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Chimerism. 2010 Jul-Sep; 1(1): 30–35.
PMCID: PMC3035115

Epigenetic approaches for the detection of fetal DNA in maternal plasma


The presence of fetal DNA in the plasma of pregnant women has opened up new possibilities for noninvasive prenatal diagnosis. Over the past decades, different types of fetal markers have been developed, initially based on discriminative genetic markers such as male-specific signals or paternally-inherited polymorphisms, and gradually evolved to the detection of fetal-specific transcripts or epigenetic signatures. This development has extended the coverage of the application of cell-free fetal DNA to essentially all pregnancies, regardless of the gender of the fetus or its polymorphic status. In this review, we present an overview of the development of noninvasive prenatal diagnosis through epigenetics. We introduce the basis of how fetal DNA could be detected from a large background of maternal DNA in maternal plasma based on fetal-specific DNA methylation patterns. We evaluate the methodologies involved and discuss the factors that affect the robustness of the detection. We review the progress in adopting fetal epigenetic markers for noninvasive prenatal assessment of fetal chromosomal aneuploidies and pregnancy-associated disorders. We conclude with comments on the future directions regarding the search for new fetal epigenetic markers and the clinical implementation of epigenetic approaches for noninvasive prenatal diagnosis.

Key words: DNA methylation, epigenetics, fetal DNA, plasma DNA, noninvasive prenatal diagnosis, fetal chromosomal aneuploidies


During normal human pregnancy, cells from the growing fetus are released into the maternal circulation and co-exist with that of the mother.13 This phenomenon is regarded as a form of fetal-maternal microchimerism. Previous studies suggested that it may have significant clinical implications in autoimmune diseases.48 On the other hand, this phenomenon also inspired scientists to explore the clinical potential of using the fetal cells in the blood of the mother to develop noninvasive prenatal diagnostic tests.3,912 Conventionally, the collection of fetal genetic materials are done with invasive procedures, which are associated with a small but finite chance of fetal loss.13 Therefore, noninvasive prenatal diagnosis is an important goal in medical genetics. In 1997, it was found that apart from fetal nucleated cells, extracellular DNA of fetal origin is also detectable in the plasma of pregnant women.14 Comparing to fetal cells, cell-free fetal DNA is relatively more abundant in the maternal plasma and thus is a promising molecular tool for noninvasive prenatal assessment.1518 The successful detection of circulating fetal DNA marked a significant step forward in this area, and soon became the focus of research for the development of noninvasive prenatal diagnostic tests.

Over the past decades, the clinical potential of cell-free fetal DNA has been demonstrated as a qualitative marker for the prenatal assessment of fetal rhesus D status, sex-linked disorders, β-thalassemia, myotonic dystrophy and congenital adrenal hyperplasia.1927 Moreover, quantitative aberrations of cell-free fetal DNA have also been reported in a number of pregnancy-associated disorders, such as pre-eclampsia, preterm labor, fetal chromosomal aneuploidies and other placental disorders.2836

The major challenge for the development of noninvasive diagnostic tests is that fetal DNA only constitutes around 3–10% of the total amount of plasma DNA in the maternal circulation.16,37 To differentiate the fetal-derived sequences from that of the mother, the most intuitive targets for the detection of fetal DNA were based on absolute discriminative genetic markers, such as Y-chromosome-specific loci or paternally-inherited polymorphic loci that are either absent or different in the maternal genome.23,28,31,3841 Many early applications were developed based on these types of fetal markers, but they were associated with certain limitations in practice. Firstly, diagnostic tests developed based on Y-specific targets could only be applied to pregnancies involving male fetuses. Moreover, negative tests results would need to be interpreted with extra precautions, as a lack of Y-chromosome signals in the maternal plasma would either imply that the fetus was a female; or that the fetal DNA proportion was below the detection limit of the assay or was not being extracted from the plasma properly. Secondly, the detection of a paternally inherited polymorphism requires prior knowledge of the polymorphic status of the parents, and could only apply to a subset of individuals who possessed that particular polymorphism.

Therefore, it would be desirable to develop a type of marker that allows confident differentiation of the fetus and the mother, and yet is independent of the gender or polymorphic status of the fetuses. An area that had attracted much attention is the use of epigenetic modifications as fetal-specific signatures to detect fetal DNA from circulating plasma DNA.

