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Letters to the Editor |
Departments of1
Chemical Pathology, and3
Obstetrics and Gynaecology
2 Centre for Research into, Circulating Fetal Nucleic Acids, Li Ka Shing, Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR
aAddress correspondence to this author at: Department of Chemical Pathology, Room 38023, 1/F, Clinical Sciences Building, Prince of Wales Hospital, 30–32 Ngan Shing Street, Shatin, Hong Kong SAR. Fax +852-2194 -6171; e-mail loym{at}cuhk.edu.hk.
To the Editor:
Cell-free fetal nucleic acids exist not only in the maternal circulation (1) but also in amniotic fluid samples (2). Evidence suggests that DNA in maternal plasma that bears the fetal genotype is predominantly derived from the placenta (3). Although the cells in amniotic fluid are widely known to have originated from fetal urogenital, respiratory, and alimentary tracts, skin and amnion, the origin of cell-free fetal DNA in amniotic fluid remains unclear (2). It has been speculated that the latter might originate from the same sources as amniotic fluid cells or the placenta (2). Here, we applied the recent developments in epigenetics to this area of research (4)(5).
The promoter of RASSF1A has been shown to be hypermethylated in the placenta but essentially unmethylated in maternal blood cells and other fetal tissues (4). Chan et al. (5) adopted hypermethylated RASSF1A as a placental-specific epigenetic marker and developed an assay for its detection in maternal plasma as a sex-independent fetal DNA marker. The assay involved digestion by BstUI, a methylation-sensitive restriction enzyme, to eliminate the background maternal plasma unmethylated RASSF1A DNA contributed by the maternal blood cells. Hypermethylated RASSF1A sequences in maternal plasma, previously confirmed to carry the fetal genotype (5), would be resistant to digestion and therefore quantifiable by real-time PCR. Another assay targeting unmethylated ß-actin (ACTB) was included to control for the completeness of enzyme digestion. We adopted this assay system to determine whether cell-free DNA in amniotic fluid is derived from the placenta.
Fourteen pregnant women (17–21 gestational weeks) clinically indicated for amniocentesis were recruited from the Prince of Wales Hospital with informed consent and institutional ethical approval. Maternal blood was collected into EDTA-containing tubes before amniocentesis, and plasma was harvested by centrifugation at 1600g for 10 min at 4 °C, with the plasma portion recentrifuged at 16 000g for 10 min at 4 °C. Two milliliters of amniotic fluid were drawn and centrifuged at 16 000g for 10 min at 4 °C. The supernatant was removed and filtered by a 0.22-µm filter to obtain the cell-free portion. The cell pellet was washed once with phosphate buffered saline and resuspended in 200 µL of PBS [7.4 (1x) liquid (Gibco), consisting of 1.06 mmol/L monobasic potassium phosphate, 155.17 mmol/L sodium chloride, and 2.97 mmol/L dibasic sodium phosphate at pH 7.4]. For each case, DNA was extracted from 1.6 mL of maternal plasma, 400 µL of cell-free amniotic fluid, and 200 µL of the resuspended cell pellet with the QIAamp DNA Blood Mini Kit (Qiagen). All of the DNA samples were divided into 2 aliquots and subjected to either no treatment or BstUI digestion followed by real-time PCR (5).
In all cases RASSF1A was detected with and without BstUI digestion in the maternal plasma and cell-free and cell-pellet portions of the amniotic fluid. The median concentrations after BstUI digestion were 120, 54, and 124 copies/mL in the 3 corresponding sample types, respectively. The results were further expressed as a fraction of the total RASSF1A DNA concentration obtained without BstUI digestion, and corresponded to 5.69% (interquartile range: 2.36%–8.72%), 0.19% (interquartile range: 0%–0.59%), and 0.15% (interquartile range: 0%–0.28%) in the maternal plasma and the cell-free and cellular portions of amniotic fluid, respectively (Table 1
). The absolute and fractional concentrations of digestion-resistant RASSF1A in maternal plasma were comparable to the previous data (5). Complete BstUI digestion was confirmed in all samples by the absence of postdigestion ACTB DNA (data not shown).
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Our previous data showed that RASSF1A was hypermethylated only in the placenta and not other fetal tissues (4). The proportion of digestion-resistant RASSF1A in maternal plasma was similar to the fractional concentrations of circulating fetal DNA previously determined using Y-chromosomal markers (1). These findings suggest that fetal DNA in maternal plasma is predominantly derived from the placenta. On the other hand, cell-free fetal DNA concentration in amniotic fluid was reported to be >100-fold higher than that of maternal plasma (2). Yet, here we show that the proportion of digestion-resistant RASSF1A was some 30-fold lower in the cell-free portion of amniotic fluid than maternal plasma. These observations suggest that the placenta is unlikely to be the main source of amniotic fluid cell-free DNA. The majority of RASSF1A sequences in amniotic fluid, either from the cell-free or cellular portions, were digestible by BstUI and thus were unmethylated, and were probably derived from the nonplacental part of the fetus.
Our study has demonstrated the use of tissue-specific epigenetic signatures in determining the origin of cell-free fetal DNA in amniotic fluid. Cell-free DNA in amniotic fluid is mainly derived from the fetus proper. Conversely, amniotic fluid is unlikely to be a significant contributor of fetal DNA in maternal plasma.
Acknowledgments
Grant/funding support: This work was supported by a Central Allocation Grant (CUHK 01/03C) from the Research Grants Council of the Hong Kong Special Administrative Region (China). Y.M.D.L. is supported by the Chair Professorship Scheme of the Li Ka Shing Foundation.
Financial disclosures: F.M.F.L., R.W.K.C., and Y.M.D.L. have filed patent applications on aspects of fetal nucleic acid analysis from maternal plasma.
References
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