Non-invasive Prenatal Trisomy test

Leon Liang

BGI Health Europe

A safe prenatal testing for fetal chromosomal aneuploidy

Common fetal aneuploidy

• Trisomy 21 (1/700-750)• 80% reach to adulthood

Down syndrome

• Trisomy 18 (1/6000)• <10% reach to 1 year old

Edwards syndrome

• Trisomy 13 (1/10000-20000)• Most die after birth

Patau syndrome

Others: Turner syndrome (XO), Klinefelter syndrome (XXY), triple X syndrome (XXX), etc.

Current screening & diagnostic tests

Method 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% miscarriagePermission required

Chorionic villi sampling (CVS) Invasive 10-13 1-2% miscarriagePermission required

Percutaneous umbilical blood sampling (PUBS) Invasive 18-24 1-2% miscarriage

Permission required

* Data present in 5% false positive rateFergal D, Jacob A, et al. The New England Journal of Medicine, 2005

Screening tests Diagnostic tests

• Serum biochemical test; ultrasound scan

• Non-invasive• Cheap• Less accurate • Low detection rate • High false positive rate

• Karyotyping (G-banding or FISH)

• Invasive• Expensive• Highly accurate • High detection rate • Low false positive rate

NIFTY

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

Accurate

Safe

Early

Optimization

Simple

New detection technology based on NGS;Sensitivity and specificity > 99%

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

NIFTY

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

TA

G

CC

TA

G

CC

1. incorporation 1. Incorporation2. Elute and scan

1. Incorporation2. Elute and scan 3. Cleavage

Sanger

NGS

Principle of NIFTY

Chr6Chr18ChrXChr21Chr7Chr13Chr11ChrY…

..

Plasma DNA

Total cfDNA sequencing

Reads count

Normal T-21

50 bp

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 YBioinformatics

analysis

– 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

Clinical validation in 3464 samples

3464 singleton pregnancies with high risk of T21, T18, T13

Plasma DNA from maternal peripheral blood

NIFTY test

Karyotyping by AF, CVS, Cord blood

Double blind

Calculate sensitivity and specificity

Bioinformatics analysis

• Study design and aim:

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)

Unable to produce results (n=79; failed DNA extraction, library construction, or sequencing)

Pregnant women with NIFTY results (n=11105)

Positive (n=190) Negative (n=10915)

0.7% of all samples

99.3% of all samples

• 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

Shan Dan, et al., Prenatal Diagnosis, 2012: p. 1-8.

NIFTY results in 11105 samples

No. of casesPositive Results

Negative resultsT21 T18 T13

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.

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

Other rare aneuploidies

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

Pre-test counseling

Blood collection Bar-coding Plasma

preparation

Laboratory testing

Bioinformatics analysis

Report delivery

Post-test counseling

Hospital

BGI Clinical Laboratories: 10 days

22

Sample Management System

Unique Identification Sample Location Storage Capability

Effective lab space separation

Progressively decreased pressure

Restricted traffic flow

Laboratory System

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 abnormalitiese.g. duplication, deletion, translocation, etc.

If the pregnant woman receives allogenic DNA importation prior to NIFTYe.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 re-sampling 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

Detection of T21, T18, T13 √ √ √Gender √ √ √Twin pregnancies √ √ √Sex chromosome abnormality

× √ √

Microdeletion & Microduplication

× × √

Monogenic disease × × √Turn around time 6 days 14 working days 14 working days

NIFTY express

DataChr21(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)

Performed on Ion Proton platform, extremely fast

Performed in Czech Republic BGI-GENNET co-lab. EU based service.

Similar price

Validated on 1647 samples

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

Non-invasive • 5ml maternal

blood• No risk of

intrauterine infection and miscarriage

Accurate • Sensitivity

>99%• Specificity

>99%

Turnaround time• 10 days

Early detection• 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-HZ, 2001

BGI-Beijing, 1999

BGI-WH, 2010

BGI-SZ, 2007

BGI-HK, 2009

BGI-Americas, 2010

BGI-Europe, 2010

Platforms of BGI

40

Computing and bioinformatics platform

Sequencing platform

Agricultural genomics platform

Cloning platform

Healthcare platform

Microbiology platform

Proteomics platform

Research collaborators worldwide