Non-Invasive Prenatal Testing/Diagnosis

Question 1

What is the current gold-standard test for prenatal genetic testing the UK and how many of these tests are performed?

Answer 1

• Invasive sampling is currently the gold standard for prenatal diagnosis in the UK
• Annually about 25,000 of the 700,000 (~3.5%) pregnant women in the UK undergo invasive testing.

Question 2

What discovery led to a paradigm shift in how prenatal genetic testing could be performed?

Answer 2

• The landmark discovery of cell-free fetal DNA (cffDNA) in maternal blood during pregnancy was published in 1997 (Lo et al)
• Cell-free fetal nucleic acid (cffNA i.e. DNA and RNA (cffRNA) testing is being used in some expert centres for selected clinical applications, but it does not yet form part of the recognised standard care offered to all through the NHS.

Question 3

What are the three key non-invasive sources of fetal material in the maternal blood stream?

Answer 3

Sources of fetal material in maternal circulation:

1. Intact fetal cells
2. cffDNA
3. cffRNA

Question 4

Is there a noninvasive sources of fetal tissue?

Answer 4

Intact Fetal Cells

• Only ~1 to 2 fetal cells per ml of maternal blood
• Lack distinct cell markers for easy enrichment.
• Fetal cells can persist for years ?potential false-positive results in subsequent pregnancy

Question 5

What are the properties of noninvasive sources of fetal DNA

Answer 5

cffDNA

• First detected in 1997.
• 3-20% total cell-free DNA is fetal.
• Detectable form 4-5 weeks.
• Increases with gestation, sharp increase in 3rd trimester.
• Rapidly cleared from circulation within an hour after delivery
• Size distribution is shorter than maternal cell-free DNA - 80% <200bp

Question 6

What are the properties of cell-free fetal mRNA (cffmRNA)?

Answer 6

• Stable fetal mRNA (cffmRNA)
• cffmRNA is stable in maternal blood due to association with trophoblast derived microparticles which seem to protect cffDNA and cffmRNA from nuclease degradation.
• Total levels do not increase throughout gestation
• Relative abundance of different transcripts dose change during pregnancy
• Rapidly cleared from circulation within an hour after delivery
• Genes only expressed in the placenta can be used as fetal specific markers

Question 7

What are the technical challenges with using cffDNA

Answer 7

• Testing cffDNA offers the most direct approach, and is therefore likely to be preferable source of noninvasive material for the foreseeable future.
• Cells and proteins require careful handling and may be extremely challenging both technically and logistically
• In contrast DNA is relatively easy to handle/use/store, but there are still significant technical challenges associated with cffDNA testing
• Concentration of all cell-free DNA in blood is relatively low
• Fetal DNA molecules are substantially outnumbered by maternal DNA molecules
• Half of the fetus DNA is inherited from the mother, making it indistinguishable from maternal cell-free DNA

Question 8

What are “fetal specific markers”, give two examples.

Answer 8

• Can be utilised simply to confirm the presence of fetal DNA in the maternal plasma (positive-control) but can also have a range of diagnostic applications
• Fetal epigenetic markers e.g. methylation of genes that are specifically expressed in the placenta can be used to confirm the presence of fetal DNA
• Advantage of non-fetal specific cfDNA is that the background of maternal cfDNA does not mask the result
• SERPINB5 (Chr 18) is hypomethylated in placental tissue but hypermethylated in maternal blood cells (methylation-specific PCR)
• RASSF1 (Chr 3) is hypermethylated in fetal tissues. The maternal hypomethylated RASSF1 sequences can be removed using methylation sensitive restriction enzyme digestion.

Question 9

What are the applications of fetal specific markers?

Answer 9

• Papageorgiou 2011., used epigenetic markers for detection of chromosomal aneuploidy.
• Hypermethylated cffDNA enriched using MeDIP then examined by RT-PCR to diagnose T21.
• Main limitation of epigenetic markers: based on bisulfite conversion and enzyme digestion which results in massive degradation of input DNA (up to 95%), detrimental in NIPT as molecules limited
• Maternally hypomethylated markers are valuable as methylation-sensitive restriction enzymes selectively destroy maternal sequences, leaving fetal in tact.

