6/20/2014
Noninvasive Prenatal
Testing (NIPT):
Separate But Not Equal
Charles (Buck) Strom MD, PhD, FAAP, FACMG, HCLD
Senior Medical Director, Genetics
Quest Diagnostics Nichols Institute
San Juan Capistrano, CA
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
Normal Male = 46,XY
• 23 pairs of
chromosomes
• 23rd pair, sex
chromosomes
• Males: XY
• Females: XX
• Each pair
• 1 maternal
• 1 paternal
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
Normal Female = 46,XX
3
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
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6/20/2014
Normal Conception
46
Meiosis
23
sperm
46
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
Definitions
• Disomy = normal number of chromosomes; 46,XX
• Trisomy = gain of 1 chromosome; 47,XY,+21
• Monosomy: loss of one chromosome, 45,XY,-16
• Triploidy: extra entire set of chromosomes: 69,XXY
• Euploidy: a normal set of chromosomes (46)
• Aneuploidy: An abnormal number of chromosomes but
not an abnormal set of chromosomes (all trisomies and
monosomies)
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
47, XX, +21; Trisomy 21 = Down Syndrome
•
•
•
•
•
•
Mental retardation
Facial features
Short stature
Heart problems
Intestinal problems
Other birth defects
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
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6/20/2014
47,XX, + 18;Trisomy 18 = Edwards Syndrome
• Reduced survival
(<10% beyond first
year)
• Severe to profound
mental retardation
• Rocker bottom feet
• Heart defects
• Clenched hands
• Other birth defects
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
47,XX, + 13; Trisomy 13 = Patau Syndrome
• <5-10% survive beyond one
year
• Severe to profound mental
retardation
• Extra fingers/toes
• Cleft lip
• Brain malformation
• Heart defects
• Other birth defects
45,X; Monosomy X = Turner Syndrome
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6/20/2014
NIPT: A New Method for Prenatal Aneuploidy Screening
Prenatal
Screening
MSS &
Ultrasound
Prenatal
Diagnostics
CVS or Amniocentesis
NIPT
Karyotype
CGH or SNP
prenatal
FISH
• NIPT detects cell-free fetal DNA (cfDNA) from the plasma of pregnant
women to screen for fetuses affected with trisomy 21, 18 and 13
• NIPT currently is considered as screening test; not diagnostic
• False positives and negatives do occur, meaning an invasive test is still
required to confirm any positive result
• Unlike CVS or amniocentesis, it is a non-invasive test by using the
mother’s blood to test fetal aneuploidy
Cell-Free Fetal DNA in Maternal Circulation
Discovery of cell-free fetal
DNA in the maternal
circulation in 1997*
Initial use:
• RhD blood group
• Fetal gender
• Single-gene disorders
2008 publications drove
implementation of this new
technology into clinical
practice
* Lo YM, et al., Lancet 1997; 350:485-7
How Does NIPT Work?
• Maternal blood (1-2 tubes) collected in special (Streck™) tubes
• Centrifuged to remove cells and cellular debris
• Free floating DNA is analyzed
– 4 different methods
• Quantitative (Z score method)
– Sequenom
» Shotgun whole genome sequencing with fetal fraction assessment
– Verinata
» Shotgun (deeper) whole genome sequencing without fetal fraction assessment
– Ariosa
» Targeted amplification plus fetal fraction by SNP frequency
• Non-quantitative SNP Based
– Natera: B allele frequency analysis with fetal fraction determination
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
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6/20/2014
NIPT Approaches and Methodologies
Targeted Sequencing
MPSS
Sequenom
(MaterniT21)
Verinata
(Verifi)
PerkinElmer
Ariosa
(Harmony)
LabCorp
QUANTITATIVE METHOD (Z‐Score)
Targeted Sequencing
Natera
(Panorama™)
Quest Diagnostics
SNP
METHOD
Dial-in: (888) 283-3954; International: (773) 756-0601; Passcode: 3805089
What is a Z Score?