Fetal-Specific Epigenetic Profiles as Noninvasive Biomarkers

Epigenetic modifications refer to the molecular changes that affect gene expression without changing the sequence context. They are stably transmitted through cell division and are potentially reversible.4244 DNA methylation is one of the most widely studied epigenetic mechanisms, particularly in human cancers, in which an aberrant DNA methylation pattern is usually associated with disrupted gene expression.45,46 In 1999, it was first demonstrated that the specific DNA methylation signatures of tumor DNA are detectable in the plasma of cancer patients, which raised the possibility of using them as noninvasive biomarker.4749 Soon after such developments, various attempts have been made to detect fetal DNA from maternal plasma based on differential methylation patterns between the fetus and the mother.5052

Parent-of-origin-specific methylation pattern.

DNA methylation is one of the mechanisms that regulates the establishment of genomic imprinting in humans.53,54 Fetal epigenetic markers may be developed based on an imprinted region, in which the DNA methylation patterns are inherited in a parent-of-origin-specific manner.55,56 Theoretically, if a pregnant woman has inherited the methylated copy of an imprinted region from her father, when she passed this copy on to her growing fetus, it would become unmethylated. The methylation status of this region should then be distinguishable between the fetus and the mother in an allele-specific manner. In 2002, Poon et al. proved this hypothesis by targeting the imprinted region between the insulin-like growth factor 2 and H19 genes to detect fetal-specific methylation from maternal plasma.50 They confirmed the results by genotyping a biallelic polymorphism within the differentially methylated region.50 Using this method, for the first time, the detection of a stretch of DNA that a fetus has inherited from the mother was achieved.

This work was based on an imprinted locus, and serves to demonstrate the principle of noninvasive prenatal testing using epigenetics. However, it would be relatively complicated to use as a routine fetal marker. In other words, a marker that can be applied universally regardless of the gender or polymorphic status of the fetus is needed. Investigators then explored another approach that was based on a placenta-specific methylation profile.

Placenta-specific methylation patterns.

Previous studies have suggested that the human placenta carries a specific pattern of DNA methylation with respect to other somatic tissues.5763 At the time of the investigation, many researchers in the field believed that the majority of the fetal nucleic acids in the maternal plasma were derived from the placenta, while the maternal counterpart was predominantly derived from the maternal hematopoietic cells.6468 Therefore, theoretically, a genomic region that is differentially methylated between the placenta and the maternal blood cells can be adopted to differentiate placenta-derived fetal DNA from the maternal background DNA in plasma. This hypothesis was first proven in 2005, when a region on the maspin (SERPINB5) gene promoter was found to be hypomethylated in the placenta while heavily methylated in the maternal blood cells.51 The authors also demonstrated that the unmethylated version of the SERPINB5 gene sequences was detectable in maternal plasma throughout the course of pregnancy, and its level dropped significantly after delivery. This was the first universal fetal marker that can be used in all pregnancies. Its main advantage over an imprinted locus is that no prior knowledge of the fetal or parental polymorphic status is needed. This feature allows the development of a single, simple test to determine the presence of fetal DNA in the maternal plasma with greater simplicity and coverage. Other examples of fetal epigenetic markers that were developed based on placenta-specific methylation pattern include the first exon of RASSF1A (Ras association domain family 1A) gene and the gene promoter of HLCS (holocarboxylase synthetase).52,69 Both of them are hypermethylated in the placenta while predominantly unmethylated in the maternal blood cells. The approaches used for the detection of these markers are variable, depending on whether the placental-derived sequences are hypermethylated or hypomethylated with respect to the maternal blood cells.

The Detection of Fetal Epigenetic Markersin Maternal Plasma

In general, to detect fetal epigenetic markers in maternal plasma, the first step is to differentiate methylated and unmethylated sequences via bisulfite modification of the template DNA or differential cleavage by restriction enzymes. The fetal-specific methylation pattern is then quantified by, for example, quantitative methylation-specific PCR, quantitative real-time PCR or other methods.

Differentiation of methylation patterns by bisulfite-dependent approaches.

