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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, University of Basel, Basel, Switzerland.
Address reprint requests to: Sinuhe Hahn, PhD, Department of Obstetrics and Gynecology, University of Basel, Schanzenstrasse 46, Basel CH 4031, Switzerland, E-mail: shahn{at}uhbs.ch
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Abstract |
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Methods: Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR).
Results: In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold.
Conclusion: The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.
Circulating extracellular fetal DNA has been detected recently in the plasma of pregnant women.1 This observation opens a new avenue for noninvasive prenatal diagnostics, as it provides another source of fetal genetic material. A caveat with respect to this method, however, is that because circulating free extracellular DNA is detectable in blood plasma and serum of normal individuals,2 it is only possible to detect fetal genetic sequences that are absent from the maternal genome in this way.1,3 Male fetal DNA was initially detected by the polymerase chain reaction (PCR) for Y chromosome–specific sequences.1 Subsequently, this approach was used to determine the fetal rhesus D status in pregnancies at risk for hemolytic disease of the fetus and newborn,4–8 because the rhesus D gene is absent in rhesus D-negative individuals.9
The advent of real-time PCR technology has permitted reproducible and sensitive quantification of the PCR reaction, which enables us to determine the amount of DNA template present in the starting material.10 This PCR process uses two specific primers and a fluorescently labeled probe to amplify the sequence. During the amplification process this labeled probe is displaced by the action of the polymerase, thus generating a light signal. By monitoring the amount of fluorescence generated in each amplification cycle, it is possible to calculate accurately the efficacy of the PCR reaction and to quantify the amount of template present in the initial reaction.
Use of this technology has recently shown that the quantity of circulating male fetal DNA was generally low in the first trimester and increased approximately tenfold near the end of pregnancy.11 Significantly elevated levels of circulating fetal DNA were detected in certain pregnancy-related disorders, such as preeclampsia12 and the onset of preterm labor.13
Of potential interest for prenatal diagnosis was the recent observation that the concentration of fetal DNA was shown to be increased by a factor of almost twofold in women with fetuses with trisomy 21,14 a feature we have confirmed in an independent masked study.15 These results suggested that quantitative PCR analysis of circulating fetal DNA could be used for screening purposes.16
The results of such a test would be severely compromised if the analyte measured varied considerably in the same individual over time, so we examined blood samples taken consecutively over 3 days to determine whether the levels of free fetal DNA fluctuated or were stable.
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Materials and Methods |
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Blood samples were processed immediately by centrifugation at 800 g for 10 minutes. The circulating free extracellular DNA was then extracted from 400 µL of plasma using the QIAamp Blood Kit (Qiagen AG, Basel, Switzerland) according to the manufacturer’s protocol and as described previously.11 Strict anticontamination procedures were used throughout all analytic steps, including the use of aerosol resistant tips (ART, Molecular Bio-Products, San Diego, CA) and ultraviolet crosslinking of the pipetting instruments (Stratagene Stratalinker, San Diego, CA). Multiple negative controls were included in each analysis. The DNA preparations were eluted in 50 µL of elution buffer (10 mM Tris HCl pH 7.4:1 mM EDTA), of which 2 µL was used as a template for the PCR reaction.
The TaqMan real-time PCR analysis was performed using a Perkin Elmer Applied Biosystems 7700 Sequence Detector (Perkin Elmer, Branchburg, NJ). Primers and dual-labeled probes, designed with the aid of Primer Express software (Perkin Elmer), were used.
The SRY locus, which is specific for the sex-determining gene on the Y chromosome (GenBank accession no. L08063), was used to ascertain the amount of male fetal DNA present in the sample. The size of the PCR fragment analyzed was 78 base pairs. For this gene the following primers and dual-labeled TaqMan probe were used: (forward) 5' TCC TCA AAA GAA ACC GTG CAT 3', (reverse) 5' AGA TTA ATG GTT GCT AAG GAC TGG AT 3', and (probe) 5' (FAM) CAC CAG CAG TAA CTC CCC ACA ACC TCT TT (TAMRA) 3'.
