DNA (cffDNA) is fetal
DNA which circulates freely in
the maternal blood. Maternal blood is sampled by venipuncture.
Analysis of cff
DNA is a method of non-invasive prenatal diagnosis
frequently ordered for pregnant women of advanced maternal age.
2 Laboratory methods
2.1 Separation of cffDNA
2.2 Analysis of cffDNA
2.2.1 Quantitative real-time PCR
2.2.2 Nested PCR
2.2.3 Digital PCR
2.2.4 Shotgun sequencing
2.2.5 Mass spectrometry
2.2.6 Epigenetic modifications
3.1 Prenatal sex determination
3.2 Single gene disorders
3.3 Hemolytic disease of the fetus and newborn
4 Future perspectives
5 See also
Cell free fetal
DNA sheds into the maternal blood circulation
DNA originates from placental trophoblasts. Fetal
fragmented when placental microparticles are shed into the maternal
blood circulation (figure 1).
DNA fragments are approximately 200 base pairs (bp) in length. They
are significantly smaller than maternal
DNA fragments. The
difference in size allows cff
DNA to be distinguished from maternal DNA
Approximately 11 to 13.4 percent of the cell-free
DNA in maternal
blood is of fetal origin. The amount varies widely from one pregnant
woman to another. cff
DNA is present after five to seven weeks
gestation. The amount of cff
DNA increases as the pregnancy
progresses. The quantity of cff
DNA in maternal blood diminishes
rapidly after childbirth. Two hours after delivery, cff
DNA is no
longer detectable in maternal blood.
Analysis of cff
DNA may provide earlier diagnosis of foetal conditions
than current techniques. As cff
DNA is found in maternal blood,
sampling carries no associated risk of spontaneous
DNA analysis has the same ethical and
practical issues as other techniques such as amniocentesis and
chorionic villus sampling.
Some disadvantages of sampling cff
DNA include a low concentration of
DNA in maternal blood; variation in the quantity of cff
individuals; a high concentration of maternal cell free
to the cff
DNA in maternal blood.
New evidence shows that cff
DNA test failure rate is higher, fetal
fraction (proportion of fetal versus maternal
DNA in the maternal
blood sample) is lower and PPV for trisomies 18, 13 and SCA is
decreased in IVF pregnancies compared to those conceived
A maternal peripheral blood sample is taken by venesection at about
ten weeks gestation.
Separation of cffDNA
Blood plasma is separated from the maternal blood sample using a
laboratory centrifuge. The cff
DNA is then isolated and purified.
In 2007, a standardized protocol for doing this was written by Legler
et al through an evaluation of the scientific literature. The highest
yield in cff
DNA extraction was obtained with the "QIAamp DSP Virus
Addition of formaldehyde to maternal blood samples increases the yield
of cffDNA. Formaldehyde stabilizes intact cells, and therefore
inhibits the further release of maternal DNA. With the addition of
formaldehyde, the percentage of cff
DNA recovered from a maternal blood
sample varies between 0.32 percent and 40 percent with a mean of 7.7
percent. Without the addition of formaldehyde, the mean percentage
DNA recovered has been measured at 20.2 percent. However, other
figures vary between 5 and 96 percent.
Recovery of cff
DNA may be related to the length of the
Another way to increase the fetal
DNA is based on physical length of
DNA fragments. Smaller fragments can represent up to seventy percent
of the total cell free
DNA in the maternal blood sample.
Analysis of cffDNA
In real-time PCR, fluorescent probes are used to monitor the
accumulation of amplicons. The reporter fluorescent signal is
proportional to the number of amplicons generated. The most
appropriate real time PCR protocol is designed according to the
particular mutation or genotype to be detected. Point mutations are
analysed with qualitative real time PCR with the use of allele
specific probes. insertions and deletions are analyzed by dosage
measurements using quantitative real time PCR.
DNA may be detected by finding paternally inherited
via polymerase chain reaction (PCR).>
Quantitative real-time PCR
In 2010, Hill et al analyzed the sex-determining region Y gene (SRY)
Y chromosome short tandem repeat "DYS14" in cff
DNA from 511
pregnancies using a quantitative real-time PCR (RT-qPCR). In 401 of
403 pregnancies where maternal blood was drawn at seven weeks
gestation or more, both segments of
DNA were found.
In 2001, Al-Yatama et al evaluated the use of nested polymerase chain
reaction (nested PCR) to determine sex by detecting a Y chromosome
specific signal in the cff
DNA from maternal plasma. Nested PCR
detected 53 of 55 male fetuses. The cff
DNA from the plasma of 3 of 25
women with female fetuses contained the Y chromosome-specific signal.
The sensitivity of nested PCR in this experiment was 96 percent. The
specificity was 88 percent.