• Knowing the size of the chromosomes, one can predict
the frequency that any sequence will occur in the
genome. For example, how many times a chromosome
21 sequence should occur when compared to a
sequence on a non-variant chromosome
• A Z-score calculates the ratio of observed sequences
from a given chromosome versus a non-variant
chromosome
• In a Trisomy 21 conception, the number of times a
chromosome sequence is increased, leads to a larger Zscore
Different Z Scores for Different Folks
• Whole genome shotgun sequencing
– Sequenom and Verinata
• Targeted sequence analysis (non-polymorphic)
– Ariosa
• All Z score (quantitative) suffer from 2 limitations
– Sequencing bias
• Chromosome 13 and 18 have high GC content and are
underrepresented in Illumina sequencing - leads to lower
sensitivity and specificity for those chromosomes
– With low fetal fractions can give undetected false negative
results
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Counting “Z Score Method”
Chromosome 3
Chromosome 21
6.2% of genome
1.5% of genome
Counting
Chromosome 21
Chromosome 3
1.5% of genome
6.2% of genome
Expected Ratio: 20% 80% Observed Ratio:
25% 75%
Counting “Z” Score Method
• Reasons for a “normal” count for chromosome 13
– Diploid (normal) fetus
– Trisomy 13 fetus
• Under amplification / sequencing of chromosome 13
• Low fetal fraction
– Reasons for an increased count for chromosome 13
• Trisomy 13
• Over correction for sequencing bias for chromosome 13
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Accuracy of Z Score Falls with Decreasing Fetal Fraction
Relative amount of DNA mapping to chromosome of interest
120
Z Score
100
Fetal Disomy
Fetal Trisomy
Maternal
80
60
40
20
0
15%15%
10%10%
8%
5% 5%
8%
3% 3%
Low Fetal cfDNA Fractions Decrease Detection Rates
•
In this data set (average gestational age 15
weeks) , 10-15% of women had fetal fractions
between 4-8%.1
•
In our internal data, we found that 25% of
women at gestational ages between 9-14
weeks had fetal fractions between 4-8%.2
Panorama
Down
Syndrome
Detection
Rate3
>10%
>99%
>99%
4-8%
75%
>99%
% Fetal DNA
Fraction
35
Fetal Fraction (%)
Counting
Down
Syndrome
Detection
Rate1
40
30
25
20
15
10
5
0
8
10
12
14
16
18
20
22
24
Gestational Age (weeks)
1. Palomaki GE et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med. 2011
Nov; 13(11):913-20
2. Natera internal data
3. Zimmermann, et al. "Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y using targeted sequencing at polymorphic
loci." Prenatal Diagnosis, 2012.
Natera Panorama™ Non-Quantitative
• Amplifies ~ 20,000 POLYMORPHIC SNP’s
• Mother’s DNA is genotyped using buffy coat
• When mother is AA or BB and fetus is AB
– Fetal fraction is directly measurable by frequency of
non-maternal allele in sample
• Mother AA, fetus AB, 10% fetal fraction
–A allele: 1000 (maternal) + 100 (fetal) = 1,100
–B allele: 100 (fetal, paternal)
–% fetal fraction = 100 / 1100 – 100) = 0.10 = 10%
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Natera Panorama™ Non-Quantitative
• For each locus
– Maternal genotype known
– Fetal genotype AA, AB, or BB
– Hypothesis test performed for trisomy, disomy, and monosomy X
• Advantages of non-quantitative technique
– Sequencing bias irrelevant because looking at the same SNP for
maternal and fetal B allele frequency, so accuracy of test does not
decrease for trisomy 13 and trisomy 18
– Accurate measurement of fetal fraction
– Paternal genotyping can “rescue” case with low fetal fraction
– Can provide accurate results with fetal fractions as low as 4% -6%
Advantages of a SNP-based Approach
• SNP = Single nucleotide
polymorphism
• SNP methodology with
19.5k Multiplex PCR probes
• Produces higher quality data
• Ability to achieve high accuracy
results at lower fetal fractions
How Does Panorama Work?
A simplification of Natera’s non-invasive prenatal aneuploidy test
SNP
Plasma = Sequencing
Maternal + Fetal DNA
Maternal blood
SNP
Sequencing
Buffy coat = Maternal DNA
Maternal + Fetal Genotype
Target Fetal
DNA Signal
Fetal Genotype Maternal Genotype 8
6/20/2014
Next-generation Aneuploidy Test Using SNPs (NATUS)
Genotypic data
from Mom (+/- Dad)
Multiple hypotheses for
each chromosome
Data from Human
Genome Project
(HapMap)
+
Each hypothesis describes the
expected sequencing data
for that case
Sub-hypotheses
with different
crossover points
What is Fetal Fraction? Why is it Important?
Fetal fraction is the percent of the cell-free DNA in the mother’s blood that is
from the fetus.
•At 13 weeks gestation, over 20% of pregnancies have a fetal DNA fraction <10%1
•Low fetal DNA fractions decrease detection rates of counting technologies
•Reporting of fetal fraction allows clinician to assess accuracy of the test result
% Fetal DNA
Fraction
Counting Down
Syndrome
Detection Rate2
Natera Down
Syndrome
Detection Rate3
>10%
>99%
>99%
<10%
91%
>99%
<8%
75%
>99%
1. Lo et al. BMJ, Fetal DNA concentration found in maternal plasma among 314 pregnancies with euploid male fetuses.
2011;342:c7401
2 .Palomaki GE et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation
study. Genet Med. 2011 Nov; 13(11):913-20.
3. Zimmermann, et al. "Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y using targeted
sequencing at polymorphic loci." Prenatal Diagnosis, 2012.
