Non-invasive Testing - Part 1

Question 1

What is the RAPID programme?

Answer 1

• Reliable Accurate Prenatal non-Invasive Diagnosis.
• 5 year programme from 2008.
• Technology development.
• Clinical evaluation.
• Economic modelling.
• Patient consultation.
• Objective - implementation of NIPND in the NHS.

Question 2

What are the current techniques for sampling cells that represent the foetus?

Answer 2

• CVS and Amniocentesis - both are invasive and involve about 1% risk of miscarriage.
• Could also sample foetal cells.

Question 3

Describe the use of whole foetal cells in screening and the associated problems.

Answer 3

WHOLE FOETAL CELLS:

• Foetal cells in maternal circulation were discovered in the 1960’s (Wakanowska 1969) - foetal lymphocytes in maternal peripheral blood.
• Also erythroblasts, trophoblastic cells and leucocytes - .
• Foetal cells detectable from about 7 weeks gestation.

PROBLEMS:

• Only several foetal cells per ml of maternal blood.
• Ten year multi centre study: male foetal cells in whole maternal blood detected in 41% pregnancies with male foetuses, with a false positive rate of 11% - i.e. trying to use the cells for sexing has a very high error rate.
• Persists for years after pregnancy (>27 years) - cannot be sure the cells you are looking at are from the current pregnancy.

Question 4

Describe the use of cell free foetal DNA in screening.

Answer 4

CELL FREE FOETAL DNA:

• Identified in 1997 by Lo et al.
• Approximately 5-10% of total cell free DNA in the maternal plasma represents DNA from the foetus. This increases during gestation (10-15%). the proportion does vary during gestation and is inversely related to maternal size.
• The DNA is thought to come from trophoblasts coming from the placenta.
• Detectable from about 4 weeks and increases with gestation.
• The DNA is actually quite small - DNA from the foetus is actually smaller than the cell free DNA from the mother. 99% = <300bp, most <200bp. This can be used to help increased the relative proportion of cell free foetal DNA when one is testing.
• Cleared from circulation within 2 hours of delivery.

Question 5

Outline the preparation of cfDNA.

Answer 5

• cfDNA is prepared from plasma from the mother. The blood is sampled and then spun to remove the red cells and white cells.
• To do this one needs fresh maternal blood which is taken using fresh EDTA tubes and is processed quite quickly. This is because the maternal white cells will degrade over time leading to an increase in the proportion of maternal cell free DNA.
• Need to think about logistics,
• There are tubes available that have cell stabilising agents within them.

Question 6

Outline the initial uses of NIPT.

Answer 6

1) . Foetal Sexing
- X-linked disorders
- DMD - avoid CVS x female foetus
- CAH - dexamethasone for female foetus to prevent virilisation, avoid side effects if male.

2) . RhD status of foetus
- Currently used in high risk (sensitised women) in the UK.

Question 7

Why might you want to look at the RhD status of a foetus via NIPT?

Answer 7

• Rhesus blood group system involves antigens on the surface of RBCs of which D is the most important - D is present in 85% of UK population, 99% of African population.
• May get a situation where you have a RhD negative mother and RhD positive child.
• Foetal cells can get into the maternal circulation during foetal maternal bleeding- amniocentesis, birth etc.
• This can cause the mother to become sensitised and produce an immune response against the foetus - 13% of at risk pregnancies.
• In later pregnancies maternal antibodies may cross the placenta and destroy RhD positive red blood cells - this can cause haemolytic disease of the newborn.
• Currently all RhD negative pregnant women are offered injections of anti-D antibodies to prevent this happening (bind and neutralise foetal RhD antigens) - following amnio/CVS + 3rd trimester + following birth.
• This has reduced the incidence of haemolytic disease in newborns from about 5% in children born to caucasian RhD negative women, now 0.5%.
• However in 40% of cases the injections are unnecessary because the RhD negative women turns out to be having a RhD negative child.
• Saves treatment and money if we test the RhD status of the child. Financial incentive, prevents exposure to blood products, limited risk x anti-D therapy, risk of false negatives.

