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Technical Briefs |
1
Laboratory for Prenatal Medicine, Department of Obstetrics and Gynaecology, and
2
Medical Genetics, Department of Clinical Biological Sciences, University of Basel, CH 4031 Basel, Switzerland
aaddress correspondence to this author at: Laboratory for Prenatal Medicine, Department of Obstetrics and Gynecology, University of Basel, Schanzenstrasse 46, CH 4031 Basel, Switzerland; fax 41-61-325-9399, e-mail shahn{at}uhbs.ch
The detection of gross chromosomal abnormalities is a major focus of invasive prenatal diagnosis testing, of which the most common cytogenetic anomaly in live births is trisomy 21 (Down syndrome). Classically these examinations are lengthy procedures relying on karyotypic analysis of cultured amniocytes or chorionic mesenchyme. More rapid alternatives, such as fluorescence in situ hybridization and quantitative fluorescent PCR on uncultured cells, are time- and labor-intensive. Here we describe a novel real-time PCR (1)(2) assay for the detection of trisomy 21 that is readily amenable to automation and high-throughput screening.
Real-time PCR can determine subtle alterations in gene dosage, such as hetero- or homozygosity of the RhD locus (3) and protooncogene amplification in breast cancer patients (4). We examined whether a real-time PCR assay could determine chromosomal ploidy, in particular for the prenatal detection of Down syndrome (trisomy 21). We examined the coamplification of a genetic locus (amyloid gene) in the Down’s region of chromosome 21 and a control locus [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] on chromosome 12. The amyloid gene locus on chromosome 21 was chosen because aberrant expression of this protein has been implicated in the physiologic lesions associated with Down syndrome (5). Furthermore, use of this locus should enable the detection of unbalanced Robertsonian translocations involving the Down’s region of chromosome 21, which occur in 
4% of Down syndrome cases (6). No such cases were available during in this pilot study.
We used a multiplex real-time PCR assay in which amplification of both loci was simultaneously monitored in the same reaction vessel. In this way, we could be sure that any alterations were not attributable to well-to-well variation. The following primers and probe were used for the amyloid gene on chromosome 21: forward, 5'-GGG AGC TGG TAC AGA AAT GAC TTC-3'; reverse, 5'-TTG CTC ATT GCG CTG ACA A-3'; and probe, 5'-(FAM) AGC CAT CCT TCC CGG GCC TAG G (TAMRA)-3', where FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine. The sequences for the GAPDH primers and the probe were as follows: forward, 5'-CCC CAC ACA CAT GCA CTT ACC-3'; reverse, 5'-CCT AGT CCC AGG GCT TTG ATT-3'; and probe, 5'-(VIC) AAA GAG CTA GGA AGG ACA GGC AAC TTG GC (TAMRA)-3'. In the TaqMan analysis, we used 25-µL reaction volumes containing 2 µL of the extracted DNA, 300 nmol/L each of the primers, 150 nmol/L each of the dual-labeled TaqMan probes, and further components supplied in the TaqMan Universal PCR Master Mix (Perkin-Elmer), corresponding to 3.5 mmol/L MgCl2, 100 µmol/L dNTPs, 0.025 U/µL AmpliTaq Gold, and 0.01 U/µL Amp Erase. Cycling conditions were as follows: incubation for 2 min at 50 °C, to permit Amp Erase activity, and for 10 min at 95 °C for AmpliTaq Gold activation and DNA denaturation, followed by 40 cycles of 1 min at 60 °C and 15 s at 95 °C.
In proof-of-principle experiments we examined DNA extracted from amniocyte cultures obtained from 10 trisomy 21 fetuses. As controls we used DNA samples obtained from 11 apparently healthy individuals. These samples included peripheral blood as well as amniocyte cultures. DNA was extracted from 400-µL samples, using the QIAamp DNA Blood Mini Kit according to the manufacturer’s recommendations (Qiagen). We included several dilutions of these DNA samples because we were concerned that the assay should be applicable over a broad range of DNA concentrations. In this small series of experiments we were able to show that the ratio of the two loci in these samples, as determined by the difference in threshold cycle value (
CT), distinguished trisomy 21 from karyotypically normal tissue (Fig. 1
). Furthermore, we ascertained that clear segregation of karyotypically normal and trisomic samples was possible over a wide sample concentration range, in that we still observed optimal results when we used 10-fold diluted DNA samples and DNA concentrations as low as 10 mg/L.
