Prenatal foetal genetic diagnosis using maternal blood sample ppt

Prenatal Foetal genetic Diagnosis using maternal
blood sample
Dr.Shivamurthy H.M.
Prof in OBG SNMC, Bagalkot
Karnataka. INDIA .

Introduction
• Prenatal diagnosis forms the cornerstone of Fetal
Medicine.
• The ability to obtain fetal genetic material by invasive
techniques, carry an inherent risk of miscarriage.
• Last 15 years have seen a dramatic and rapid
advances in the field resulting, in alternative non-
invasive methods of obtaining and investigating fetal
genetic material
• Extremely fast progress occuring, so many clinicians
are not been able to keep pace with these advances.
Lo YM. Lo ES, Watson N, et al. Two-way cell traffic between mother and fetus:
biologic and clinical implications. Blood 1996;.

GIST of presentation
An overview of the current state of tests used to
aid Non-invasive Prenatal Diagnosis and Non-
invasive Prenatal Testing (NIPT) and the
implications for clinical practice.

• A scientific breakthrough in 1997,showed maternal
plasma contained fetal cell-free DNA (c f DNA)
Hence no need for intact fetal cells.
• c f DNA proven mainly to originate from fetal
trophoblast and consists of relatively short fragments
of fetal DNA (143 base pairs on average).
• Recent work has shown, this cfDNA represents the
whole of the fetal genome.
Important observations /developments
YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum.
Lancet 1997;.

• It is more suitable for prenatal diagnosis than
retriving Intact Fetal Cells and has more useful
implications.
• Firstly, cfDNA of fetal origin is present in relatively
large quantities, representing up to 20% of the total
maternal plasma DNA in later pregnancy.
• It is also found from early in pregnancy, even from
4-5 weeks of gestation.
• Also it is very rapidly cleared from the maternal
circulation (with a half-life of 16 minutes) making it
no longer detectable only hours after delivery
Important observations / developments
Lo YM. Tein MS, Lau TK. et al. Quantitative analysis of fetal DNA In maternal plasma and serum
: implications for noninvasive prenatal diagnosis 18. Am J Hum Genet 1998;.

• Fetal DNA forms only a small component of mixture
DNA in maternal circulation, it’s separation is a
major technical challenge.
• Early tests detected genes known only to be present
in the fetus such as Y chromosome Rh factor.
• Advances like digital PCR, and MPS (Massively
Parallel Sequencing), detect genes in CfDNA
precisely ,make the far more difficult challenge now
becoming a reality.
Important observations /developments
Lo YM. Zhang J, Leung TN. et al. Rapid clearance of fetal DNA from maternal plasma.
Am J Hum Genet 1999:.

The applications of this developments
• Fetal Sex Determination
• Rhesus Status Determination
• Aneuploidy Detection( Non-invasive Prenatal Testing-N I P T)

Fetal Sex Determination
NIPT KIMS 20-2-20

• Subjecting cfDNA for fetal sex determination is best
established, in case of carriers of X-linked genetic
disorders, in which a male fetus is at particular risk.
• Early fetal sexing will enable appropriate counselling
and an informed decision making.
• This test has also important application in sex
determination in case of ambiguous genitelia
detected by USG.
Finning KM. Chitty LS. Non-invasive fetal sex determination: impact on clinical practice. Semin Fetal
Neonatal Med 2008 NIPT KIMS 20-2-20

• The fetal sexing relies upon the detection of Y
chromosome sequences in cfDNA of male foetus
• There have been two genes targeted for this test,
Single copy SRY Gene
Multi copy Dys14 Gene
• First trim 95.0% sensitive 98.8% specific
Sec trim 98.2% sensitive 99.5% specific
• Before 5-7 weeks, the test is not as reliable due to
the lower levels of fetal DNA ( false negative results)
Zimmermann B, El-Sheikhah A. Nicolaides K, et al. Optimized real-time quantitative PCR measurement
of male fetal DNA in maternal plasma 3. Clin Chem 2005: NIPT KIMS 20-2-20