Microfluidic devices allow the quantification of cff
DNA segments in
maternal plasma with accuracy beyond that of real-time PCR. Point
mutations, loss of heterozygosity and aneuploidy can be detected in a
single PCR step. Digital PCR can differentiate between
maternal blood plasma and fetal
DNA in a multiplex fashion.
High throughput shotgun sequencing using tools such as Solexa or
Illumina, yields approximately 5 million sequence tags per sample of
maternal serum. In 2008, Fan et al identified aneuploid pregnancies
such as trisomy when testing at the fourteenth week of gestation. In
2010, fetal whole of genome mapping by parental haplotype analysis was
completed using sequencing of cff
DNA from maternal serum. Chiu et
al. in 2010 studied 753 pregnant females, using a 2-plex massively
parallel maternal plasma
DNA sequencing and trisomy was diagnosed with
z-score greater than 3. The sequencing gave sensitivity of 100
percent, specificity of 97.9 percent, a positive predictive value of
96.6 percent and a negative predictive value of 100 percent.
Matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometry (MALDI-TOF MS) combined with single-base extension after
PCR allows cff
DNA detection with single base specificity and single
DNA molecule sensitivity.
DNA is amplified by PCR. Then, linear
amplification with base extension reaction (with a third primer) is
designed to anneal to the region upstream from the mutation site. One
or two bases are added to the extension primer to produce two
extension products from wild-type
DNA and mutant DNA. Single base
specificity provides advantages over hybridization-based techniques
TaqMan hydrolysis probes. In 2008, when assessing the technique,
Ding et al found no false positives or negatives when looking for
DNA to determine fetal sex in sixteen maternal plasma samples.
In 2010, Akolekar et al correctly detected the sex of ninety of
ninety-one male foetuses using MALDI-TOF mas spectrometry. The
technique had accuracy, sensitivity and specificity of over 99
Differences in gene activation between maternal and fetal
DNA can be
exploited. epigenetic modifications (heritable modifications that
change gene function without changing
DNA sequence) can be used to
detect cffDNA. The hypermethylated RASSF1A promoter is a
universal fetal marker used to confirm the presence of cffDNA. In
2012, White et al described a technique where cff
DNA was extracted
from maternal plasma and then digested with methylation-sensitive and
insensitive restriction enzymes. Then, real-time PCR analysis of
RASSF1A, SRY, and DYS14 was done. The procedure detected 79 out of
90 (88 percent) maternal blood samples where hypermethylated RASSF1A
RNA transcripts from genes expressed in the placenta are detectable
in maternal plasma. In this procedure, plasma is centrifuged so an
aqueous layer appears. This layer is transferred and from it
RT-PCR is used to detect a selected expression of RNA. For
Human placental lactogen
Human placental lactogen (hPL) and beta-hCG m
RNA are stable
in maternal plasma and can be detected. (Ng et al. 2002). This can
help to confirm the presence of cff
DNA in maternal plasma.
Prenatal sex determination
X-linked genetic disorder
The analysis of cff
DNA from a sample of maternal plasma allows the
determination of fetal gender. Whether the sex of the fetus is male or
female allows the determination of the risk of a particular X-linked
recessive genetic disorder in a particular pregnancy, especially where
the mother is a genetic carrier of the disorder. Most X-linked
diseases are evident in males because of the lack of the second
X-chromosome that can compensate for the disease allele. Common
X-linked recessive disorders include Duchenne muscular dystrophy,
fragile X syndrome and haemophilia.
In comparison to obstetric ultrasonography which is unreliable for sex
determination in the first trimester and amniocentesis which carries a
small risk of miscarriage, sampling of maternal plasma for analysis of
DNA is without risk. The main targets in the cff
are the gene responsible for the sex-determining region Y protein
(SRY) on the
Y chromosome and the DYS14 sequence.
Congenital adrenal hyperplasia
In congenital adrenal hyperplasia, the adrenal cortex lacks
appropriate corticosteroid synthesis, leading to excess adrenal
androgens and affects female fetuses. There is an external
masculinization of the genitalia in the female fetuses. Mothers of
at risk fetuses are given dexamethasone at 6 weeks gestation to
suppress pituitary gland release of androgens.
If analysis of cff
DNA obtained from a sample of maternal plasma lacks
genetic markers found only on the Y chromosome, it is suggestive of a
female fetus. However, it might also indicate a failure of the
analysis itself ( a false negative result). Paternal genetic
polymorphisms and sex-independent markers may be used to detect
cffDNA. An high degree of heterozygosity of these markers must be
present for this application.
DNA paternity testing is commercially available. The test can
be performed at nine weeks gestation.
