Potential application of fetal epigenetic markers on the non-invasive prenatal detection of chromosomal abnormality.

Identification of fetal chromosomal aneuploidy is a predominant reason for pregnant women to undergo prenatal testing, most of which are invasive and carry a risk for fetal loss. The presence of fetal DNA in maternal circulation has offered an opportunity for non-invasive prenatal detection [1]. However, this fetal DNA exists only as a minor fraction among the co-existing background of maternal DNA [2, 3]. Hence, the use of fetal DNA in maternal plasma to detect fetal aneuploidy is technically challenging. With the advent of massively parallel genomic sequencing (MPGS) to shotgun (non-specifically) sequence all the fetal and maternal DNA molecules, non-invasive prenatal detection of fetal trisomy 21 could now be achieved at high sensitivity and specificity [4–6]. However, using MPGS for non-invasive prenatal detection of fetal aneuploidy requires a turn-around-time of 7–10 days [7], the use of expensive equipment and reagents and the use of relatively complex bioinformatics methods. Thus, investigators have been seeking alternatives for the non-invasive prenatal detection of aneuploidy. Most of these alternatives rely on the fetal-specific nucleic acid species or polymorphisms for analyzing the fetal DNA in maternal plasma. For instance, the existence of epigenetic (DNA methylation) signatures that are specific to the fetus, but not its mother, has facilitated the development of fetal epigenetic markers in maternal plasma for the non-invasive analysis of the fetal chromosome of interest. In this issue of Clinical Chemistry and Laboratory Medicine, Lim and colleagues have demonstrated that a fetal-specific DNA methylation pattern (fetal epigenetic marker) on chromosome 21 could potentially be used for such purpose [8]. The use of epigenetic marker to specifically identify the fetal DNA in maternal plasma was first demonstrated in 1999 [9]. However, that fetal epigenetic marker was polymorphism-dependent and could only be applied in certain fetal-maternal pairs. The first polymorphism-independent fetal epigenetic marker was the unmethylated form of the serpin peptidase inhibitor, clade B (ovalbumin), member 5 (also known as maspin) gene (U-SERPINB5 or U-maspin), as discovered by a candidate gene approach [10]. Since U-SERPINB5 is located on chromosome 18, it has been further demonstrated for the first time that noninvasive detection of fetal trisomy 18 could be achieved using fetal epigenetic marker [11]. Seeing this promising demonstration, various investigators have launched screening efforts at high resolution and wide genome coverage to systematically identify more fetal epigenetic markers [12–17]. CpG islands (CGIs), which harbor a high density of CpG sites, often undergo DNA methylation. The first study to systematically investigate CGIs on chromosome 21 for fetal epigenetic markers at single-nucleotide resolution has covered 114 (76% of all the 149 CGIs defined by bioinformatics criteria) and involved the use of the Epityper platform, cloning and conventional Sanger sequencing techniques [12]. This study has provided the first empirical evidence that the fetal (placental) and the maternal (blood cell) genomes harbor a lot of DNA methylation differences. Since the placenta and maternal blood cells are the respective sources of fetal and maternal DNA in maternal plasma, these DNA methylation differences could be developed into fetal epigenetic markers. In that study, a panel of 22 (19% of 114 analyzed CGIs) fetal epigenetic markers have been identified, including the unmethylated form of the phosphodiesterase 9A gene (U-PDE9A), which has been developed by Lim and colleagues as a potential noninvasive prenatal test for trisomy 21 in 2011 [18]. Later, using more sophisticated screening techniques, namely combined bisulfite and restriction analysis (COBRA), investigators have screened beyond the CpG islands for fetal epigenetic markers on 51 regions on gene promoters located on chromosome 21 [14]. This study has discovered the methylated form of the holocarboxylase synthetase gene (M-HLCS) as a fetal epigenetic marker. To compare the relative chromosome dosage, the concentrations of this fetal epigenetic marker M-HLCS were normalized against those of a fetal genetic marker on chromosome Y, zinc finger protein, Y-linked (ZFY ). Hence,