Non-invasive Prenatal Trisomy test
A safe prenatal testing for fetal
chromosomal aneuploidy
Leon Liang
BGI Health Europe
Common fetal aneuploidy
Down
syndrome
• Trisomy 21 (1/700-750)
• 80% reach to adulthood
Edwards
syndrome
• Trisomy 18 (1/6000)
• <10% reach to 1 year old
Patau
syndrome
• Trisomy 13 (1/10000-20000)
• Most die after birth
Others: Turner syndrome (XO), Klinefelter syndrome (XXY), triple X syndrome (XXX), etc.
Current screening & diagnostic tests
Invasive or
Noninvasive
Pregnancy
(weeks)
Risks and detection rate
First-trimester serum screening
Non-invasive
11-13
65%-70% detection rate*
Second-trimester serum
screening
Non-invasive
15-19
69%-81% detection rate*
Fetal nuchal translucency (NT)
Non-invasive
11-13
64%-70% detection rate*
Serum integrated screen
Non-invasive
11-13 & 15-19
85%-88% detection rate*
Serum integrated screen + NT
Non-invasive
11-13 & 15-19
94%-96% detection rate*
Amniocentesis
Invasive
16-21
0.5-1% miscarriage
Permission required
Chorionic villi sampling (CVS)
Invasive
10-13
1-2% miscarriage
Permission required
Percutaneous umbilical blood
sampling (PUBS)
Invasive
18-24
1-2% miscarriage
Permission required
Method
* Data present in 5% false positive rate
Fergal D, Jacob A, et al. The New England Journal of Medicine, 2005
Screening tests
• Serum biochemical test;
ultrasound scan
• Non-invasive
• Cheap
• Less accurate
• Low detection rate
• High false positive rate
Diagnostic tests
• Karyotyping (G-banding
or FISH)
• Invasive
• Expensive
• Highly accurate
• High detection rate
• Low false positive rate
What’s NIFTY test?
Non-Invasive Fetal TrisomY
A superior screening test
High detection rate and low false positive rate
Non-invasive method based on NGS and bioinformatics
Analysis of fetal cell free DNA in maternal plasma
Evaluate the likelihood of fetal trisomy 21, 18, and 13
Features of NIFTY
New detection technology based on NGS;
Sensitivity and specificity > 99%
Accurate
Safe
Early
Optimization
Simple
No risks of intrauterine infection
and miscarriage
Fetal trisomy risk can be evaluated as
early as 12 gestational week
Reduce clinical pressure of unnecessary
invasive tests
Only 5ml of peripheral blood is needed;
easy to handle in clinical practice
Scientific discovery
Fundamental Features of Cell-Free Fetal DNA
• Short fragments of 145-200bp, derived from placental
trophocytes
• 970 times greater than fetal cells DNA in maternal
blood.
• Detectable in maternal plasma from the 5th week of
gestation.
• Concentration increase as the gestation age grows
• Disappears soon after childbirth.
Sequencing revolution
NGS
Sanger
1. incorporation
C
C
1. Incorporation
2. Elute and scan
3. Cleavage
C
C
T
A
G
1. Incorporation
2. Elute and scan
T
A
G
Principle of NIFTY
Normal
T-21
Plasma DNA
50 bp
Total cfDNA
sequencing
Chr6
Chr18
ChrX
Chr21
Chr7
Chr13
Chr11
ChrY
Bioinformatics
…..
Reads alignment
Normal
T-21
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
Reads count
Bioinformatics
analysis
Clinical validation in 3464 samples
• Study design and aim:
3464 singleton pregnancies
with high risk of T21, T18, T13
Plasma DNA from maternal
peripheral blood
– Validation of the NIFTY in predicting the fetal
risk of trisomy 21, 18, and 13 in high risk
population by a double blind test
– Evaluation of sensitivity and specificity of the
NIFTY by comparing to karyotyping result
(clinical gold standard)
– provide supports for large-scale test in real
clinical setting
NIFTY test
Double blind
Bioinformatics analysis
Calculate sensitivity and
specificity
Karyotyping by AF, CVS, Cord
blood
NIFTY results in 3464 samples
T21
T18
T13
NIFTY positive
189
64
10
Karyotyping positive
188
63
10
False positive No.
1*
1*
0
False Negative No.
0
0
0
Sensitivity
100.00%
100.00%
100.00%
Specificity
99.97%
99.97%
100%
False Positive Rate
0.03%
0.029%
0.00%
False Negative Rate
0.00%
0.00%
0.00%
Positive Predictive Rate
99.49%
98.44%
100.00%
*caused by insufficient sequencing depth
Large scale clinical test (2009-2011)
Qualified maternal blood samples (n=11184)
•
Gestational week from 9 – 28 weeks, averagely 20 weeks
•
Maternal age from 18 – 45 years, averagely 31 years
•
4522 screening test high risk pregnancies
•
2426 screening test low risk pregnancies
•
2720 other high risk factors (AMA, abnormal NT, previous abnormal pregnancy, etc.)
•
1387 screening not done
0.7% of all samples
Unable to produce results (n=79; failed DNA extraction,
library construction, or sequencing)
99.3% of all samples
Pregnant women with NIFTY results (n=11105)
Positive (n=190)
Shan Dan, et al., Prenatal Diagnosis, 2012: p. 1-8.
