CIRCULATION NUCLEAR ACID
AND
NIPT
Outlines
■ Cell-free fetal DNA (cffDNA)
■ Non-invasive prenatal testing (NIPT)
– Methodological approaches
– Clinical implementation
o False-negative rates
o False-positive rates
– Analytical aspects
■ Other diagnostic applications of circulating cffDNA
Fetal Cells in Maternal Blood
■ Schmorl observed the presence of trophoblasts in the lung tissues of
women who died of preeclampsia in 1893
■ Lo, et al detection of chromosome Y DNA sequences in the blood of
women pregnant with male fetuses in 1989
■ Intact fetal cells are rarely present in maternal circulation, with about
just one cell per milliliter of maternal blood
■ To date, the identification and isolation of intact fetal cells in maternal
circulation have remained difficult
Cell-Free Fetal DNA in Maternal Plasma
■ Lo, et al detected chromosome Y DNA in the plasma and serum of
women who were pregnant with male fetuses but not in women with
female fetuses in 1997
Biological Properties of cffDNA
■ cffDNA is a by-product of placental cell turnover
– epigenetic markers specific to the placenta are detectable in
maternal plasma
– chromosomal abnormalities confined to the placenta are
detectable in maternal plasma
Biological Properties of cffDNA
■ cffDNA is detectable in early pregnancy
■ It is most reliably detected from the late first trimester onward
■ cffDNA has rapid clearance kinetics and an apparent half-life of 1
hour
Biological Properties of cffDNA
■ cffDNA coexists in maternal plasma with a major background of
maternal DNA
■ cffDNA amounts to some 10% to 20% of the total DNA in maternal
plasma
Biological Properties of cffDNA
■ cffDNA is highly fragmented and is generally less than 200 bp long
NIPT
(NON–INVASIVE PRENATAL TESTING)
■ Testing of cffDNA from maternal blood during
pregnancy or trisomy 21, 18 and 13
Methodological Approaches
Methodological Approaches
% of Genome
Relative Size of Chromosomes
8%
7%
6%
5%
4%
3%
2%
1%
0%
1
3
10 13 18 20 21
Chromosome
X
Y
Counting
Chromosome
21
Chromosome
3
Counting
Chromosome 21
Expected Amount:
Observed Amount:
20%
25%
Chromosome 3
80%
75%
Clinical Implementation
Screening test
Test info
First Trimester Screening MA, NT, PAPP-A, betahCG
Integrated prenatal
MA, NT, PAPP-A, AFP,
screening
uE3, hCG
Quad/maternal serum
MA, AFP, uE3, total
screening
hCG, inhibin
Quad Screening Serum
MA, PAPP-A, AFP, betaIntegrated Prenatal
hCG/total hCG, uE3,
Screen
inhibin
NIPT
+/-MA, cfDNA
1 Prenatal Screening Ontario
2 Bianchi et al 2014 N Engl J Med 370:9
3 Norton et al 2015 N Engl J Med 372:17
4 Gil et al 2015 Ultrasound Obstet Gynecol 45
Detection rate /
Sensitivity for T211,4
False Positive Rate
for T211,4
80-85%
3-9%
85-90%
2-4%
75-85%
5-10%
80-90%
2-7%
>99%
<0.1%
Positive predictive value
for T212,3
~4%
~80.9% for all populations
(high and low risk women)
Clinical Implementation
Aneuploidy
Detection Rate
False Positive Rate
Trisomy 18
Trisomy 13
96.3%
91.0%
0.13%
0.13%
Monosomy X (Turner syndrome)
90.3%
0.14%
Other sex chromosome
aneuploidies
93.0%
0.14%
Clinical Implementation
■ cffDNA assessment is a screening procedure and not a
definitive diagnosis
■ All clinical guidelines recommend that cffDNA tests with
positive findings suggestive of chromosomal aneuploidy
should be confirmed by tests on fetal genetic material
collected by conventional invasive methods such as
chorionic villus sampling or amniocentesis.
False-negative results
■ Low fetal fraction
– Between 9 to 13 weeks of gestation, 2% of pregnancies show fetal DNA
fractions that are below 5%
■ Mosaicism
– In genetics, mosaicism, involves the presence of two or more populations of
cells with different genotypes in one individual, who has developed from a
single fertilized egg
■ absence of the abnormality in the placenta
False-positive results
■ Statistical reasons
– choice of a cutoff value for aneuploidy detection influences the
theoretical false-positive rate of the test
■ Confined placental mosaicism
■ maternal DNA abnormalities
– 8.6% of the sex chromosome aneuploidies detected by cffDNA
testing occur when the maternal blood cell DNA showed the same
finding
– other non-pregnancy-related diseases
NIPT Indications
■
Increased maternal age
■ Increased risk on Combination or triple test
■ Anxiety for invasive procedure (AC / CVS)
NIPT Contraindications
■ Fetal anomalies on ultrasound
■ A triplet pregnancy
■ Known genetic anomalies that cannot be diagnosed by
NIPT
Advantages of NIPT
■ High sensitivity (few false-negatives)
■ High specificity (few false-positives)
■ Non-invasive : no fetal risk
– CVS : Risk of miscarriage : 1-2 %
– AC : Risk of miscarriage : 0.5 %
Disadvantages of NIPT
■ Expensive (750 – 1200 $)
■ Only testing 3 chromosomes, and gender
■ Failure rate : < 1 %
Analytical Aspects
■ Maternal Blood Sample Collection and Processing
– maximizing the fetal DNA fraction in the maternal sample is a key
factor
■ maternal plasma is preferred over maternal serum
■ EDTA is preferred than heparin and citrate
■ manufacture blood collection tubes containing proprietary cell-stabilizing
reagents
■ two-step centrifugation protocols are recommended
Analytical Aspects
■ Circulating Cell-Free Fetal DNA Analysis
 Improve the detection of fetal DNA
o Used size-selection methods to increase the proportional representation of
short DNA within the sample or dataset
 Consider approaches to further minimize the effect of the
maternal DNA interference.
 Include an internal control to indicate the presence of fetal DNA or
measure the fetal DNA fraction.
 Maximize the signal-to-noise ratio of the massively parallel
sequencing protocols used for cffDNA analysis
Fetal Sex Assessment
■ Useful for the prenatal management of diseases with sexlinked patterns of inheritance and conditions
– congenital adrenal hyperplasia
– hemophilia and Duchenne muscular dystrophy
Fetal Rhesus D Status Determination
Rh Incompatibility
Fetal Rhesus D Status Determination
■ The great majority of RhD-negative individuals lack the RhD gene, due
to gene deletion.
■ Advantages
– Free from the risk of inducing fetomaternal hemorrhage
– Reduce further sensitization
– Minimize the unnecessary use of the scarce and expensive anti-D
immunoglobulin
Single-Gene Diseases
■ Noninvasive prenatal diagnosis of autosomal dominant diseases of paternal origin
may be achieved in a similar manner to fetal rhesus D genotyping
■ When a paternal mutation is detected in the plasma of a mother known not to share
the same mutation as the father, this may imply that the fetus has inherited the
paternal mutation
Take Home Messages
■ Cell-free fetal DNA in the maternal circulation are a reliable and
noninvasive source of fetal genetic material
■ NIPT led to a major reduction in the number of amniocenteses
performed worldwide, causing a paradigm shift in prenatal diagnosis
■ cffDNA analysis has the potential to detail the entire fetal genome
noninvasively before the birth of the child