DNA methylation in the human genome predominantly takes place at the cytosine nucleotide mainly in the context of CpG dinucleotides.70 The process of bisulfite conversion changes unmethylated cytosine residues into uracil, leaving methylated cytosine unchanged.71 This important technique translates the apparently identical DNA sequences into distinguishable species according to individual epigentic profiles, which are then detected by various molecular techniques.

For qualitative measurement, the bisulfite-converted DNA can be differentially amplified by methylation-specific PCR (MSP), which amplifies the DNA depending on the methylation status of the regions where the primers bind.72 Alternatively, the methylation status can be ascertained by sequencing the bisulfite-converted DNA.71,73 The work by Poon et al. has adopted these approaches for detecting the fetal-specific methylated alleles.49 When applied to maternal plasma analysis, one can confirm the detection specificity by genotyping a polymorphic site within the target region by direct sequencing or primer-extension PCR.50,51

For quantitative measurement, bisulfite-converted DNA can be quantified by quantitative MSP (qMSP), in which MSP is performed in the presence of a fluorescence probe. The fluorescence signal is compared against a series of calibration standards. This method has been used to quantify methylated biomarkers in plasma of cancer patients.47 The work by Chim et al. has demonstrated that using qMSP, it was feasible to quantify fetal-specific methylation patterns in maternal plasma from early gestation, during which the concentration of circulating fetal DNA is less abundant than that in the later stage of pregnancy.16 Alternatively, it is also possible to quantify the bisulfite-converted target in plasma with a variety of single molecule based approaches for methylation analysis.74,75

However, the major drawback of this technique is that the chemical bisulfite is known to degrade >90% of the template DNA.76 This is undesirable for the detection of fetal DNA, which is present at low abundance in maternal plasma, particularly during early gestation. As a result, less DNA molecules would be available for measurements, and the accuracy of the test would be affected. Therefore other approaches were developed to bypass the use of bisulfite.

Differentiation of methylation patterns by methylation-sensitive restriction enzymes.

A variety of restriction enzymes are sensitive to methylation at bases in their recognition sequence, such as BstU I or HpaII. The cleavage may be blocked completely, but sometimes the effect may be partial, depending upon the duration of digestion or the amount of enzymes that is used.77 For markers that are hypermethylated in the placenta, like RASSF1A and HLCS, if there are recognition sites of methylation-sensitive restriction enzymes within the differentially methylated region, one could treat the plasma DNA with such enzymes to remove the unmethylated maternal DNA, then quantify the digestion-resistant (methylated) fetal DNA by quantitative methods such as real-time PCR or digital PCR.52,69 For markers that are hypomethylated in the placenta, like SERPINB5, after treatment with the methylation-sensitive restriction enzymes, the placental derived unmethylated SERPINB5 molecules would be cleaved. One could then amplify the short fragments of cleaved unmethylated DNA by using stem-loop primers, a technique that has been developed for amplifying microRNA.78,79 Compared with bisulfite conversion, this digestion-based method introduces less damage to the plasma DNA. Therefore, more DNA molecules are available for quantitative assessment.

Factors that affect the robustness of detection.

The robustness of detection of fetal epigenetic markers may be affected by a number of factors: Firstly, it depends on the difference of methylation levels between the placenta and the maternal blood cells. The more divergent the two are, the better is the differentiation. Chim and coworkers have proposed a set of criteria to define a particular methylated CpG site as a potential target for fetal epigenetic marker development.80 The methylation profiles are usually confirmed by cloning and bisulfite sequencing to assess the percentage of methylation at single molecule precision to the resolution of each CpG site.51,59,69,80 Secondly, in general, detection methods that do not depend on the use of bisulfite result in less degradation and thus allow more precise quantification of fetal-specific molecules, and thus allowing more robust detection of disease status.69 The loss of template DNA due to the degradation by bisulfite would need to be compensated by, for example, increasing the amount of plasma present or using a more sensitive analytical platform.75,81

Fetal Epigenetic Markers for Noninvasive Prenatal Assessment

Noninvasive determination of fetal gender and quantification of fetal DNA in maternal plasma samples.

