Group 5

The nature of free fetal DNA and

detection by real time PCR in prenatal

tests

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: Conditions:

Denaturation

94-96˚C

30 secs

Annealing

50-65 ˚C

30 secs

Extension

70-72 ˚C

1 minute

• Reaction buffer

• Oligonulceotides

• Template DNA

• dNTPs

• Thermostable DNA

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-Márquez, M., Rubio-Calvo, A., Carmona-González, M., Martin-Sánchez, J., Pérez-Simón, 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( 3–4), 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