or multiple pregnancy
History of (<3months)
maternal blood transfusion
stemcell therapy
immunotherapy
transplantation

US anomalies in foetus (

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NIPT - Non-invasive prenatal testing

array)

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Questions?

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