Fetal epigenetic markers can be used as qualitative markers to indicate the presence of fetal DNA or as quantitative markers to quantify the amount of fetal DNA, in a particular maternal plasma sample. As a qualitative marker, it can be used to detect the potentially false negative results obtained based on genetic markers when performing fetal rhesus D genotyping, fetal gender determination or the detection of paternally-inherited polymorphisms.52 This application addresses the problem of false negative results associated with the use of fetal genetic markers. As a quantitative marker, it can be used to determine the fractional concentration of circulating fetal DNA in a maternal plasma sample. Such information is useful for interpreting the results of diagnostic tests performed using plasma DNA. For example, when one measures the relative mutation dosage in maternal plasma for the prenatal diagnosis of monogenic diseases, the fractional fetal DNA concentration would affect the number of DNA molecules required for one to achieve the confident detection of disease status.82 Another example is that when one measures the proportional representation of different chromosomes in maternal plasma using massively parallel DNA sequencing, the fractional fetal DNA concentration would affect the degree of sequencing that is required for an accurate assessment.8385

Noninvasive detection of the quantitative aberrations of plasma fetal DNA in pregnancy-associated disorders.

Previous studies have shown the association between quantitative aberrations of circulating fetal DNA with the development of pregnancy-associated disorders. For example, the plasma concentration of SRY (sex-determining region Y) sequences is significantly elevated in the plasma of pregnant women who suffered from pre-eclampsia.28,29 Such aberrations can also be detected using epigenetic markers, for example, by detecting the hypomethylated SERPINB5 sequences or hypermethylated RASSF1A sequences in the maternal plasma from pre-elcamptic pregnancies, therefore pregnancies bearing female fetuses are also covered.51,86 We envision that fetal epigenetic markers may also be used for prenatal assessment of other pregnancy-associated disorders in which quantitative aberrations of plasma fetal DNA has been reported.30,31,3436

Noninvasive prenatal detection of fetal chromosomal aneuploidies.

Fetal chromosomal aneuploidies may cause severe birth defects or even early childhood death.87 This disease is the main reason why pregnant women seek prenatal diagnosis. The three commonest autosomal aneuploidies at birth are the trisomies of chromosome 21, 18 and 13, respectively.

A number of reports have demonstrated the feasibility of using fetal epigenetic markers for the prenatal detection of fetal trisomy. For example, Tong et al. have adopted SERPINB5, which is on chromosome 18, to deduce the fetus's trisomy 18 status noninvasively by an approach based on epigenetic allelic ratio (EAR) analysis.81 This approach is based on the detection of a polymorphic site within the fetal-derived hypomethylated SERPINB5 sequences. When a heterozygous fetus has an extra copy of chromosome 18, the resultant allelic imbalance can be detected in maternal plasma by qMSP and primer-extension PCR. The main drawback of this approach is that it involves the use of bisulfite, and thus a large volume of plasma is needed to compensate for the degradation of the template DNA to confidently deduce the disease status.81 Moreover, this approach is only applicable to fetuses that are heterozygous for this particular polymorphism, and thus more of such markers on the aneuploid chromosome would be needed to maximize the population coverage.

To overcome this problem, the same group then developed another approach, which is called epigenetic-genetic chromosome dosage analysis (EGG).69 They have chosen trisomy 21 as a model to evaluate this approach, using the hypermethylated HLCS sequences on chromosome 21 as the fetal epigenetic marker. They removed the unmethylated maternal HLCS sequence by methylation-sensitive restriction enzymes, and then performed digital PCR to measure the concentration of hypermethylated HLCS sequences in maternal plasma. They inferred the concentration of the fetal chromosome 21 and compared it to the concentration of a fetal genetic marker on chromosome Y. The relative dosage of fetal chromosome 21 in maternal plasma was shown to be elevated in pregnant women carrying trisomy 21 fetuses compared to those carrying euploid fetuses. Using this approach, fetal trisomy 21 can be detected noninvasively even during the first trimester.69 In the same report, the authors also evaluated whether it is feasible to extend the population coverage by replacing the Y-specific genetic marker with another epigenetic marker which is not over-represented in the concerned aneuploidies, such as RASSF1A on chromosome 3, as the reference for relative chromosome dosage analysis. The authors commented that the accuracy of such an approach would be adversely affected by the variability of the levels of DNA methylation of individual fetal-derived epigenetic markers, and have shown that it was inferior to the EGG approach.69 To extend this approach to female fetuses, one could use a paternally-inherited polymorphism as the reference instead of the Y-specific marker. Unlike the Epigenetic Allelic Ratio or EAR approach in which the polymorphic site needs to be located within the differentially methylated region on the aneuploid chromosome, any fetal-specific SNP alleles located anywhere in the human genome could be used as a reference marker for the EGG approach. Using a genetic marker as the reference for dosage comparison would be more stable than using an epigenetic marker, unless one is able to identify an epigenetic marker that demonstrates a highly stable DNA methylation levels across individual fetal-derived molecules.


