Prenatal diagnostic applications

The first diagnostic applications using fetal DNA in maternal plasma have focused on the detection of sequences that a fetus has inherited from the father but which are absent in the genome of the mother. Examples of such sequences involve those that are present on the Y chromosome. Several groups have reported the detection of fetal-derived Y chromosomal sequences in maternal plasma for prenatal gender determination for sex-linked diseases, such as hemophilia. Another application of prenatal gender determination using this approach is in congenital adrenal hyperplasia in which prenatal dexamethasone treatment for the prevention of virilization is only indicated for female fetuses.

Another early diagnostic application of fetal DNA in maternal plasma is for the prenatal determination of fetal RhD status in RhD-negative pregnant women. This application has been validated by several large-scale studies.

Due to the robustness with which fetal gender and RhD status can be determined from maternal plasma, these applications have been used diagnostically in several centers, initially in Europe and more recently in the United States.

Application to the hemoglobinopathies

Fetal DNA in maternal plasma was first applied to the hemoglobinopathies for the prenatal diagnosis of β-thalassemia. Chiu and colleagues developed a real-time PCR assay to detect the 4–base pair (bp) deletion in codons 41/42 of the human β-globin gene ( HBB ), which is the most common β-thalassemia mutation in China. They studied families in which the father was a carrier of this mutation whereas the mother was a carrier of another HBB mutation. Thus, the primary use of this approach is in the exclusion of β-thalassemia in the fetus, in which case the paternal mutation is not detected in maternal plasma. Provided that the assay has been optimized to have a high sensitivity for the detection of circulating fetal DNA, a negative signal indicates that no further prenatal testing is needed for a particular case. Because the diagnostic significance of this lack of detection of the paternal mutation is great, several investigators in the field have proposed developing a positive control for the detection of fetal DNA in maternal plasma (discussed later).

Conversely, if the paternal mutation is detected in maternal plasma, the only conclusion is that the fetus has inherited the paternal mutation. It is unclear, however, if a fetus is only a carrier or also has inherited the maternal mutation. This is a more challenging issue because the maternal mutation is present in maternal plasma after the release by the mother’s own cells, irrespective of fetal mutational status. Recent advances in highly quantitative digital counting technologies, such as digital PCR, have allowed the solution of this problem (discussed later).

Several investigators have studied the detection other paternally inherited HBB mutations in maternal plasma. Unlike the 4-bp deletion (described previously), many of these other mutations are caused by point mutations. Hence, the specificity of the detection method, with regard to the target sequence, needs to be high. One approach with which this can be done is using variants of allele-specific or single-allele extension reactions followed by mass spectrometry. This has been successfully implemented for the IVS2 654 (C →T), nt -28 (A → G), and CD 17 (A → T) mutations causing β-thalassemia and the (GAG→AAG) missense mutation in codon 26 of the HBB gene causing hemoglobin E disease. Furthermore, it has been shown that through the detection of a paternally inherited single nucleotide polymorphism (SNP) allele that is linked to the nonmutant HBB gene of the father, β-thalassemia major can be excluded. Several groups have demonstrated that arrayed primer extension assays can be used for the detection of paternally inherited fetal β-thalassemia mutations and linked SNPs in maternal plasma. Another promising approach is the use of single-molecule techniques, such as digital PCR (discussed later).

Another strategy that has been attempted by investigators is developing technologies that allow the selective enrichment of fetal DNA in maternal plasma. For this approach to work, physical or biochemical characteristics that could differentiate fetal from maternal DNA in maternal plasma have to be found. One characteristic is the size of the DNA molecules, because Chan and colleagues first reported that fetal DNA molecules in maternal plasma are shorter than their maternally derived counterparts. Li and colleagues were able to obtain a relative enrichment of the circulating fetal DNA by the use of a gel-based electrophoretic system and to selectively extract the short DNA molecules from the gel. Using this approach, Li and colleagues performed noninvasive prenatal detection of four HBB mutations in maternal plasma. This gel-based system is complex, however, and potentially prone to contamination to be used in a routine diagnostic context. Thus, it is hoped that future developments of more rapid and efficient size separation systems will allow this strategy to be used on a routine basis.