Question 10

How can cffDNA be utilised for fetal sex determination?

Answer 10

• Used in pregnancies at risk of a sex-linked disease (e.g. DMD, Haemophilia, SCID) and DSDs where intra-uterine treatment is available (CAH)
• Available from 7 weeks gestation, through detection of male Y chromosome DNA in mat plasma by qPCR3.
• If no Y detected then ‘inferred’ as female (can use HLA-B gene to confirm presence of fetal DNA)
• Unnecessary invasive testing can then be avoided in these pregnancies
• Ultrasound scan still recommended to confirm findings

Question 11

How is cffDNA utilised for Fetal Rhesus D antigen typing?

Answer 11

• RhD -ve mothers carrying a RhD +ve fetus are at risk of haemolytic disease, if already sensitised to a RhD+ pregnancy
• RhD -ve mothers can avoid anti-D prophylaxis if fetal also RhD -ve = cost saving
• Similar testing to sex determination

Question 12

How is cffDNA utilised for desting for Autosomal Dominant single gene disorders?

Answer 12

• Referred to as NIPD as considered ‘diagnostic’ i.e. no invasive test required to confirm result
• Been available for paternally transmitted dominant and de novo disorders for some time e.g. Skeletal dysplasias (Achondroplasia, Aperts, Thanataphoric).
• Technically easy as just looking for fetal specific sequencing that are not present in the mother.
• NIPD has clinical utility as very different prognosis for different skeletal dysplasias which may appear similar on USS.

Question 13

How can cffDNA used in a simple way for Autosomal Recessive single gene disorders?

Answer 13

• Recessive disorders are much more challenging due to presence of maternal background.
• Exclusion of paternal mutation in AR conditions where parents are comp het is possible and was first available in the UK for CF. But invasive still needed if pat mutation is detected as risk of being affected rises to 50%

Question 14

What more complex methods enable cffDNA to be utilised for Autosomal Recessive single gene disorders?

Answer 14

• Lo et al developed relative mutation dosage (RMD) to assess contribution of mat wt v mutant allele and in turn decipher whether the mat mutant allele has been transmitted to fetus
• RMD requires digital PCR which has limitations so this is not a routinely offered clinical service
• Relative Haplotype Dosage (RHDO) also developed by Lo, utilises MPS of SNPs to track fetal inheritance of parental haplotypes rather than direct mutation assessment.
• RHDO also has limitation but these are considered more manageable and as such clinical services using RHDO are becoming available in the UK for SMA, CF, CAH.

Question 15

Why is NIPT for aneuploidy considered a ‘screening’ test?

Answer 15

• Sensitivity is high but specificity is slightly lower because other genetic causes for a ‘positive result’ e.g. maternal cancer, CPM etc
• As such the PPV of NIPT for common aneupoidies is only ~50%
• Therefore this information alone can not be used as the basis for diagnosis in the fetus
• All positive results must be confirmed by invasive testing

Question 16

What different approaches can cffDNA be used to detect Fetal Aneuploidy?

Answer 16

1. fetal specific markers
2. Relative Chromosome Dosage (RCD)
3. targeted MPS
4. MPSS of the WGS is by far the most popular

Question 17

How is MPSS used to detected fetal aneuploidies?

Answer 17

• MPSS - Reads aligned and counted per chromosome.
• If fetal aneuploidy present then would expect an increase in the number of ‘counts’ for that chr.
• First demonstrated by Chiu 2008 and Fan 2008.
• Initially problems with T13 due to GC content but methods have been extensively refined and now very high levels of sensitivity and specificity (near 100%) for all common aneuploidy is possible.
• Many large scale validation studies have replicated findings and proven the clinical utility of MPSS.
• Some important limitations to be aware of, hence need for invasive confirmation of +ve results

Question 18

What are the benefits of NIPD/NIPT

Answer 18

1. Safer: Dramatically reduced number of invasive tests (1% miscarriage), plus no risk of infection or physical trauma to mother.
2. Less expertise required to perform a blood test rather than an invasive test.
3. Can be offered earlier than invasive testing and in many instances have shorter TaT. Gives parents time to make a decision about pregnancy, i.e. facilitates reproductive choice.
4. Likely to be substantially cheaper than current invasive diagnostic testing in the long run
5. Improve the quality of antenatal care (e.g. better targeting of anti-D therapy)

Question 19

What are the limitations of NIPD/NIPT?

