Free Fetal DNAProf 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 situationCurrent 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 allwomen 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 determinationPregnancies 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 diagnosisCurrently 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 notin 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 reductionCommunication Skills.Potential simplicity of NIPD.CommercialisationFetal 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

They used this technology to determine the fetal RHD status in 1788 RhD-negative women at 28 weeks of gestation and demonstrated a 95.7% accuracy compared with postnatal serological RhD typing with a 0.2% false negative rate. If this technique performs well in early pregnancy, bearing in mind that the relative proportion of cffDNA is lower earlier in gestation, then there is the potential to decrease the use of anti-D immunoglobulin in RhD-negative women whoare found to be carrying an RhD-negative baby. The clinical validity and utility of this high-throughput methodology to routinely identify the fetal RhD type in all RhD-negative women in early pregnancy is now being investigated in pilot sites in the UK

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