The nature of free fetal DNA and

detection by real time PCR in prenatal

tests
Group 5

Cell free fetal DNA (cffDNA)

1969- Discovery of fetal cells

in maternal circulation
(Walknowska J et al)
1997- Discovery of cffDNA in
maternal blood (Lo YMD et
al)
Sampled by venipuncture on
the mother. Analysis of
cffDNA provides a method of
non-invasive prenatal
diagnosis.

cffDNA originates from the trophoblasts making up the placenta.

Fetal sex determination using cffDNA is a well-established prenatal test that
can be carried out.
The most common clinical indication for early fetal sex determination is for
carriers of X-linked genetic disorders.

Advantages of cffDNA
Safer than current invasive approaches
Can also be performed much earlier in pregnancy, from as little as 6-7

weeks in the first trimester (compared with second trimester for CVS and
amniocentesis), making it a highly desirable tool for clinical genetics and
other antenatal-related services
Could allow improved (safer, earlier and cheaper) antenatal screening
for many serious genetic diseases.
Enable doctors to monitor pregnancies much more effectively for serious
complications that can affect the health or survival of both the fetus and
the mother.
Allows planning for further tests, and/or possible termination. Also helps
determine whether an invasive test is needed.
Allow diagnosis of aneuploidies (Downs syndrome, Edwards syndrome,
and patau syndrome are most common ones) without invasive routes.
However , the fetal DNA is fragmented, so that complete fetal genotyping
may not be possible, and genetic diseases that involve large expansions
of DNA would not be amenable to diagnosis using free fetal DNA.

Cell free fetal DNA

Why it has replaced use of foetal cells for NIPT?
Less false positives during screening

When was it discovered?

1997

What is it?
Circulating fetal DNA in maternal blood stream

What size is it?

286 28 bp

Where does it originate from?

Trophoblastic cells

When is free fetal DNA found?

ffDNA is detectable from 18 days after embryo

transfer in assisted reproduction (Lo et al, 1998)

The ffDNA increases as the pregnancy progresses
(Lo and Rossa, 2007)
It cannot be acquired after delivery
Some research had found that cffDNA is more
amplified in the second trimester in down syndrome
male fetuses (Farina et al, 2003)

How much ffDNA is found?

Makes up 3-6 per cent of the total cell free DNA in

the maternal circulation

Many studies find that ffDNA is higher in abnormal
pregnancies.
However the results in the Alberry et al paper
contradict this.
(Alberry et al, 2007) (Lo and Rossa, 2007)

Alberry et al, 2007

What is it used for?

For non-invasive pre-natal diagnosis
Identification of pregnancies that are at risk of

HDFN (haemolytic disease of the fetus and

newborn)
Sexing for mothers who are carriers of x-linked or
other genetic diseases
Confirm structural abnormalities on scans. E.g.
achondroplasia
Identifying one gene or chromosome abnormalities
Only possible to look at specific mutation due to
limited volume of ffDNA

Why is ffDNA used in comparison to

ffRNA
ffRNA is a possible candidate for producing new

biomarkers in aneuploidies
Fetal RNA or ffRNA may be favoured over ffDNA
because it removes gender and inheritance
limitations
It also demonstrates which genes are actually
expressed and not just the make up of genes
Gene expression
(Maddocks et al, 2009)
Post genomics paper

What is real time PCR?

Polymerase Chain Reaction

In vitro amplification of DNA

Two reasons
To create multiple copies of a rare piece of DNA
More commonly, to compare 2 different samples of DNA

to see which is the more abundant

Stages of PCR:
Denaturation
94-96C
30 secs

Annealing

Conditions:
Reaction buffer
Oligonulceotides
Template DNA

50-65 C

dNTPs

30 secs

Thermostable DNA

Extension
70-72 C
1 minute

polymerase

How does it differ to end point PCR?

After amplifying your gene it is possible to run the amplified DNA out on an agarose gel and stain it with a
dye which makes it visible. The brighter the visible band, the more copies of your target you have created.

Disadvantages of end point PCR:

Very time consuming. Results may not

be obtained for days

Results are based on size
discrimination poor precision
The end point is variable from sample
to sample
Low sensitivity
Short dynamic range < 2 logs
Low resolution - about 10 fold
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not
very quantitative
Post PCR processing

Why is real time PCR used in an antenatal setting?

Fetal rhesus-D genotyping
Fetal sexing for X-linked disorders
Paternally inherited genetic diseases
Pregnancy-associated conditions such as

preeclampsia

What are the different assays utilised?