Answer 19

1. Not possible to tell which fetus affected in twin pregnancies.
2. Increased maternal BMI reduces sensitivity of testing
3. Invasive test still be required to confirm an abnormal result
4. Source of cffDNA is placenta – CPM an issue in a small proportion of cases.
5. False negatives can be the result of failure to extract or detect sufficient material (time to process sample is critical in NIPT), or due to the low fetal fraction
6. False positives: many causes, technical e.g. contamination, or clinical e.g. vanishing twin, maternal constitutional abnormality, maternal neoplasm.
7. As a result PPV of NIPT for common aneuploidies ~50%. Therefore NIPT should only ever be used as a ‘high-performance’ screen.

Question 20

List some future possibilities/considerations for NIPD

Answer 20

1. Enhancement of the MPSS method has now enabled the noninvasive detection of micro-deletions
2. Many more applications for cffDNA are being published. For example noninvasive paternity testing has been demonstrated.
3. Whole noninvasive-fetal genome sequencing has also been demonstrated both with and without the paternal DNA sample for analysis. Lo 2010.
4. Interest in fetal cells has also resurfaced. Can provide a pure source of fetal DNA, and the single cells screening methods already in place for PGS/D.

Question 21

List some Ethical, legal and social concerns of NIPT/D

Answer 21

1. New developments do raise ethical, legal and social issues which need to be address
2. There are concerns that as cffDNA screening techniques become mainstream patients may not receive suitable counselled before opting for what appears to be ‘just another blood test’.
3. Safeguarding patient autonomy
4. Ensuring informed consent provided by DTC services
5. Ensuring equity of access where there’s a clinical need.
6. Avoiding adoption in new clinical areas without sufficient consideration/validation

Question 22

What projects have driven the evaluation and implementation of NIPT and NIPD in the UK?

Answer 22

RAPID project: Reliable Accurate Prenatal non Invasive Diagnosis

• Improve the quality of NHS prenatal diagnostic service by evaluating non invasive options
• Develop standards and recommendations for the use of NIPD/NIPT in the NHS
• 5 year UK national programme funded by the NIHR.

NIPSIGEN: Translation of NIPD for selected Single Gene Disorders into clinical Practice

• Wellcome Trust and DoH translational funding scheme
• 2013-2016 in Birmingham

Question 23

What is the UK strategy for implementation of NIPT/D into routine clinical care?

Answer 23

January 2016: UK NSC recommended that NIPT be introduced as part of the NHS funded fetal anomaly screening programme

• Initially only as an additional screening test for those at high risk of trisomy 21, 13 and 18 following existing primary screening.
• Women with a risk of >1/150 offered NIPT.
• NIPT+ confirmed by invasive test.
• This evaluation programme was offered at 8 centres.

November 2016: the DoH announced that NIPT would be made available as part of NHS maternity care.

• A staged roll out will be carried out over 3 years, starting from 2018, to allow time for training of midwives, counsellors etc

Question 24

What technological advances are expected to come with NIPR/D.

Answer 24

• Hui et al 2017 published a “Universal Haplotype-Based Noninvasive Prenatal Testing for Single Gene Diseases”
• This approach works similarly to RHD but does bypasses the need for mutation-specific assays and is not dependent on the availability of DNA from other affected family members.
• Thus, the approach is universally applicable to pregnancies at risk for the inheritance of a single gene disease.
• The whole fetal genome is represented in mothers blood so there is the potential for WGS/exome sequencing