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Technical Briefs |
Departments of1 Chemical Pathology and 3 Obstetrics & Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR;2 Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, United Kingdom
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F Clinical Sciences Bldg., Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong Special Administrative Region, China; e-mail loym{at}cuhk.edu.hk
The recent demonstration of detectable circulating fetal RNA in maternal plasma (1)(2) has led to the development of new, noninvasive prenatal diagnostic opportunities (3)(4). Unlike fetal DNA measurements in maternal plasma/serum, quantitative analysis of circulating fetal RNA has the advantage of being applicable to all pregnant women irrespective of fetal gender and genetic polymorphism status. In addition, the unexpected stability of circulating RNA has enhanced the practicality of this approach (2)(5)(6). Previously, we developed a real-time quantitative reverse transcription-PCR (RT-PCR) assay for measuring the concentration of human chorionic gonadotropin ß-subunit (ßhCG) mRNA in plasma samples from healthy pregnant women (2). We conducted a case–control study to investigate whether abnormal concentrations of ßhCG mRNA might be detectable in the serum of mothers carrying fetuses with trisomy 21 and trisomy 18.
We sought informed consent from pregnant women who presented for aneuploidy screening at the King’s College Hospital London in the United Kingdom between January and August 2003. Ethics approval was obtained from the Institutional Review Board. Among women who underwent chorionic villous sampling for fetal karyotyping as a result of clinical indications, 149 women consented to blood sampling for ßhCG mRNA measurements. Maternal blood samples were collected into plain tubes immediately before chorionic villous sampling. The blood samples were centrifuged at 1600g for 10 min at 4 °C. The serum was carefully transferred into plain polypropylene tubes, and 3.2 mL was immediately stored in 4 mL of Trizol and kept at –80 °C until RNA extraction. Serum RNA was extracted from 1.6 mL of serum with use of a modified RNeasy RNA Mini Kit (Qiagen) as described previously (2). Total RNA was eluted with 30 µL of RNase-free water and stored at –80 °C until real-time quantitative RT-PCR analysis. DNase treatment was carried out to remove any contaminating DNA (RNase-Free DNase Set; Qiagen).
One-step real-time quantitative RT-PCR was used for ßhCG mRNA quantification, as described previously, without knowledge of the karyotyping results (2). The RT-PCR reactions were set up in a reaction volume of 25 µL. The primers and fluorescent probe were used at concentrations of 300 and 100 nM, respectively, and 6 µL of extracted serum RNA was used for amplification. The thermal profile used for the analysis was as follows: the reaction was initiated at 50 °C for 2 min for the included uracil N-glycosylase to act, followed by reverse transcription at 60 °C for 30 min. After a 5-min denaturation at 95 °C, 40 cycles of PCR was carried out with denaturation at 94 °C for 20 s and annealing/extension at 57 °C for 1 min. The sensitivity, linearity, and precision of the assay have been established as described previously (2). We were able to detect down to 100 copies of the synthetic oligonucleotide in the reaction mixture. Concentrations of serum ßhCG mRNA are expressed as copies/mL of serum. Because no recovery experiments had been done, the reported concentrations (copies/mL) are minimum estimates.
Among the 149 pregnant women recruited, trisomy 21 and trisomy 18 were confirmed by fetal karyotyping in 15 and 11 pregnancies, respectively. The remaining 123 cases had euploid fetuses and served as controls. The median gestational age of the controls was 12.5 (range, 11.2–14.3) weeks, and the median gestational ages of the trisomy 21 and trisomy 18 cases were 12.5 (12.1–14.2) weeks and 12.3 (11.4–14.1) weeks, respectively. No significant difference of the gestational age was observed among the three cohorts (Kruskal–Wallis, P = 0.706).
Maternal serum samples from the 149 studied cases were subjected to ßhCG mRNA quantification. ßhCG mRNA could be detected in the maternal serum of 140 of 149 pregnancies (94%). In the control cohort, the detection rate of ßhCG mRNA was 97% (119 of 123). For the trisomy 21 and trisomy 18 cohorts, the detection rates were 93% (14 of 15) and 64% (7 of 11), respectively. The median serum ßhCG mRNA concentrations of the three cohorts were 6108 (interquartile range, 2867–19 249) copies/mL for the control cohort, 13 165 (4403–25 265) copies/mL for the trisomy 21 cohort, and 652 (0–11 662) copies/mL for the trisomy 18 cohort (Fig. 1
). The median ßhCG mRNA concentrations differed significantly among the three cohorts (Kruskal–Wallis, P = 0.024). Pairwise multiple comparisons were performed and showed that differences were significant between the trisomy 18 and control cases (Dunn test, P <0.05) and between the trisomy 18 and trisomy 21 cases (Dunn test, P <0.05). No statistically significant difference was observed between the serum ßhCG mRNA concentrations in trisomy 21 and control cases (Dunn test, P >0.05).
