NIPT

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
NIPT - Non-invasive prenatal testing
Indications and limitations
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
History (2)
Late 1980, early 1990 : introduction of

second-trimester maternal serum markers
Marker
AFP
Originpregnancies
Tr21
Ca. 2/3Fullname
of Down syndrome
diagnosed
4%
alphaYolksac/fetal
of fetoprotein
IPD had chromosomal
abnormalities
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
09-05-2014
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 pregnancie s
09-05-2014
History (4)
Late 1990, early 2000 : combined first-trimester

testing
Marke
r
Ca. 90% of Down syndrome

Fullname
Origin pregnancies diagnosed
Tr21
HCG
beta-humanchorionic
gonadotropin
PAPP-A
Pregnancy-associated trophoblastanddeciplasmaproteinA
dualizedendometrial
stromacells
6% of IPD had chromosomal abnormalities

Trophoblast
(MoM:1,8)
(MoM:0,4)
Ultrasoundmeasurement
NT
Nuchaltranslucency
thickness
(MoM:2-2,5)
09-05-2014
History (5)
09-05-2014
History (6)
Trisomy 21
Normal
Disadvantage : inter-operator differences
Advantage : useful in twins, elevated in other trisomies

Fluid filled space (cardiac failure, structural malformation,
delayed/abnormal development of lymphatic system)
09-05-2014
History (7)
Factors influencing 1st trimester testing :
10
09-05-2014
Current first trimester screening

Curtosy Dr. Anniek Vorsselmans :
11
09-05-2014
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 :
12
Results are available earlier

in pregnancy
Less parental anxiety
methods for
NIPT -Earlier/safer
Non-invasive prenatal testing
pregnancy termination
09-05-2014
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)
13
09-05-2014
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
14
09-05-2014
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
15
Risk
for overgrowth
with
NIPT - Non-invasive
prenatal testing
maternal cells
09-05-2014
Example Maternal Cell Contamination
Prenatal sample
Mother
For biochemistry analysis, MC

detection is more difficult : comparison
in enzymatic activities decidua/villi
-glucuronidase
-hexosaminidase
16
Father
09-05-2014
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
17
09-05-2014

Karyotype
FISH
>5 Mb dup/del
AC lower error rates than CVS :

Confined Placental Mosaicism (CPM)
MC
Banding resolution
18
QF-PCR
MLPA
aCGH (+ SNP
array)
09-05-2014
Molecular DNA analysis

Wide range of molecular test/techniques can
be applied if DNA is available
Restriction digestion
Southern blotting
Fragment analysis
Sequencing
19
09-05-2014
Non-invasive testing
Detection of cfDNA
Detection of cell-free fetal DNA

in maternal plasma in 1997 1
Shedding of trophoblast cells
microparticles of fragmented DNA
maternal bloodstream
short
half life (2 h clearance)
Pro : multigravid pregnancy
1.
2.
21
Median prevalence of 3% (real time

PCR) to 10% 2 (digital PCR) in 1st
and 2nd trimester
Reliable detection from 11-12
on N, Chamberlain PF, Rai V, Sargent IL, et al. (1997) Presence of fetal DNA in
Loweeks
YMD, Corbetta
maternal
plasma and
serum.
Lancet
350:
485487.
Increasing
%
during
2
and
3rd
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.
trimester
09-05-2014
Introduction - Media
22
09-05-2014
NIPT - Methodologies
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
parallel Sequencing)
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
23
SNP based
qPCR
Examination of differentially
expressed genes (trophoblast
vs. Maternal RNA)
Currently being
investigated.
09-05-2014
Proof-of-principle was
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
24
SNP based
qPCR
09-05-2014
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 :
25
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
09-05-2014
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
26
SNP based
qPCR
09-05-2014
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 :
27
Selection/optimization of test
is tedious (selection of DMRs)
Multiple DMRs needed to
obtain sufficient statistical
power
Not
highly multiplexable
Laborious
09-05-2014
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
28
SNP based
qPCR
09-05-2014
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
29
09-05-2014
NIPT SNP based approaches (2)
Advantages
High accuracy (also for sex
chromosomes)
Ability to detect polyploidy
Disadvantage
30
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
NIPT
- Non-invasive
prenatal testing
with
Sequenom
No information on other
09-05-2014
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
31
SNP based
qPCR
09-05-2014
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)
32
prenatal testing
SparksNIPT
AB,- Non-invasive
Struble CA,
Wang ET, Song K, Oliphant A.
Noninvasive prenatal detection and selective analysis
09-05-2014
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
33
09-05-2014
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
34
09-05-2014
NIPT
Digital PCR
s-MPS cfDNA based
(Shotgun Massive
Clinical
utility
cfRNA based
RNA based
approaches
t-MPS
(Targeted Massive
35
SNP based
qPCR
09-05-2014
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)
36
Verinata : T21, T18, T13, X/Y aneuploidy
09-05-2014
NIPT sMPS (2)