Fetal Fraction vs. Gestational Age
45%
Fetal Fraction
40%
35%
30%
25%
20%
FF+1SD
FF-1SD
15%
10%
5%
0%
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Gestational Age (weeks)
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6/20/2014
Counting Versus SNPs
Z Score
– More false negatives at fetal fractions < 10%
– Sequencing bias issue for chromosomes 13 and 18
– Requires reference chromosome(s)
– Does not detect triploidy
SNP (NATUS)
– Sensitivity not affected by fetal fraction down to 4%
– Not affected by sequencing bias
– Fetal fraction accurately determined and reported
– Detects triploidy
Comparison of NIPT Technology Claims
Sensitivity
False Positive
Rate
Methodology
Trisomy 21
(Down Syndrome)
Sequenom
MaterniT21
plus
MarterniT21 plus
Verinata
Verifi
MPSS
MPSS with
SAFeR
Ariosa
Harmony
Natera
PanoramaTM
Targeted Sequencing
with FORTE
Targeted Sequencing
with SNPs
>99.9%
0.2%
>99.9%
0.2%
>99%
<0.1%
>99%
0%
Trisomy 18
(Edwards Syndrome)
>99.9%
0.3%
97.4%
0.4%
>98%
<0.1%
>99%
0%
Trisomy 13
(Patau Syndrome)
91.7%
0.9%
87.5%
0.1%
80%
<0.1%
>99%
0%
Not evaluated
95.0%
1.0%
Not evaluated
>99%
0%
45,X
(Monosomy X)
(1)
(2)
(3)
(4)
Zimmermann et al. Prenat Diag 2012 (2) Natera Internal Data (3) Rabinowitz et al. Presented at ASHG 2012
Downloaded on February 5, 2013 http://www.sequenomcmm.com/Home/Health-Care-Professionals/Trisomy21/Performance-Data
Downloaded on February 5, 2013 http://www.verinata.com/providers/provider-overview/
Downloaded on February 5, 2013 http://www.ariosadx.com
Limitations of NIPT Testing
• Accuracy Reduced for Mosaic Fetuses (All)
• Not applicable to twins (All but Sequenom)
• Not applicable to gestations >=3 (All)
• Confined Placental Mosaicism Potential Problem
– All: Similar to CVS
• Can’t use with donor egg pregnancy (Natera)
– (donor will be detected)
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Professional Recommendations for NIPT (1)
• ACOG / Society for MFM – Joint Statement –
December 2012
– NIPT should be an informed patient choice after pretest
counseling and should not be part of routine prenatal
laboratory assessment. It should not be offered to low-risk
women or women with multiple gestations because it has
not been sufficiently evaluated in these groups. A negative
test result does not ensure an unaffected pregnancy.
A patient with a positive test should be referred for genetic
counseling and should be offered invasive prenatal
diagnosis for confirmation.
Professional Recommendations for NIPT (2)
• NSGC – 2012
– Supports NIPT as an option for patients at an increased
risk for certain chromosome abnormalities. NSGC urges
that NIPT only be offered in the context of informed
consent, education, and counseling by a qualified provider.
Patients whose NIPT results are abnormal, or who have
other factors suggestive of a chromosome abnormality,
should receive genetic counseling and be given the option
of standard confirmatory diagnostic testing.
Professional Recommendations for NIPT (3)
• ISPD – October 2011
– Accepts that, with suitable genetic counseling, NIPT can be
helpful for women who may have been determined to be
high risk by one of the previously recommended screening
strategies. The ISPD does not endorse the ad hoc use of
MPS testing in women at lower risk, outside a formal
protocol that considers the overall best combination of
tests, their impact on screening performance, and patient
acceptability.
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6/20/2014
NIPT: Current Status
• Recommendations that NIPT can be offered as an
alternative to invasive prenatal diagnosis to couples at
risk for aneuploidies
– Advanced maternal age (AMA, >35 yrs at delivery)
– High-risk maternal serum screening test
– Abnormality on ultrasound
– Previous child or fetus with aneuploidy
– Robertsonian translocation carriers
• Recommend invasive prenatal diagnosis for all positives
Positive Predictive Value
• Varies with Specificity and Prevalence
• Assume 99.9% Specificity
– Tri 21*: Prevalence 1: 270:
PPV = 75%
– Tri 18*: Prevalence 1: 470:
PPV = 67%
– Tri 13*: Prevalence 1: 1,500
PPV = 37%
*
Gardner et al, Chromosomal Abnormalities and Genetic Counseling, 4th
Edition, Oxford Press, NY, 2012
Expanding Content - Microdeletions
• Natera and Sequenom have added DiGeorge
and other microdeletions to NIPT
• Ariosa and Verinata do not offer
• Rapidly moving target.
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6/20/2014
Thank Your Attention
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