Fetal Sex Determination Contd……..
• There is update of methods to ensure adequate
quantities of fetal DNA, in sample obtained to
reduce this false negative rate.
• Acurate dating of the pregnancy is required to
ensure the test is not undertaken too early.
• Also rule out twin pregnancy to rule a false
positive result.
NIPT KIMS 20-2-20

• Economic analysis shown these tests to be cost
neutral and reducing the risk of miscarriage in
invasive tests.
• NIPT is widely available in the UK,for women at
high risk of sex-linked disorders
(UK Genetic Testing Network).
NIPT KIMS 20-2-20

Rh status Determination Foetus in Utero
NIPT KIMS 20-2-20

Rh-determination
• RhD status is determined by the presence of the RhD
gene on chr 1, which is inherited in an autosomal
dominant manner.
• Administration of anti-D to RhD negative mothers is done
during pregnancy, to avoid maternal immunisation
• However, anti-D has theoretical risks inherent in the use
of blood products .
• If Anti D is given to all Rh negative mothers in a Caucasian
population, it is not needed in 40% as they have Rh
negatve babies.
Hill M, Taffinder 5, Chitty LS. et at. Incremental cost of non-invasive prenatal diagnosis
versus invasive prenatal diagnosis of fetal sex in England. Prenat Diagn 2011;
NIPT KIMS 20-2-20

Rh determination
• In Rh negative mothers the Rh gene is normally
deleted and entirely absent.
• In Rh negative mothers, if there is Rh poisitive
gene by cfDNA test the baby is for sure Rh
positive.
• If the examination reveals no Rh genes, it is
assumed the fetus is Rh negative.
NIPT KIMS 20-2-20

Rh determination
• These techniques similar to those used for fetal
sex determination.
• Indeed high yielding systems using PCR have
proven to be effective.
• However, alternative approaches like Mass
Spectrornetric Assays are also possible and may
result in higher output
• Accuracy -99.5% -100%.
NIPT KIMS 20-2-20

Rh determination
• The problem arises from false negative results, as
these women would not be given anti-D and are at
risk of becoming sensitised and future pregnancies
could be affected .
• False negative rates are low around 0.l2% .
Van der Schoot CE, Soussan AA, Koelewijn J, et al. Non-invasive antenatal RhD typing.
Transfus Clin Biol 2006; 13:53—57 .
NIPT KIMS 20-2-20

• Significant improvements made to reduce these
incorrect results.
• Rh pseudogenes occur here an individual will have
an intact Rhgene but are serotypically Rh negative.
• This is seen in women of African descent (found in
66% who are RhD negative.)
• In light of these observations, additional PCR
reactions should be used, appropriate to the patients
ethnicity.
Rh Determination
BK, Green CA, Avent ND. et aI.The presence of an RhD pseudogene containing a 37 base pair duplication and a
nonsense mutation in africans with the RhD-negative blood group phenotype. Blood 2000:95:12—18.

Rh determination
• Fetal RhD status from cfDNA has led to their
widespread use in clinical practice.
• They have virtually replaced invasive diagnostic
procedures to determine fetal RhD status where
ever fesible
Daniels G, Finning K, Martin P. et al. Fetal blood group genotyping: present and future
. Ann NY Acad Sci. 2006;.
NIPT KIMS 20-2-20

• The area where uncertainty still remains is testing
strategy in a low-risk population.
• The integration of screening into Individual
National Health Care and the cost benefit still to be
explored.
• Denmark Iroutine testing at 25 weeks’ gestation has already been
successfully introduced into antenatal care.
• In the UK recent guidance from the NICE( National Institute of Health and
Clinical Excellence) has recommended the exploration of routine antenatal
fetal RhD typing .
Rh status Determination
Clausen FB, Christiansen M, Steffensen R, et al. Report of the first nationally implemented
clinical routine screening for fetal RhD in D-pregnant women to ascertain the requirement for
antenatal RhO prophylaxis. Transfusion, 2012: NIPT KIMS 20-2-20

