PRENATAL COUNSELING
Unlike intact fetal cells, ffDNA fragments are present in the maternal plasma in sufficient quantities to allow extensive investigation. While most maternal free DNA is composed of longer DNA fragments, shorter DNA fragments of fetal origin appear as the pregnancy advances and in some studies are first detectable as early as 32 days after conception.
Free fetal DNA increases throughout gestation, representing 3% of total DNA in maternal plasma during the second trimester, and increasing to 6% in the third trimester. Free fetal DNA fragments are cleared rapidly by the renal system, with a half-life of 16 minutes and no discernable levels as soon as 2 hours after delivery.
We now understand that ffDNA fragments are continuously deposited in the maternal circulation from early in pregnancy, perhaps even before fetal circulation develops. We also know that maternal levels of ffDNA depend on 2 forces: rate of deposition and rate of removal.10
Trophoblastic origin?
A placental source is suggested by evidence that ffDNA can be retrieved from maternal plasma prior to the development of fetal circulation. A trophoblastic origin is supported by identification in maternal plasma of fetal mRNA with specificity for genes expressed by the placenta. Moreover, ffDNA has been detected in maternal circulation as early as 14 days after conception, corroborating a trophoblastic origin, with programmed apoptosis of placental cells a likely mechanism.
Further increases in ffDNA throughout gestation may reflect direct contributions from the fetal circulation that are transferred to maternal circulation via the placenta. In later gestation, destruction of fetal cells within the maternal circulation may contribute to the pool of ffDNA in maternal plasma. The exact proportions of each contribution are unknown.11
ffDNA may help diagnose these disorders
Placental abnormalities
Recent work suggests sufficient quantities of ffDNA can be obtained for both quantitative and qualitative assessments.12 Used quantitatively, ffDNA reflects placental integrity, an active area of investigation.
Autosomal trisomies, preeclampsia, and fetal growth restriction are conditions thought to involve abnormal placental function. Increased levels of ffDNA have been found in these entities. Increases have been documented even before onset of preeclampsia.10
Gene defects
Use of ffDNA to identify specific gene defects is also under study. Sensitive microarray technology will likely be needed to assess fetal chromosome aneuploidy from maternal plasma.
The detection of single gene defects from ffDNA has been reported for paternally inherited myotonic dystrophy, Huntington disease, and achondroplasia.
For autosomal recessive disorders, genetic testing of ffDNA may be a first step to exclude inheritance of a paternal allele. For this application, discordant parental alleles will be needed so that exclusion of the paternal mutation in the ffDNA signifies an unaffected fetus or a heterozygotic carrier of the maternal allele. If the paternal allele is detected by ffDNA, further genetic testing by chorionic villus sampling or amniocentesis would be needed to differentiate heterozygotic carriers of the paternal mutation from homozygotic, affected fetuses.
RhD genotyping
Since 2001, ffDNA has been used clinically in the United Kingdom for fetal blood group genotyping in isoimmunized gravidas with heterozygous partners, through the International Blood Group Reference Laboratory (part of the National Blood Service), which brings us to the highlighted study. Gautier and colleagues added data affirming that the RhD genotype can be detected through ffDNA with high sensitivity and specificity. Among 285 RhD-negative women, the fetal RhD genotype was determined in 283. In 2 cases, the maternal RhD-negative phenotype did not result from a complete gene deletion; thus, the genotypes of fetus and mother could not be differentiated. Among the women with RhD-negative genotypes, all fetuses were accurately genotyped through ffDNA.
This study differs from prior investigations in its use of RhD-negative women who were not already sensitized, and suggests that ffDNA genotyping in RhD-negative women is sensitive enough to be incorporated into the distribution of Rh immune globulin.
2 problems
As Moise points out in an editorial accompanying the study, a robust, automated system for ffDNA assessment prior to administration of Rh immune globulin likely would be cost-effective. The Moise editorial also points out these 2 concerns:
False positives are a real possibility, as the 2 cases in the Gautier study illustrate. Free fetal DNA analysis for RhD genotyping assumes that the serologic finding that indicates RhD-negative status (lack of RhD on the fetal red blood cells) is due to deletion of the RhD locus. Thus, when RhD DNA fragments are detected in maternal plasma, they are presumed to be fetal in origin. However, we now know that pseudogene regions of the RhD locus occur with relatively high frequency—in particular, in more than half of African Americans, who serologically type as RhD-negative. Such pseudogenes cause a stop codon that effectively diminishes production of RhD antigen. Serologic typing of such individuals indicates an RhD-negative phenotype. Because the most common pseudogenes are within exon 4, inclusion of primers that assess multiple exons can reduce these false positives.