Question 8

Describe the technical aspects of foetal sexing using NIPT.

Answer 8

• Technically demanding technique because of the low proportion of cfDNA from the foetus.
• Various extraction methods used to make sure DNA has been extracted properly - some labs use >1 method.
• For foetal sexing many labs us real time PCR which is a sensitive technique. They use a Y-specific probe for SRY or DYS14 (multicopy) to detect the male foetus.
• Commonly occurs from around 7 weeks gestation to allow the levels of cfDNA to build up.
• Many labs use multiple replicates so as to avoid the chance of a false negative result because of failure of the PCR.
• Some labs use the PAP technique to increase the sensitivity.
• Need to control for correct extraction of the DNA.
• Potential need to control for the presence of foetal DNA.
• False positives have been found - probably due to vanishing twins - 0.3-0.7% pregnancies - can be detected via USS in the first 7 weeks.

Question 9

Outline the use of Real Time PCR.

Answer 9

• PCR with a forward and reverse primer but with a sequence that binds in between those two primers with a reporter fluorescent molecule at one end of the probe and a quencher at the other end of the probe.
• When the Taq polymerase synthesises a new strand it breaks the probe between the reporter and the quencher and increases the fluorescence in the tube.
• As the PCR proceeds you get an increase in fluorescence in real time. This tells you that the PCR is working.

Question 10

Outline the use of the PAP system in the testing of cffDNA.

Answer 10

• Pyrophosphorolysis activated polymerisation.
• Originally developed to enhance the specificity of allele specific PCR for detection of known mutants in an excess of wild type allele.
• Enhances specificity of PCR.
• Involves PCR where the primers have a dideoxynucleotide at the 3’ end.
• When the primer is fully annealed to the target DNA the DNAP removes dideoxynucleotide by pyrophosphorolysis.
• Any mismatch within 15 nucleotides of the 3’ end of the primer will prevent extension.

Question 11

What controls are used in cffPCR?

Answer 11

• Have controls to test there is DNA in the PCR.
• Also want controls specific for foetal cffDNA.
• One method of achieving this is to have polymorphic markers that are specific to DNA polymorphisms only seen in the father.
• Potential problem of informativeness with this approach.
• Another way that has been examined is to actually look at the genes where there are differences in methylation between the maternal genome and the placenta e.g. maspin and RASSF1A.
• For RASSF1A the promoter is hypermethylated in the placenta and hypomethylated in maternal blood cells.
• By digestion with a methylation sensitive restriction enzyme before RT-PCR we can digest away the hypomethylated maternal DNA and we will only get RASSF1A copies from the hypermethylated foetal (placenta-derived) cells.
• Need a control for complete digestion = a gene that is unmethylated in both tissues.

Question 12

Is sex determination via cffDNA available in the UK?

Answer 12

• Now available from several UK labs.
• Audits have been performed - available from 7 weeks gestation - in 511 pregnancies 99.5% concordance, no results in 4%.
• Gene dossier approved for serious X linked diseases and CAH.

Question 13

Outline the use of NIPT for single gene disorders.

Answer 13

The main difficulty for this type of testing for single gene disorders would be when you are trying to find a mutation in the foetus which is also present in the mother.

It is easier when:

• AD and father has the mutation
• AR and different mutations in parents (further testing required if inherited paternal mutation).
• Can use paternal-specific polymorphisms linked to the disease locus.
• Difficulty to detect SNPs in foetal DNA by RT-PCR with high analytical certainty.
• Digital PCR or NGS are more sensitive techniques.

Use of NIPT has been reported for several genes including:
- Achondroplasia, thanatophoric dysplasia, apert syndrome, DM, HD, CF, beta thalassaemia.

RAPID at GOS are using NIPT for NHS testing.