Recently, a proof-of-concept report has shown that fetal DNA in maternal plasma can be used for the noninvasive prenatal exclusion of hemoglobin Bart’s disease. The sensitivity reported in this preliminary report, however, which is based on microsatellite polymorphisms, is only 33.3%. Because an established ultrasound-based approach is already used for this purpose, it is currently unclear if the DNA-based approach offers any additional advantage over the more conventional ultrasound-based method.

Application to the hemoglobinopathies

Fetal DNA in maternal plasma was first applied to the hemoglobinopathies for the prenatal diagnosis of β-thalassemia. Chiu and colleagues developed a real-time PCR assay to detect the 4–base pair (bp) deletion in codons 41/42 of the human β-globin gene ( HBB ), which is the most common β-thalassemia mutation in China. They studied families in which the father was a carrier of this mutation whereas the mother was a carrier of another HBB mutation. Thus, the primary use of this approach is in the exclusion of β-thalassemia in the fetus, in which case the paternal mutation is not detected in maternal plasma. Provided that the assay has been optimized to have a high sensitivity for the detection of circulating fetal DNA, a negative signal indicates that no further prenatal testing is needed for a particular case. Because the diagnostic significance of this lack of detection of the paternal mutation is great, several investigators in the field have proposed developing a positive control for the detection of fetal DNA in maternal plasma (discussed later).

Conversely, if the paternal mutation is detected in maternal plasma, the only conclusion is that the fetus has inherited the paternal mutation. It is unclear, however, if a fetus is only a carrier or also has inherited the maternal mutation. This is a more challenging issue because the maternal mutation is present in maternal plasma after the release by the mother’s own cells, irrespective of fetal mutational status. Recent advances in highly quantitative digital counting technologies, such as digital PCR, have allowed the solution of this problem (discussed later).

Several investigators have studied the detection other paternally inherited HBB mutations in maternal plasma. Unlike the 4-bp deletion (described previously), many of these other mutations are caused by point mutations. Hence, the specificity of the detection method, with regard to the target sequence, needs to be high. One approach with which this can be done is using variants of allele-specific or single-allele extension reactions followed by mass spectrometry. This has been successfully implemented for the IVS2 654 (C →T), nt -28 (A → G), and CD 17 (A → T) mutations causing β-thalassemia and the (GAG→AAG) missense mutation in codon 26 of the HBB gene causing hemoglobin E disease. Furthermore, it has been shown that through the detection of a paternally inherited single nucleotide polymorphism (SNP) allele that is linked to the nonmutant HBB gene of the father, β-thalassemia major can be excluded. Several groups have demonstrated that arrayed primer extension assays can be used for the detection of paternally inherited fetal β-thalassemia mutations and linked SNPs in maternal plasma. Another promising approach is the use of single-molecule techniques, such as digital PCR (discussed later).

Another strategy that has been attempted by investigators is developing technologies that allow the selective enrichment of fetal DNA in maternal plasma. For this approach to work, physical or biochemical characteristics that could differentiate fetal from maternal DNA in maternal plasma have to be found. One characteristic is the size of the DNA molecules, because Chan and colleagues first reported that fetal DNA molecules in maternal plasma are shorter than their maternally derived counterparts. Li and colleagues were able to obtain a relative enrichment of the circulating fetal DNA by the use of a gel-based electrophoretic system and to selectively extract the short DNA molecules from the gel. Using this approach, Li and colleagues performed noninvasive prenatal detection of four HBB mutations in maternal plasma. This gel-based system is complex, however, and potentially prone to contamination to be used in a routine diagnostic context. Thus, it is hoped that future developments of more rapid and efficient size separation systems will allow this strategy to be used on a routine basis.

Recently, a proof-of-concept report has shown that fetal DNA in maternal plasma can be used for the noninvasive prenatal exclusion of hemoglobin Bart’s disease. The sensitivity reported in this preliminary report, however, which is based on microsatellite polymorphisms, is only 33.3%. Because an established ultrasound-based approach is already used for this purpose, it is currently unclear if the DNA-based approach offers any additional advantage over the more conventional ultrasound-based method.