Aneuploidy detection / Non-invasive
Prenatal Testing (NIPT)
NIPT KIMS 20-2-20

Aneuploidy detection / NIPT
• The previously described uses of cfDNA analysis
is not difficult / challenging, because the DNA is not
present in the mother.
• The detection of fetal aneuploidy (using cfDNA ) is
more difficult amongst the cfDNA mixed reservoir,
which also contains the maternal DNA (80%)
Lun FM. Chiu RW. Allen Chan KC, et at. Microfluidics digital PCR reveals a
higher than expected fraction of fetal DNA in maternal plasma. Clin Chem. 2008;NIPT KIMS 20-2-20

Aneuploidy detection /Non-invasive Prenatal
Testing
• In a woman carrying a trisomy 21 fetus, cfDNA
forms only the minority of DNA present in maternal
blood(20%).
• Hence a very precise technique is required to
detect these small and different fractions reliably
or
• A technique is required to enrich the fetal
cf DNA fraction.
NIPT KIMS 20-2-20

Fetal cfDNA enrichment
NIPT KIMS 20-2-20

Fetal cf DNA enrichment
• A range of approaches have been attempted to
selectively enrich the fetal cfDNA fraction.
• Natural - there appears to be a physiological
enrichment of cfDNA in pregnancies with Down’s
syndrome fetuses (38).
• Artificial enrichment methods available to separate
cfDNA from mat DNA bsed on cfDNA fragments
being typically smalI in size
Li V. Zimmermann B, Rusterholz C, et al. Size separation of circulatory DNA in maternal plasma
permits ready detection of fetal DNA polymorphisms. Clin Chem. 2004;

Fetal cf DNA enrichment
• Chemical purification techniques and
Immunoprecipitation methods based on differential
DNA methylation (MeDIP-qPCR) have been used.
• Over time, these methods will become integrated
as part of commonly used screening techniques.
Papageorgiou EA, Fiegler H, Rakyan V. et at. Sites of differential DNA methylation between
placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies.
Am J Pathol 2009; 174:1609—1618.

The foetal cfDNA detection techniques
(sequencing techniques)

The DNA detection by sequencing
Massively Parellel Sequencing (MPS)
1 Shotgun Massively Parellel Sequencing
2 Targetted Massively Parellel Sequencing

MAPS (MASSIVE PARELLEL SEQUENCING ) TO DETECT
FOETAL CHROMOSOMAL ANEUPLOIDY
Maternal blood sample
containing both MATERNAL (80%)
and FOETAL (20%) DNA fragments
SUBJECT FOR MPS TO
IDENTIFY THE CODONS
SUBJECT THE CODONS TO COUNT
THE NUMBER CHROMOSOMES
MATERNAL
DNA
FRAGMENTS
FOETAL DNA
FRAGMENTS
( 20%)

Massively Parallel Sequencing (MPS)
• MPS does sequence identification by reference to the known
human genome sequence.
• There is only a small increase in the amount of cfDNA from a
particular abnormal chromosome, which is seen in seen
fetal aneuploidy.
• Hence extremely large numbers of cfDNA fragments present
in cfDNA must be counted.
• MPS analyses millions of DNA molecules, on a single
run,and do detect and quantify the fragments.

2 Shotgun Massively Parallel Sequencing
approaches
• The ‘shotgun’ approach involves a non-selective
sequencing of all fragments.
• Here only a few chromosomes of interest are
detected .
. Boon EM, Faas BH. Benefits and limitations of whole genome versus targeted approaches
for noninvasive prenatal testing for fetal aneuploidies. Prenat Diagn 2013;.

1 Shotgun MPS contd……
• This approach was the first MPS technology
reported and has been widely used.
• The studies by Fan and Chiu have demonstrated
that fetal aneuploidy with very high sensitivity and
specificity using this technology.
• This approach is now available on a clinical basis
through a number of companies worldwide.

1 Shotgun MPS contd….
• Challenges - cost and practicalities of such testing
approaches.
• Costs can be improved by running multiple samples
at the same time, by adding a ‘bar-code’ to each
patient sample.
• Chiu et al. demonstrated this as if the mean mappable sequence reads was
reduced from 2.3 million (running two samples in parallel) to 0.3 million (running
eight samples in parallel) there was a corresponding drop in test sensitivity from
100% to 79% for the detection of trisomy 21.