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession no. J04038) gene is common to all genomes. This primer and probe combination was used to determine the total amount of DNA in the plasma and served as a control that DNA was indeed present in the sample analyzed. The length of the PCR fragment analyzed was 97 base pairs. The following primers and probe were used for the GAPDH gene: (forward) 5' CCC CAC ACA CAT GCA CTT ACC 3', (reverse) 5' CCT AGT CCC AGG GCT TTG ATT 3', and (probe) 5' (FAM) AAA GAG CTA GGA AGG ACA GGC AAC TTG GC (TAMRA) 3'.
Because most circulatory DNA in maternal plasma is of maternal rather than fetal origin,11 the data obtained by this assay are indicative of the level of circulatory maternal DNA.
For the TaqMan PCR analysis, we used 25 µL of reaction volumes containing 2 µL of the extracted DNA, 300 nM of each amplification primer, 100 nM of the dual-labeled TaqMan probe, and the necessary components provided in the TaqMan PCR Core Reaction kit (Perkin Elmer). This corresponded to 2.5 µL of 10x Buffer A, 3.5 mM MgCl2, 100 µM deoxyribonucleotides, 0.025 U per µL of AmpliTaq Gold, and 0.01 U per µL of Amp Erase. The uracil N-glycosylase activity of the latter was used to prevent contamination by the carryover of PCR products. In addition, because the Taq-Man system is a closed system, this greatly reduces the risk of contamination by the carryover of PCR amplicons.
Because the analysis was designed to use identical thermal profiles for both the SRY and GAPDH TaqMan assay, the DNA samples were analyzed for those two markers on the same plate in the same analytic run. These reactions were carried out using a 2-minute incubation at 50C to permit Amp Erase activity, followed by an initial denaturation step at 95C for 10 minutes, which facilitates activation of the AmpliTaq Gold polymerase activity, followed by 40 cycles of 1 minute at 60C and 15 seconds at 95C.
To determine the number of copies of total circulating DNA in the plasma sample, a standard dilution curve using a known concentration of male genomic DNA was used. For the conversion to the number of copies or genome equivalents, 6.6 pg was used as described previously.11 This curve was also used to calculate the amount of circulating male fetal DNA in the plasma samples obtained from pregnant women. All samples were analyzed in duplicate and scoring was masked. The amount of maternal circulatory DNA was determined in an analogous manner using a standard curve for the GAPDH gene.
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Results |
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Because little is known about the physiologic mechanisms leading to the release of free DNA into the circulation or about the stability of this material in the blood system, we first examined whether the total levels of free extracellular DNA varied or remained constant in the blood of normal healthy nonpregnant individuals. For that purpose we examined the levels of total circulating DNA in plasma samples that had been drawn once or twice daily for 3 days from eight female and five male volunteers. The analysis of these samples showed that differences as high as 67.9-fold could occur in the quantity of total circulating DNA in a given individual (Table 1
and Figure 1). These data also showed that differences up to tenfold could be measured in samples taken 8 hours apart (Figure 1).
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). The concentration of male fetal circulatory DNA in the ten maternal plasma samples taken from pregnancies with male fetuses also varied over the same period, with a mean difference of 2.2-fold (range 1.4–4.5; Table 3). A surprising finding of this analysis was that the quantities of fetal and maternal circulatory DNA varied independent of each other (Figure 2).
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Discussion |
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Real-time quantitative PCR technology10 accurately determined the quantity of circulatory DNA.11 This technique has shown that levels of free fetal DNA increase considerably during gestation11 and that significant increments in the levels of this fetal DNA were observed in pregnancy-related disorders.12,13 Of particular interest is the twofold increase in free fetal DNA level that was observed in pregnancies with aneuploid fetuses, especially those with trisomy 21.14,15 These findings suggest that quantitative PCR assays that measure levels of free circulatory DNA can be used for prenatal screening.16
Our data indicate that the levels of total free circulatory DNA were not constant and that they varied considerably both in the circulation of each of the 13 healthy individuals and in each of the 16 healthy pregnant women. Furthermore, these fluctuations were not restricted to maternal free DNA but were also observed for free fetal DNA in each of the ten pregnancies examined with a male fetus using a Y chromosome–specific quantitative PCR assay. The examination in this study of pregnancies from a wide range of gestational ages indicated that fetal DNA levels varied throughout pregnancy. Although there might be a tendency for greater fluctuations to occur around 25 weeks’ gestation (2.4- to 4.5-fold), there was no clear increase in variation toward the end of pregnancy. Clarification of this issue will require the study of a larger number of cases.
