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Detection of Chromosome 21-encoded mRNA of Placental Origin in Maternal Plasma

Departments of
¹ Clinical Chemistry and
² Obstetrics and Gynaecology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

^aAuthor for correspondence. Fax 31-20-444-3895; e-mail cbm.oudejans{at}vumc.nl.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Background: mRNA of placental origin (i.e., human placental lactogen and ß-human chorionic gonadotropin) has been demonstrated to be easily detectable in maternal plasma. We tested whether detection of chromosome 21-encoded mRNA of placental origin is possible in maternal plasma obtained during the first trimester.

Methods: Plasma samples were obtained from pregnant women between weeks 9–13 of pregnancy. RNA was isolated from 800 or 1600 µL of plasma by silica-based affinity isolation and, after on-column DNase treatment, was subjected to two-step, one-tube reverse transcription-PCR with gene specific primers.

Results: Three chromosome 21-encoded genes located within the Down syndrome critical region with overexpression in trisomy 21 placentas were screened for expression in early placental tissue to select their potential use for RNA based plasma screening. One of the chromosome 21-encoded genes (LOC90625) showed strong expression in first trimester placenta similar to CSH1 (human placental lactogen) and was selected for plasma analysis. The RNA isolation assay was validated with CSH1 mRNA, which could be detected in the plasma of all women tested in weeks 9–13 of pregnancy. RNA from the chromosome 21-encoded, placentally expressed gene, LOC90625, was present in maternal first-trimester plasma and could be detected in 60% of maternal plasma samples when 800 µL of plasma was used and in 100% of samples when 1600 µL of plasma was used.

Conclusion: The detection of chromosome 21-encoded mRNA of placental origin in maternal plasma during the first trimester may allow development of plasma-RNA-based strategies for prenatal prediction of Down syndrome. LOC90625 is a candidate gene for this purpose.

	Introduction

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Recently, it has been demonstrated that mRNA of placental origin can be detected easily in maternal plasma (1). Central to this breakthrough in the field of noninvasive prenatal diagnostics was the understanding that fetal RNA circulates in a protected form and is predominantly placental in origin (1)(2)(3)(4). The protected form of circulating fetal RNA not only allows its detection, but also necessitates adaptation of the RNA isolation methods used that require some form of denaturation (2). The understanding that fetal RNA in maternal plasma is predominantly placental in origin with easy and reliable detection of human placental lactogen (hPL)¹ mRNA in all pregnancy stages is in accordance with the fact that the major cell type circulating in the maternal blood is of trophoblastic, in particular extravillus, origin (5). The apparently physiologic manner by which components (i.e., nuclei and mRNA) (1)(6)(7) of these cells appear in the maternal plasma is unknown, although programmed cell death (apoptosis) seems to be involved (6)(7).

We believe that given the predominantly placental origin of fetal RNA in maternal plasma, new markers could be developed for Down syndrome screening using the placental RNA in maternal plasma if they fulfill the following criteria: (a) the genes analyzed should be encoded by chromosome 21; (b) they should be located within the Down syndrome critical region (DSCR); (c) they should be expressed in healthy early placental tissue; (d) they should be overexpressed by the placenta in trisomy 21 pregnancies; and (e) they should be detectable in maternal plasma during early pregnancy. Gene expression profiling of trisomy 21 placentas has identified at least two genes that meet the first four criteria (8). We therefore tested these genes and one additional gene from the DSCR (9) with placental expression to determine whether one or more of these genes is expressed in the first-trimester placenta and, if so, whether it or they also meet the last criterion, i.e., detection of the gene-encoded RNA in maternal plasma during the first trimester.