2 Targeted MPS( Massively Parallel Sequencing )

2 Targeted MPS (Massively Parallel Sequencing )
• Here, sequencing techniques used to target the at
risk chromosomes found in aneuploidy, instead of
sequencing across the whole genome.
• In this approach methods include those selectively
amplify specific regions on chromosome (Eg 21 and 18,)
• This improves efficiency as unimportant information
is reduced, and hence reduces costs.

2 Targeted MPS
• The relative merits and limitations of targeted v/s
shotgun approaches have been summarised in
the Table.

Large validation studies using massively parallel sequencing
based approaches for cfDNA screening for fetal trisomy (21)
Study Method OF
MPS
Detection rate(%)
OF ANEUPLOIDY
False positive rate
(%)
No result *
Chiu et al. Shotgun 39/39 (100) 3/146 (2.1) 11/764 (1.4)
Ehrich et al. Shotgun 209/212 ( 98.6) 1/410 (0.2) 18/467 (3.9)
Palomaki et al. Shotgun 209/212( 98.6%) 1/1471 (0.2) 1 3/1686 (0.8)
Bianchi et al.** Shotgun 90/90 (100%) 6/41 0 (1.5) 16/532 (3.0)
Sparks et al.]
Targeted 36/36 (100%) 1 /123 (0.8) 8/338( 2.4)
Ashoor et al. Targeted 50/50 ( 100%) 0/297 (0) 3/400 (0.8)
Norton et al. Targeted 81/81 (100%) 3/2888 (0.1 ) 148/3228 (4.6)
Total 591/594 (99.5%) 17 /5745 (0.3) 217/7415 (2.9)
* Due to low fetal DNA levels or test failure ** Results returned as unclassified and
require further testing.

2 Targeted MPS
• These studies show that Targeted MPS method
has shown to perform well, with similar results
across both approaches.
• Therefore, T. MPS is also offered clinically by a
number of companies.

MPS as a whole
• Currently, shotgun MPS approaches are more
extensively validated than targeted approaches for
detection of aneuploidy.
• Although, targeted approaches are currently
cheaper, they have the limitation of only being able
to study the region being targeted by the assay.

Other methods in sequencing involve
enrichment for single nucleotide
polymorphism loci, which have also been
shown to be effective in identifying the
common trisomies.
. Norton ME, Brar H. Weiss J. et al. Non-invasive chromosomal evaluation (NICE) study: results
of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18.
Am J Obstet Gynecol 2012;

Intellectual / patency/ disputes issues
• The clinical translation of NIPT has largely been driven
by industry and the huge commercial stakes involved.
• Technologies for isolation and genetic analysis of
cfDNA have been patented by a small number of
companies.
• These patents are currently the subject of on going
disputes and legal action between these companies
that are yet to he resolved.
• How these patents will influence the clinical
implementation of NIPT remains to be seen.
Agarwal A, Sayres IC, Cho MK, et al. Commercial landscape of noninvasive prenatal testing
in the United States. Prenat Diagn 2013;

Clinical use of cfDNA testing for
fetal aneuploidy

Clinical use of cfDNA testing for fetal aneuploidy
Certain specific caveats to cfDNA testing for fetal
aneuploidy in clinical practice to consider are:
• These tests despite having demonstrated good test
accuracy,but should not be considered fully diagnostic,
and therefore do not currently offer a replacement for
amniocentesis and CVS.
• Women should therefore be advised that although false
positive do occur and women with a positive test result
should at this time be offered diagnostic invasive testing.
.
Benn P. Borell A. Chiu R, et al. Position statement from the Aneuploidy Screening Committee
on behalf of the Board of the International Society for Prenatal Diagnosis. Prenat Diagn 2013;.