What is more pertinent, however, is that the 2.2-fold mean variation in free fetal DNA levels was determined to be comparable to the twofold increases in free fetal DNA in pregnancies with aneuploid fetuses. Hence, these data suggest that caution should be taken when considering the quantification of free fetal DNA as the basis of a screening assay to detect fetal aneuploidies.
A further interesting aspect of this study is that the alterations in the amount of circulatory fetal DNA occurred independently of the variation in the quantity of maternal DNA. This finding implies that the release of fetal DNA into the maternal circulation is not coupled with the release of maternal DNA in normal pregnancies and that these two factors are regulated independently. It will be important to determine whether this phenomenon holds true for pregnancy-related pathologies.
It is also likely that the results presented here will have wider application because elevated levels of extra-cellular circulating DNA also have been detected in the plasma of patients with cancer,2 pulmonary embolism,17 and systemic lupus erythematosus.18 The quantitation of circulatory DNA has been proposed for monitoring of engraftment in transplant patients and to assess levels of drug-induced cytotoxicity.19,20 The potential diagnostic use of this technology in these disorders will have to be addressed carefully.
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Footnotes |
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Received December 27, 1999. Received in revised form May 24, 2000. Accepted June 22, 2000.
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References |
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2. Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nature Med 1996;2:1033–5.[Medline]
3. Hahn S, Sant R, Holzgreve W. Fetal cells in maternal blood: Current and future perspectives. Mol Hum Reprod 1998;4:515–21.
4. Faas BH, Beuling EA, Christiaens GC, von dem Borne AE, van der Schoot CE. Detection of fetal RHD-specific sequences in maternal plasma. Lancet 1998;352:1196.[Medline]
5. Lo YM, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734–8.
6. Bischoff FZ, Nguyen DD, Marquez-Do D, Moise KJ Jr, Simpson JL, Elias S. Noninvasive determination of fetal RhD status using fetal DNA in maternal serum and PCR. J Soc Gynecol Invest 1999;6: 64–9.[Medline]
7. Hahn S, Zhong XY, Bürk MR, Troeger C, Holzgreve W. Multiplex and realtime quantitative PCR on fetal DNA in maternal plasma: A comparison with fetal cells isolated from maternal blood. Ann N Y Acad Sci 2000;906:148–55.
8. Zhong XY, Holzgreve W, Hahn S. Detection of fetal rhesus D and sex using fetal DNA from maternal plasma by multiplex PCR. Br J Obstet Gynaecol 2000;107:766–9.
9. Mouro I, Colin Y, Cherif-Zahar B, Cartron JP, Le Van Kim C. Molecular genetic basis of the human rhesus blood group system. Nature Genet 1993;5:62–5.[Medline]
10. Heid C, Stevens J, Livak K, Williams P. Real time quantitative PCR. Genome Res 1996;6:986–94.
11. Lo DYM, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: Implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.[Medline]
12. Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45:184–8.
13. Leung TN, Zhang J, Lau TK, Hjelm NM, Lo YM. Maternal plasma fetal DNA as a marker for preterm labour. Lancet 1998; 352:1904–5.[Medline]
14. Lo YM, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45:1747–51.
15. Zhong XY, Bürk M, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn. In press.
16. Holzgreve W, Hahn S. Novel molecular biological approaches for the diagnosis of preeclampsia. Clin Chem 1999;45:451–2.
17. Riboldi P, Asero R, Marazzini L, Longhini E, Cherubini P, Pacetti M, et al. Detection of circulating DNA in plasma of patients with pulmonary embolism by counterimmunoelectrophoresis. Respiration 1984;45:45–9.[Medline]
18. Levine JS, Koh JS. The role of apoptosis in autoimmunity: Immunogen, antigen, and accelerant. Semin Nephrol 1999;19:34–47.[Medline]
19. Lo YM, Tein MS, Pang CC, Yeung CK, Tong KL, Hjelm NM. Presence of donor-specific DNA in plasma of kidney and liver-transplant recipients. Lancet 1998;351:1329–30.[Medline]
20. Bret L, Lule J, Alary C, Appolinaire-Pilipenko S, Pourrat JP, Fournie GJ. Quantitation of blood plasma DNA as an index of in vivo cytotoxicity. Toxicology 1990;61:283–92.[Medline]
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