The search for new markers.

Since the report of the first fetal epigenetic markers, the search for more markers has been under way.69,80,88 Most of these initial efforts have been focused on chromosome 21, which is involved in the commonest fetal trisomy, trisomy 21. Moreover, the technologies of the time favor the search to be focused on specific regions of the genome, such as the promoter regions that are associated with the genes of interest or CpG islands in which extreme methylation patterns in the maternal blood cells have been described.69,80,88

The recent emergence of high-throughput methylation profiling platforms has opened new avenues in the field of DNA methylation analysis. In particular, the introduction of genomic tiling array and massively parallel sequencing techniques allows scientists to study the human genome in a previously unprecedented depth and scale.89,90 In combination with various methylation-specific enrichment techniques, for example, antibody-mediated enrichment of methylated fragments by MeDIP (methylated DNA immunoprecipitation), McrBC fractionation and differential amplification via HELP (HpaII tiny fragment enrichment by ligation-mediated PCR), these new platforms could be applied to the search of epigenetic markers.9199 Moreover, the development of bioinformatic algorithms for the analysis of these data is also progressing rapidly.96,97,100,101 Taken all these together, it is expected that the coverage of the search for new fetal epigenetic markers will extend to the entire genome. In fact, investigators have already begun to launch genome-wide search for new fetal epigenetic markers, hoping to extend the application to cover the other two commonest trisomies, trisomy 18 and 13.102,103 However, given the complexity of such high-coverage studies, it is expected that any new markers being identified would need to be evaluated via a systematic scheme to verify their clinical potential.

The clinical implementation of fetal epigenetic markers.

The past few years have been a fruitful period for the development of noninvasive prenatal diagnostic tests. The emergence of sophicated analytical platforms, such as digital PCR and massively parallel sequencing, has catalyzed the development of noninvasive prenatal diagnosis (reviewed in refs. 105 and 106).82,85,104106 In particular, the sequencing-based methods allow one to detect the small quantitative changes of genomic distribution of the chromosome directly from maternal plasma in a locus-independent manner.8385 However, the high instrumental and operational costs of the sequencing-based methods might hinder their rapid implementation into the clinical setting. Before their costs decline, which is expected to happen in the near future, diagnostic tests that are based on fetal epigenetic markers or other fetal-specific targets are more economical to be implemented clinically. The demonstrated improved robustness of combining epigenetic approaches with microfluidic digital PCR system for the detection of trisomy 21 is only one among many other successful examples.69,104,107 The analytical power of these tests would need to be evaluated with large-scale trials before these approaches could be put into routine usage.

In conclusion, we envision that, in the near future, there will be more fetal epigenetic markers being discovered and adopted in the development of noninvasive prenatal diagnostic tests. With the rapid advancement in analytical power, it is expected that the diagnostic accuracy associated with these tests would be further improved in the future. After systematic evaluation of their reliability, these approaches may eventually be applied in the clinical setting as an economical choice for pregnant women who opt for safer prenatal diagnosis.

Disclosure Statement

The authors hold patents and have filed patent applications on aspects of the use of cell-free fetal nucleic acids in maternal plasma for non-invasive prenatal diagnosis. Part of the patent portfolio has been licensed to Sequenom. Y.M. Dennis Lo is a consultant to, and holds equities in, Sequenom.


The authors were supported by the University Grants Committee of the Government of the Hong Kong Special Administration Region, China, under the Areas of Excellence Scheme (AoE/M-04/06) and a sponsored research agreement with Sequenom. Y.M. Dennis Lo was supported by an Endowed Professorship from the Li Ka Shing Foundation. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.


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