3 The reliance of cfDNA tests on the proportion of
fetal cfDNA present in the plasma is only an
important consideration.
4 The proportion of cfDNA increases with gestation
and to ensure adequate cfDNA is present the test
advised to be used after 10 weeks’ gestation
5 Another situation where lower than expected
amounts of cfDNA are found is in obese mothers
Ashoor G, Syngelaki A, Poon LC, et al. Fetal fraction in maternal plasma cell-free DNA at 11—13 weeks’ gestation:
relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol 2013;

• Multiple pregnancies, lead to substantial additional
complexity.
• Determination of zygosity from cf DNA is
recommended clinically .
• False positive results could be obtained from a
vanishing twin or empty gestation sac.
• Therefore ultrasound examination prior to cfDNA
testing therefore remains important.
QuiZ, Leung TV, Jiang P, et al. Noninvasive prenatal determination of twin zygosity by maternal
plasma DNA analysis. Clin Chem 2013;

• Placental mosaicism has been well described
when fetal genetic material is obtained from CVS
samples .

Newer developments
Digital PCR

Digital PCR to increase amount of cfDNA
• Recent advance
• The principle behind digital PCR is that, the template
DNA is diluted until there is on average < 1 copy /
reaction.
• Then hundreds of individual PCR reactions are then
carried out.
• Then precise counting of the starting copy number is
possible
. Lench N, Barrett A. Fielding 5, et al. The clinical implementation of non-invasive prenatal diagnosis
for single-gene disorders: challenges and progress made. Prenat Diagn 2013

Ethical considerations involved in
these non invasive techniques

Ethical Considerations
The non-invasive testing to reduce the need for
invasive testing, there by decreasing the
inherent risk of miscarriage is a good with this
intensions.
However, this new technology does also pose
new ethical challenges.

1 Inadequate Counseling.
The simplicity of non-invasive diagnosis based on
a maternal peripheral blood sample early in
pregnancy, means that there may be inadequate
counselling .

2 Multiple disorders screened together
it may be possible to screen for multiple diseases
simultaneously,
and this poses difficulties in providing appropriate
informed consent for all the potential disorders..

3 Concern for other Disorders
The screening done not only for serious diseases,
but for other genetic traits such as increased risks
of adult cancer or late onset disorders.
Here decisions must be made by society as well
as individuals, about where the limits of
acceptable screening lie.

4 Nonmedical Concerns
The relative ease of early fetal sexing could be
abused for gender preference or paternity
testing to be conducted prenatally.

Clients must be informed that
a. Currently available tests mostly focus on detection of fetal trisomies 21, 18 and 13
b. Detection rates are high, but testing does not detect all cases of these trisomies.
c. False positive results do occur, though at a low rate, therefore, women with positive
results should be offered confirmatory chromosome analysis by invasive testing.
d. For some women cfDNA testing may not be informative and they may need to
consider a screening test and / or invasive testing.
e In particular, women with obesity are at risk of test failure
or an inconclusive result.
f In late gestational age, this may lead to delays and insufficient time for a repeat
screening test and or invasive test.

Conclusions
• The discovery of cfDNA in the maternal circulation has opened
up the possibility to investigate the fetal genome prenatally.
• This combined with the on-going revolution in molecular
genetics, including in sequencing technology, now also enables
this fetal material to be investigated in detail.
• It is predicted that within the next decade, problems of cost and
output, will be solved and prenatal diagnosis will become
predominantly a non-invasive process.

Conclusions ………..
Information should be provided to pregnant women,
wanting to undergo cfDNA testing for fetal aneuploidy
includes,
1 Detailed counselling on the benefits
and
2 Limitations of the tests, and notification
3 that these tests are still under clinical development.
Guidance from the position statement of the Board of the International Society for Prenatal Diagnosis (53) )

MAIN REFERENCE
NON INVSIVE PRENATAL DIAGNOSIS
David Lissauer, Stephanie Allen Fiaona Mackie
Mark D Kilby, recent advances in obstetrics
aand gynaecology. vol 25 p.41-52

Prenatal foetal genetic diagnosis using maternal blood sample ppt

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Prenatal foetal genetic diagnosis using maternal blood sample ppt

Similar to Prenatal foetal genetic diagnosis using maternal blood sample ppt (20)

More from Shivamurthy Hm

More from Shivamurthy Hm (8)

Recently uploaded

Recently uploaded (20)

Prenatal foetal genetic diagnosis using maternal blood sample ppt