Single gene disorders
Autosomal dominant and recessive single gene disorders which have been
diagnosed prenatally by analysing paternally inherited
cystic fibrosis, beta thalassemia, sickle cell anemia, spinal muscular
atrophy, and myotonic dystrophy. Prenatal diagnosis of single
gene disorders which are due to an autosomal recessive mutation, a
maternally inherited autosomal dominant mutation or large sequence
mutations that include duplication, expansion or insertion of DNA
sequences is more difficult.
In cffDNA, fragments of 200 – 300 bp length involved in single gene
disorders are more difficult to detect.
For example, the autosomal dominant condition, achondroplasia is
caused by the FGFR3 gene point mutation. In 2007, a study of two
pregnancies with a fetus with achondroplasia found a paternally
inherited G1138A mutation from cff
DNA from a maternal plasma sample in
one and a G1138A de novo mutation from the other.
In studies of the genetics of
Huntington's chorea using q
DNA from maternal plasma samples, CAG repeats have been detected at
normal levels (17, 20 and 24).
DNA may also be used to diagnose single gene
disorders.Developments in laboratory processes using cff
allow prenatal diagnosis of aneuploidies such as trisomy 21 (Down's
syndrome) in the fetus.
Hemolytic disease of the fetus and newborn
Incompatibility of fetal and maternal RhD antigens is the main cause
of Hemolytic disease of the newborn. Approximately 15 percent of
Caucasian women, 3 to 5 percent of black
Africa women and less than 3
percent of Asian women are RhD negative.
Accurate prenatal diagnosis is important because the disease can be
fatal to the newborn and because treatment including intramuscular
immunoglobulin (Anti-D) or intravenous immunoglobulin can be
administered to mothers at risk.
In 2010, Cardo et al reported that PCR to detect
RHD (gene) gene exons
5 and 7 from cff
DNA obtained from maternal plasma between 9 and 13
weeks gestation gave a high degree of specificity, sensitivity and
diagnostic accuracy (>90 percent) when compared to RhD
determination from newborn cord blood serum. In 2013, Aykute et al
found similar results targeting exons 7 and 10. In 2015, Svobodova
et al reported that droplet digital PCR in fetal RhD determination was
comparable to a routine real-time PCR technique.
Routine determination of fetal RhD status from cff
DNA in maternal
serum allows early management of at risk pregnancies while decreasing
unnecessary use of Anti-D by over 25 percent.
Analysis of maternal serum cff
DNA by high-throughput sequencing can
detect common fetal sex chromosome aneuploidies such as Turner's
Klinefelter's syndrome and triple X syndrome but the
procedure's positive predictive value is low.
Fetal trisomy of chromosome 21 is the cause of Down's syndrome. This
trisomy can be detected by analysis of cff
DNA from maternal blood by
massively parallel shotgun sequencing (MPSS). Another technique is
digital analysis of selected regions (DANSR). However, such tests
show inconsistent degrees of sensitivity and specificity and therefore
may be best used to confirm a positive maternal screening test such as
ultrasound markers of the condition..
Trisomy 13 and 18
Analysis of cff
DNA from maternal plasma with MPSS looking for trisomy
13 or 18 is possible
Factors limiting sensitivity and specificity include the levels of
DNA in the maternal plasma; maternal chromosomes may have
A number of fetal nucleic acid molecules derived from aneuploid
chromosomes can be detected including SERPINEB2 mRNA, clad B,
hypomethylated SERPINB5 from chromosome 18, placenta-specific 4
(PLAC4), hypermethylated holocarboxylase synthetase (HLCS) and
RNA from chromosome 12. With complete trisomy, the mRNA
alleles in maternal plasma isn't the normal 1:1 ratio, but is in fact
2:1. Allelic ratios determined by epigenetic markers can also be used
to detect the complete trisomies. Massive parallel sequencing and
digital PCR for fetal aneuploidy detection can be used without
restriction to fetal-specific nucleic acid molecules. (MPSS) is
estimated to have a sensitivity of between 96 and 100%, and a
specificity between 94 and 100% for detecting Down syndromeIt can be
performed at 10 weeks of gestational age. One study in the United
States estimated a false positive rate of 0.3% and a positive
predictive value of 80% when using cff
DNA to detect Down syndrome.
Preeclampsia is a complex condition of pregnancy involving
hypertension and proteinuria usually after 20 weeks gestation. It
is associated with poor cytotrophoblastic invasion of the myometrium.
Onset of the condition between 20 and 34 weeks gestation, is
considered "early". Maternal plasma samples in pregnancies
complicated by preeclampsia have significantly higher levels of cffDNA
that those in normal pregnancies. This holds true for
early onset preeclampsia.
New generation sequencing may be used to yield a whole genome sequence
from cffDNA. This raises ethical questions. However, the utility
of the procedure may increase as clear associations between specific
genetic variants and disease states are discovered.
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