Test

Testing for

Fetal rhesus-D genotyping

Paternally inherited allele

Rh locus - two homologous genes
RHD and RHCE closely linked on
chromosome 1p34-p36
Each gene consists of 10 exons
containing 69 kb of DNA.
Regions of exon 7 & 10 within the
RhD gene are the areas of focus.

Fetal sexing for X-linked disorders

DYS14 and SRY genes on a Y

chromosome

RT-PCR
Controls- determining sex
CCR5- positive control
SRY- specific Y probe

Sensitivity and specificity

Many studies of fetal sex determination:
98% specificity and 100% sesnititvity
RhD determining
94-99.5% specificity and 99.5% sensitivity

Low cost to perform

Save time and reagent costs

Fetal chromosome dosage assessment

Down Syndrome (Trisomy 21)
Methylated DNA immunoprecipitation (MeDiP)
Investigates DNA methylation pattern
uses an antibody specific for 5-methylcytidine to

capture methylated sites and therefore enriching for

fetal-specific methylated DNA
To provide chromosome dosage information, the

ffDNA has to be hypermethylated compared to the

maternal DNA
100% sensitivity and specificity

Novel Research
A novel Alu-based real-time PCR method for the
quantitative detection of plasma circulating cell-free
DNA: Sensitivity and specificity for diagnosis of
myocardial infraction

Lou, X. et al (2014)

 cfDNA

- prenatal screening for down syndrome (Nicolaides

KH et al, 2013 ; Johnson J et al, 2013)
Alu

- Alu-based real-time PCR may be a potentially

sensitive approach for the measurement of the human
cDNA in blood

 Aim: To determine whether Alu-based real-time PCR

can serve as an effective tool for the detection of

cfDNA
Conclusion: This Alu-based assay was reliable,

accurate and sensitive method for the quantitative

detection of cfDNA and that it is useful for studying
the regulation of cfDNA in certain pathological
conditions.

Macher et al (2012)
Standardization non-invasive fetal RHD and SRY

determination into clinical routine using a new

multiplex RT-PCR assay for fetal cell-free DNA in
pregnant women plasma
Evaluation of fetal RHD in the pregnant plasma
using multiplex real time PCR
Single and multiplex real-time PCR results were
compared with postnatal serology and sex
identification.
The assay is 100% sensitive to detect RHD positive
fetuses

References

Alberry et al (2007), Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin
is the trophoblast, Prenatal Diagnosis, 27: 415-418
Farina et al (2003), Evaluation of Cell-free Fetal DNA as a Second-Trimester Maternal Serum Marker of Down
Syndrome Pregnancy, Clinical Chemistry, 49 (2) : 239-242
Hill, M., Barrett, A., White, H. and Chitty, L. (2012). Uses of cell free fetal DNA in maternal circulation. Best
Practice & Research Clinical Obstetrics & Gynaecology, 26(5), pp.639-654.
Maddocks et al (2009), The SAFE project: towards non-invasive prenatal diagnosis, Biochemical Society
Transactions, 37(2); 460-465

Macher,H., Noguerol, P., Medrano-Campillo, P., Garrido-Mrquez, M., Rubio-Calvo, A., Carmona-Gonzlez,
M., Martin-Snchez, J., Prez-Simn, J. andGuerrero, J. (2012) Standardization non-invasive fetal RHD
and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in
pregnant women plasma: Results in clinical benefits and cost saving, Clinica Chimica Acta, 413( 34),
pp. 490-494 [Online]. Available at:
http://www.sciencedirect.com/science/article/pii/S0009898111006310

Lo and Rossa (2007) Prenatal Diagnosis: progress through plasma nucleic acids, Persceptives, 8:71-76
Lo, Y., Corbetta, N., Chamberlain, P., Rai, V., Sargent, I., Redman, C. and Wainscoat, J. (1997). Presence of fetal
DNA in maternal plasma and serum. The Lancet, 350(9076), pp.485-487.
Why: http://www.rapid.nhs.uk/guides-to-nipd-nipt/nipt-for-down-syndrome/
Discovery: http://www.bionews.org.uk/page_38015.asp
What is: http://en.wikipedia.org/wiki/Cell-free_fetal_DNA
What size: http://www.ncbi.nlm.nih.gov/pubmed/21928694
Origin: http://www.ncbi.nlm.nih.gov/pubmed/17286310
Dr. Phillipa Brice, Bionews, 2009 http://www.bionews.org.uk/page_38052.asp