N
Sensitiv 95%
Specifici 95%CI
Verinata
(website
verinata)
it
CI
ty
Sensitivit
y
95%
CI
Specifici
ty
95%
CI
Accur
a
cy
95%C
I
XX
50
8
97.6%
(243/249
)
94.899.1
99.2%
(257/25
9
)
97.299.9
98.4
%
96.999.3
XY
50
8
99.1%
(227/229
)
96.999.9
98.9%
(276/27
9
)
96.999.8
99.0
%
97.799.7
y
21
18
13
MX
500
>99.9
%
(90/90)
96100.
0
99.8%
(409/41
0)
98.7100.0
501
97.4%
(37/38)
86.2
99.9
99.6%
(461/46
3)
98.5100.0
501
87.5%
(14/16)
61.7
98.5
>99.9%
(485/48
5)
99.2100.0
508
95.0%
(19/20)
75.1
99.9
99.0%
(483/48
8)
97.6-99.7
Sequenom (website verinata)
37
Results of published clinical
09-05-2014
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)
38
09-05-2014
Overview NIPT technique

1. Phlebotomy
5. Cluster generation
39
2. Plasma isolation
6. Sequencing
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
40
09-05-2014
NIPT technique Phlebotomy (2)
gDNA release from nucleated (maternal)

cells over time (0-14 days)
41
09-05-2014

1. Phlebotomy
42
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
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)
<1% of total blood
source of maternal DNA
Ca. 55% of total blood
43
Ca. 45% of total blood
09-05-2014

1. Phlebotomy
44
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
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
maternal
short half
plasma
in bloodstream
1997 1
Shedding
of trophoblast cells
life (2 h clearance)
microparticles
of fragmented
Median prevalence
of 3% DNA
(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.
NIPTLun
- Non-invasive
prenatal
testing
09-05-2014
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.

1. Phlebotomy
46
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
7. Data-analysis
8. Reporting
09-05-2014
NIPT technique Library prep. (1)

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 :
47
End repair
Adenylation (3)
Adapter ligation
Library purification (x2)
PCR amplification
Library purification
Library validation
09-05-2014
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
48
instrument can discriminate

1. Phlebotomy
49
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
7. Data-analysis
8. Reporting
09-05-2014
NIPT technique Cluster gen. (1)

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
50
09-05-2014
Glass slide coated with DNA fragments

that recognize the adapters
Random binding of cfDNA fragments to probes on flowcell
Fetal cfDNA fragment
mm
51
09-05-2014
Amplification of each unique

fragment per cluster (clonal
amplification)
52
09-05-2014

1. Phlebotomy
53
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
7. Data-analysis
8. Reporting
09-05-2014
NIPT technique Sequencing (1)

6. Sequencing
Each cluster is interrogated for

its sequence
Create correct single stranded template for sequencing reaction
54
09-05-2014
NIPT technique Sequencing (2)
Anneal sequencing primer

Sequencing By Synthesis (SBS)
Iteration of DNA polymerisation (1
base per cycle), laser scanning and
decapping
55
09-05-2014