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In this study we confirmed that circulating ßhCG mRNA is easily and robustly detectable in the serum of first-trimester pregnant women, with a detection rate of 94%. These data are largely concordant with our previous data on first-trimester pregnancies (median gestational age, 12.3 weeks) (2). Our present data also demonstrated that the median concentration of serum ßhCG mRNA in pregnancies with trisomy 18 was 9.4-fold lower than the median concentration in the control pregnancies and that the difference was statistically significant. On the other hand, although the median concentration of serum ßhCG mRNA in pregnancies with trisomy 21 was 2.2-fold higher than the median concentration in the control pregnancies, the difference was not significantly different. Interestingly, similar relationships were demonstrated in several previous studies on placental tissue expression of ßhCG mRNA. Although some inconsistent data exist in the literature (7)(8), ßhCG mRNA concentrations in placental tissues have been shown to be significantly lower in trisomy 18 than in control pregnancies (9), whereas no difference has been reported for trisomy 21 pregnancies (10). Thus, the ßhCG mRNA concentration in maternal serum could potentially be a reflection of the placental tissue expression pattern as demonstrated in a study by Tsui et al. (11).
The data presented here demonstrate for the first time that circulating ßhCG mRNA concentrations in the first-trimester serum of trisomy 18 pregnancies is significantly lower than in non-trisomy 18 pregnancies. The mechanisms accounting for this difference require further investigation. Our findings indicate the potential diagnostic usefulness of circulating ßhCG mRNA as a marker for predicting trisomy 18 pregnancies. However, our data also show that there is an overlap in the ßhCG mRNA concentrations between the trisomy 18 and control cases. This implies that a relatively low sensitivity and specificity would result if maternal serum ßhCG mRNA measurement were used as the sole predictor for pregnancies with trisomy 18. In ROC curve analysis (using MedCalc 5.0 software), the mean (SE) area under the ROC curve was 0.734 (0.067) with a 95% confidence interval of 0.651–0.806. On the other hand, a larger scale study may be necessary to explore whether maternal serum ßhCG mRNA is a useful marker in trisomy 21 screening.
In summary, our findings provide the first evidence for the value of circulating placental mRNA measurement in the noninvasive detection of a fetal chromosomal aneuploidy. The current study is designed primarily as a proof-of-concept investigation. The main technical advantage of the mRNA technology is the relative ease with which new mRNA markers can be developed, including genes coding for proteins for which no immunoassays are currently available. We believe that the availability of microarray technology could lead to development of panels of placenta-specific mRNA markers for future fetal aneuploidy screening.
Acknowledgments
This study was supported by the Innovation and Technology Fund (ITS/195/01).
References
and ß human chorionic gonadotrophin in early trisomy 18 pregnancies. Mol Hum Reprod 1996;2:463-465. and ß subunits of human chorionic gonadotrophin in early pregnancies with Down’s syndrome. Hum Reprod 1995;10:2506-2509.The following articles in journals at HighWire Press have cited this article:
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  S. S.C. Chim, T. K.F. Shing, E. C.W. Hung, T.-y. Leung, T.-k. Lau, R. W.K. Chiu, and Y.M. Dennis Lo Detection and Characterization of Placental MicroRNAs in Maternal Plasma Clin. Chem., March 1, 2008; 54(3): 482 - 490. [Abstract] [Full Text] [PDF]
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R. W.K. Chiu, W.-b. Lui, M.-c. Cheung, N. Kumta, A. Farina, I. Banzola, S. Grotti, N. Rizzo, C. J. Haines, and Y.M. D. Lo Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma Clin. Chem., February 1, 2006; 52(2): 313 - 316. [Abstract] [Full Text] [PDF]
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B. C.K. Wong, R. W.K. Chiu, N. B.Y. Tsui, K.C. A. Chan, L. W. Chan, T. K. Lau, T. N. Leung, and Y.M. D. Lo Circulating Placental RNA in Maternal Plasma Is Associated with a Preponderance of 5' mRNA Fragments: Implications for Noninvasive Prenatal Diagnosis and Monitoring Clin. Chem., October 1, 2005; 51(10): 1786 - 1795. [Abstract] [Full Text] [PDF]
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 Y.M. D. Lo Recent Advances in Fetal Nucleic Acids in Maternal Plasma J. Histochem. Cytochem., March 1, 2005; 53(3): 293 - 296. [Abstract] [Full Text] [PDF]
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X. Y. Zhong, W. Holzgreve, I. Hoesli, and S. Hahn Circulatory Corticotropin-Releasing Hormone mRNA Concentrations Are Increased in Women with Preterm Delivery But Not in Those Who Respond to Tocolytic Treatment Clin. Chem., March 1, 2005; 51(3): 635 - 636. [Full Text] [PDF]
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S. K. R. Chinnapapagari, W. Holzgreve, O. Lapaire, B. Zimmermann, and S. Hahn Treatment of Maternal Blood Samples with Formaldehyde Does Not Alter the Proportion of Circulatory Fetal Nucleic Acids (DNA and mRNA) in Maternal Plasma Clin. Chem., March 1, 2005; 51(3): 652 - 655. [Full Text] [PDF]
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