Negative (n=10915)
NIFTY results in 11105 samples
No. of cases
Positive Results
T21
T18
T13
Negative results
NIFTY
11,105
140
42
0
10915
Karyotyping
182 + 2818
139
41
0
2818
False positive rate
0.03%
0.03%
n/a
False negative rate
0%
0%
n/a
Detection rate
100%
100%
n/a
Specificity
99.96%
99.96%
n/a
Shan Dan, et al., Prenatal Diagnosis, 2012: p. 1-8.
Other rare aneuploidies
A T21 case was missed by
biochemical screening
Sample ID: PDP10003761
Age: 31
Screening test: 1/510（Low risk）
NIFTY: T21
Karyotyping: 47, XX, +21
15
T9
Sample ID: PDB11AJ00026
Age: 41
NIFTY: T9
Karyotyping: T9
T16
Sample ID: PDB11AJ00783
Age: 30
NIFTY: T16
FISH: T16
Mosaic T21
Sample ID: PDB12AO00267
Age: 38
NIFTY: T21
Karyotyping: 47, XX, +21 (88%)
T21-T7-XXY complex placental mosaic
Sample ID: PDB12AL00732
Age: 37
NIFTY: T21-T7-XXY/XY
Karyotyping: CVS T21-T7-XXY/XY; AF euploid
NIFTY clinical pipeline
AMA,
Previous affected fetus,
Recurrent miscarriage,
Aneuploidy background
Test workflow
Hospital
Pre-test
counseling
Blood
collection
Bar-coding
Plasma
preparation
Post-test
counseling
BGI Clinical Laboratories: 10 days
Laboratory
testing
Bioinformatics
analysis
Report
delivery
Sample Management System
Unique Identification
Sample Location
Storage Capability
Laboratory System
Effective lab space
separation
Progressively decreased
pressure
Restricted traffic flow
22
NIFTY is not suitable for
Detection of balanced rearrangements and low level of mosaicism
The pregnant woman is a chromosomal aneuploidy carrier
If either of the parent has chromosomal structural abnormalities
e.g. duplication, deletion, translocation, etc.
If the pregnant woman receives allogenic DNA importation prior to NIFTY
e.g. blood transfusion, transplantation, stem cell therapy, etc.
Report
Genetic testing report
Low risk:
the fetus is unlikely to be T21, T18,
or T13. No special medical
procedure is recommended. Routine
prenatal checks is suggested.
High risk:
the fetus is likely to be T21, or T18,
or T13. diagnostic procedure such as
amniocentesis or cordocentesis is
recommended.
More than 98% of cases
Delay notification
QC:
Caused by either the experiment or
blood sample quality; need to
repeat the experiment
Data fluctuation:
Caused by high data deviation; need
to repeat the experiment
cfDNA concentration low:
Need to repeat the experiment
If repeating experiment still cannot
solve the problem, blood resampling is required.
Re-sampling notification
QC:
Caused by poor blood sample
quality
cfDNA concentration low
Previous NIFTY failed to produce
informative result, and gestational
age is more than 22 weeks
Sample requirement
Blood sampling
• 5ml in EDTA tube
• Clearly labeled
Plasma preparation
• Immediately extract plasma
• Stored at 4℃, extract in 4 hours
Packing for delivery
• Strong support in case of damage
• Enough dry ice to keep frozen
Sample storage
• -20℃ for a week, -80℃ for long term
• Avoid room temperature and
repeated thaw
BGI papers
30
More Choice
NIFTY express
NIFTY
NIFTY plus
6 days
14 working days
14 working days
Detection of T21, T18, T13
Gender
Twin pregnancies
Sex chromosome
abnormality
Microdeletion &
Microduplication
Monogenic disease
Turn around time
NIFTY express
Validated on 1647 samples
Performed on Ion Proton platform, extremely fast
Performed in Czech Republic BGI-GENNET co-lab. EU based service.
Similar price
Data
Chr21
(61 T21 cases)
Sensitivity
96.7% (59/61)
Specificity
98.4% (1561/1586)
Chr18
(16 T18 cases)
Sensitivity
100% (16/16)
Specificity
98.7% (1610/1631)
Chr13
(13 T13 cases)
Sensitivity
92.3%(12/13)
Specificity
99.1%(1619/1634)
NIFTY plus
Detection of Microdeletion syndromes
Non-invasive Monogenic disease detection
Results of other prenatal tests such as biochemical and ultrasound
tests should be considered. Diagnostic procedure such as
amniocentesis/cordocentesis is suggested.
Summary
Noninvasive
• 5ml maternal
blood
• No risk of
intrauterine
infection and
miscarriage
Accurate
• Sensitivity
>99%
• Specificity
>99%
Turnaround
time
Early
detection
• 10 days
• As early as
12 weeks
About BGI
• Largest genomics research center in the world
– 137 Hiseq2000, 27 AB Solid, 1 Roche 454, 1 Ion Torrent, 30 AB 3730
• Strong leader board and qualified employees
– More than 4000 employees including 1500 bioinformaticians
• Extraordinary super computer and cutting-edge
cloud computing technique
– 102T flops/ 10PB storage/ 20TB memory
• Numerous high-quality publications on top academic
Journals
– Nature/ Science/ …
Selected Top Publications of BGI
38
Milestones of BGI
BGI-Beijing, 1999
BGI-Americas, 2010
BGI-WH, 2010
BGI-HZ, 2001
BGI-SZ, 2007
BGI-Europe, 2010
BGI-HK, 2009
Platforms of BGI
Sequencing platform
Computing and bioinformatics platform
Proteomics platform
Agricultural genomics platform
Microbiology platform
Cloning platform
Healthcare platform
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Research collaborators worldwide