1. Phlebotomy
56
2. Plasma isolation
6. Sequencing
3. cfDNA extraction
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. 150x10 6 reads required
Higher resolution has high impact on total

cost
Range of currently implemented no. of reads
for detecting aneuploidies :
Min : 2x10 6 reads
Max : 25x10 6 reads
Yu et al, 2013, Noninvasive Prenatal Molecular Karyotyping from Maternal Plasma
57
09-05-2014
Raw Data
Fetal cfDNA fragment
GC correction
Raw sequencing data is

aligned to the genome
Counting Statistics
58
09-05-2014
Z-score calculation
Step 1 : normalize for amount of data
Sample 1 : 1x representation
Sample 2 : 2x representation
Divide no fragments for chr N with total no of

fragments
59
09-05-2014
Z-score calculation
Step 2 : determine the number of standard
deviations from mean (control SDs should be
established for comparison purpose)
60
09-05-2014
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
09-05-2014
Claimed accuracy (2)
The sensitivity/specificity of the test

varies per chromosome & with
bioinformatics corrections
1.
62
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.
09-05-2014
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
63
09-05-2014
Claimed positive predictive value
If positive predictive value , 99%

If 1/50 risk
sensitivity
and 90.8%
0.2% false positives
If 1/500 risk
64
49.7%
09-05-2014
Indications
NIPT shall replace amniocentesis

Guidelines of HC
See next slide for indications
RIZIV
No RIZIV refund for NIPT
Biology
65
NIPT will fail in certain situations : twins,

translocations, subchromosomal abberations,09-05-2014
placental mosaicism,
Indications
NIPT
Abnormal TT (>1/300) and no other US anomalies
Previous
indications for invasive
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
66
09-05-2014
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
67
Infection
09-05-2014
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
68
09-05-2014
Contra indications for NIPT
Twin or multiple pregnancy
History of (<3months)
maternal blood transfusion
stemcell therapy
US anomalies in foetus (
immunotherapy
array)
transplantation
69
09-05-2014
Questions?
70
09-05-2014

NIPT - BeSHG 2014

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Non-Invasive Prenatal Testing

Summary/introduction on invasive testing

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

NIPT - Non-invasive prenatal testing

Morris JK, Mutton DE, Alberman E. Revised estimates

Late 1980, early 1990 : introduction of

Marker levels vary with gestation week

NIPT - Non-invasive prenatal testing

Late 1990, early 2000 : combined first-trimester

Ca. 90% of Down syndrome

6% of IPD had chromosomal abnormalities

NIPT - Non-invasive prenatal testing

NIPT - Non-invasive prenatal testing

Disadvantage : inter-operator differences

Advantage : useful in twins, elevated in other trisomies

NIPT - Non-invasive prenatal testing

NIPT - Non-invasive prenatal testing

Current first trimester screening

NIPT - Non-invasive prenatal testing

Prenatal testing Invasive (1)

Chorion Villus Sampling

Results are available earlier

Prenatal testing Invasive (2)

NIPT - Non-invasive prenatal testing

Prenatal testing Invasive (3)

NIPT - Non-invasive prenatal testing

Assessing invasive results (1)

Check for maternal cell

Dependent on analysis : put

Assessing invasive results (2)

Example Maternal Cell Contamination

For biochemistry analysis, MC

NIPT - Non-invasive prenatal testing

Assessing invasive results (3)

Measure enzymatic activities via artificial

NIPT - Non-invasive prenatal testing

Assessing invasive results (4)

AC lower error rates than CVS :

NIPT - Non-invasive prenatal testing

Assessing invasive results (5)

Molecular DNA analysis

NIPT - Non-invasive prenatal testing

Detection of cell-free fetal DNA

Median prevalence of 3% (real time

NIPT - Non-invasive prenatal testing

NIPT - Non-invasive prenatal testing

NIPT - Non-invasive prenatal testing

NIPT Digital PCR

Absolute quantitation of nonpolymorphic chr1 (reference)

Only limited amount of markers

NIPT - Non-invasive prenatal testing

Different array of methods,

NIPT - Non-invasive prenatal testing

NIPT SNP based approaches (1)

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

NIPT - Non-invasive prenatal testing

NIPT SNP based approaches (2)

Not useable for donor-egg

NIPT - Non-invasive prenatal testing

NIPT tMPS (1)

NIPT tMPS (2)

DANSR : probes for both polymorphic