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We required that the following two conditions be met: that efficient amplification of both loci examined occurred and that both loci were amplified with equal efficiency over the entire exponential phase of the PCR. For this purpose we examined, on the one hand, the amplitude of the amplification curve (RN). Because this parameter is indicative of amplification efficiency and template concentration, it can be considered a rather basic endpoint measurement for quality surveillance. In our pilot study we arbitrarily selected a RN cutoff value of 0.7. It is possible that another cutoff value may be obtained in a larger series of experiments. For the second step, which was to ensure that both loci had been amplified with equal efficiency, we examined their ratio at three separate points (bottom, middle, top) along the exponential phase of the amplification reaction. Samples in which a deviation was found were disregarded.
Using this approach, we were able to correctly determine the ploidy in 9 of 11 cases with normal karyotype. Our analysis included six diluted samples in which the ploidy was also identified correctly. In the 10 samples from trisomy 21 fetuses, we were able to determine the ploidy correctly in 9 samples and in 10 separate dilutions. In those cases where we could not reliably determine fetal ploidy, we were alerted to incorrect amplification either by a RN value <0.7 or by an inconsistency in the ratio of the two loci examined at the three thresholds selected over the exponential phase of the amplification. In one case of trisomy 21, an outlier was recorded with a CT of almost 0.8. This sample was almost 3 years old. Interestingly, in two dilutions of this extraction, we obtained the expected CT values for trisomy 21.
These added precautions prevented incorrect determination of the ploidy in this series. A recent study regarding the detection of single-nucleotide polymorphisms by real-time PCR indicated that relying solely on CT values may lead to erroneous results, which could be averted by examining the corresponding RN values (7). Our small-scale pilot study, therefore, suggests that real-time PCR technology can be used for the rapid determination of trisomy 21. This technology could be easily extended to examine the most common fetal aneuploidies (13, 16, 18, X, and Y). Because real-time PCR permits the analysis of numerous samples in an automated manner, this technology may be more suited to this task than current molecular or cellular cytogenetic methods because these are considerably more time- and labor-intensive. In a large-scale setting, this method may also compare favorably with a fluorescent PCR-based approach or fluorescence in situ hybridization analysis regarding speed and price.
We caution against premature use of the described method for routine prenatal diagnosis of trisomy 21. The feasibility of this method must be explored in a large-scale prospective study before it is applied to a routine diagnostic setting.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. M. Dennis Lo and R. W. K. Chiu Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis Clin. Chem., March 1, 2008; 54(3): 461 - 466. [Abstract] [Full Text] [PDF]
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 Y. M. D. Lo, F. M. F. Lun, K. C. A. Chan, N. B. Y. Tsui, K. C. Chong, T. K. Lau, T. Y. Leung, B. C. Y. Zee, C. R. Cantor, and R. W. K. Chiu From the Cover: Digital PCR for the molecular detection of fetal chromosomal aneuploidy PNAS, August 7, 2007; 104(32): 13116 - 13121. [Abstract] [Full Text] [PDF]
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N. B.Y. Tsui, R. W.K. Chiu, C. Ding, A. El-Sheikhah, T. N. Leung, T. K. Lau, K. H. Nicolaides, and Y.M. D. Lo Detection of Trisomy 21 by Quantitative Mass Spectrometric Analysis of Single-Nucleotide Polymorphisms Clin. Chem., December 1, 2005; 51(12): 2358 - 2362. [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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 Y. Li, E. Di Naro, A. Vitucci, B. Zimmermann, W. Holzgreve, and S. Hahn Detection of Paternally Inherited Fetal Point Mutations for {beta}-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma JAMA, February 16, 2005; 293(7): 843 - 849. [Abstract] [Full Text] [PDF]
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G. Pont-Kingdon and E. Lyon Rapid Detection of Aneuploidy (Trisomy 21) by Allele Quantification Combined with Melting Curves Analysis of Single-Nucleotide Polymorphism Loci Clin. Chem., July 1, 2003; 49(7): 1087 - 1094. [Abstract] [Full Text] [PDF]
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