Free Fetal DNA

Prof Khaled
Background and Introduction

Fetal cells in the maternal circulation are a source
of fetal material for safer noninvasive prenatal
diagnosis (NIPD) but it is now recognised that this
approach is unlikely to be clinically useful.

In 1997, Cell-free fetal DNA (cffDNA) in the
maternal circulation Identified

Current prenatal diagnosis of fetal genetic
status or aneuploidy uses invasive
diagnostic tests which carry a small but
significant risk of miscarriage.

Detected from 4 weeks of gestation

Fetal component contributing around 3%
in early pregnancy, rising to 6% towards
term.

Current situation
Current methodologies do not allow complete
separation.

Current applications focus on the detection or
exclusion of genes not present in the mother, such
as Y chromosome sequences or rhesus D (RHD) in
RhD-negative women.

Following extraction of total cffDNA---- Real-time PCR
is used to amplify the gene in question.
e.g. RHD in an RhD-negative woman, or SRY or DYS14 if
undertaking fetal sex determination. A positive signal
would indicate the fetus was RhD-positive or male,
respectively, but no signal would indicate that the fetus
did not carry the target gene and was RhD-negative or
female, respectively.
Failure to obtain a signal could, of course, be a result of
a failure to amplify the fetal DNA in the sample and
work is continuing to identify universal fetal markers to
use as internal standards.
Fetal RHD typing

Since 2001, fetal RHD typing using cffDNA has
been used to direct management in RhD-women
at increased risk of haemolytic disease of the
newborn (HDN) because of a previous affected
pregnancy or elevated antibody titre.

NIPD has almost completely replaced
amniocentesis or chorionic villus.

Determination of other fetal RhC, c, E and Kell
blood groups using cffDNA has been reported.
Fetal RHD typing

Recently, the International Blood Group
Reference Laboratory in Bristol has reported the
successful development of a high-throughput
methodology using automated robotic techniques
that is potentially suitable for mass screening of all
women who are RhD-negative.
Fetal sex determination

Fetal sex can be determined by cffDNA in
maternal plasma & the identification of genes
(DYS14 or SRY) on the Y chromosome.

In the UK, it is in women at risk of X-linked
disorders where early identification of a male
fetus indicates a need for an invasive diagnostic
test to determine whether the affected X
chromosome has been inherited but no invasive
test is required if the fetus is female.

Fetal sex determination
Pregnancies at risk of (CAH) where early
treatment of pregnancies with an affected female
fetus has been shown to reduce the degree of
virilisation of the external genitalia.

NIPD reduce the uptake of invasive diagnostic
testing by nearly 50% & allowed for early
cessation of dexamethasone treatment in
pregnancies at risk of CAH where the fetus is
found to be male.
Fetal sex determination

A recent national audit demonstrated
concordance rate of 97.8% between sex reported
using cffDNA performed at or after 7 weeks and
sex confirmed at invasive testing or birth.

Testing before 7 weeks was less accurate.

Direct-to-consumer NIPD fetal sex testing is
available in the USA but w/o testing standards
from these providers.
Single gene disorder diagnosis

Single gene disorders have been reported by
detecting or excluding the paternal allele inherited
from an affected father with an autosomal
dominant condition, such as Huntingdons
disease.
Confirmation of Achondroplasia in a fetus
presenting de novo with short limbs in the third
trimester has also been reported.
Single gene disorder diagnosis
Currently available techniques are unsuitable
for the diagnosis of X-linked and most
recessive disorders.
Thalassaemia or Cystic fibrosis
if the parents carry different mutations then
exclusion of the paternal allele from the
maternal plasma indicates that the fetus
would be unaffected but, if the paternal allele
is present, an invasive test is required to
determine whether the fetus has inherited the
abnormal maternal allele and is thus affected.
Aneuploidy diagnosis

NIPD..Down syndrome not feasible.
Recently, Lo et al. described a novel method.
This approach focuses on the analysis of a cell-free
fetal messenger ribonucleic acid (cff mRNA)
derived exclusively from the fetus having no maternal
contribution.
A gene located on chromosome 21 (PLAC4) was
found to be expressed in the placenta but not
in maternal blood (that is,it is fetal specific) by
extracting cell-free RNA (cfRNA) rather than
cell-free DNA from maternal plasma.
Ethical aspects

PNID & Risk reduction
Communication Skills.
Potential simplicity of NIPD.
Commercialisation
Fetal sex determination from a blood Spot.
Regulation is required.
Fetal sex determination where the high degree of
accuracy of NIPD as early as 7 weeks of pregnancy
carries a number of socio-ethical implications,
Selective TOP.
Increasing numbers of conditions, or for paternity
testing.
Thank you