	Patients and Methods

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patients
Peripheral blood samples (n = 25) were collected from pregnant women attending the Prenatal Diagnostic Unit of the VU University Medical Center with informed consent and approval of the Ethics Committee. EDTA blood was collected between weeks 9 and 13 of pregnancy. All blood samples were obtained before invasive diagnostic procedures, i.e., chorionic villus sampling.

processing of blood samples
EDTA blood was stored at 4 °C in an upright position and processed within 24 h after collection by two sequential centrifugation steps as described previously (10). In brief, after centrifugation for 10 min at 2000g at 4 °C in a Hettich Rotanta 96R centrifuge, plasma was subjected to a second centrifugation for 10 min at 25 000g at 4 °C in a Hettich EBA12R centrifuge. Plasma was stored as aliquots at -70 °C and thawed only once. Processing of blood was done within a laminar flow hood.

rna extraction from maternal plasma
RNA was extracted from 800 or 1600 µL of maternal plasma by silica-based affinity isolation using the QIAamp MinElute Virus Spin or QIAamp MinElute Virus Vacuum system (Qiagen). Before isolation, plasma samples were thawed at room temperature, the heating block was preheated to 56 °C, carrier RNA was added to AVE buffer (1 µg/µL), and the protease was thawed. All steps were done at room temperature unless stated otherwise. We added 50 µL of protease (Qiagen) to a 1.5-mL tube, followed by of 400 µL of plasma, and 400 µL of buffer AL (with 28 µg/mL carrier RNA). After vortex-mixing for 15 s, samples were incubated for 15 min at 56 °C. After centrifugation, 500 µL of ethanol was added; samples were then vortex-mixed for 15 s and left at room temperature for 5 min. All centrifugation steps were done for 1 min at 8200g unless stated otherwise. After centrifugation, the lysate mixture was carefully loaded on a QIAamp MinElute column and centrifuged.

When the vacuum system was used, the columns were inserted into the QIAvac 24 vacuum manifold (Qiagen) according to the manufacturer’s instructions. The vacuum conditions used were -80 to -90 kPa with a 19 L/min vacuum pump (Biometra MP26). All centrifugations steps except for the final elution step described below were substituted by processing with the vacuum system. For plasma starting volumes of 800 or 1600 µL, the number of tubes needed was increased accordingly, although the total volume was loaded on a single column. After transfer of the column to a new tube, 500 µL of buffer AW1 was added, followed by centrifugation. For on-column DNase digestion, 70 µL of SDD buffer was added to 10 µL of DNase, loaded on the column, and left for 15 min at room temperature. After centrifugation, 500 µL of buffer AW2 was added, and the samples were recentrifuged. The column was placed in a new tube, 500 µL ethanol was added to the column, and the column was centrifuged. The column was then placed in a new tube and centrifuged for 3 min at 25 000g. The bound RNA was eluted by placing the column in a new tube, followed by application of 20–150 µL of RNase-free MilliQ water, incubation for 5 min at room temperature, and centrifugation for 1 min at 25 000g. Finally, samples were concentrated by use of Microcon-PCR filters according to the manufacturer’s instructions (Millipore). The samples were used all in one, i.e., the RNA obtained from 800 or 1600 µL of plasma was used for a single reverse transcription-PCR (RT-PCR) assay. For controls, plasma samples obtained from nonpregnant females of similar age and race were processed and used identically.

rt-pcr
The two-step, one-tube RT-PCR assay was performed as described previously (11) with the RNase H-negative Superscript II Platinum system (Life Technologies) in the presence of 1 M betaine. RT-PCR reactions were set up on ice within a PCR workstation (CBS Scientific). In brief, RNA was mixed with 50 pmol each of forward and reverse primers in a final volume of 10 µL in MicroAmp tubes and heated for 1 min at 95 °C, followed by immediate cooling on ice. Forty microliters of master mixture was subsequently added, giving a final concentration of 1x buffer; 1.25 mM magnesium sulfate; 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 1 M betaine (Fluka); and 1 µL of enzyme mixture containing RNase H-negative Superscript II reverse transcriptase and Taq DNA polymerase (Life Technologies). After reverse transcription for 30 min at 50 °C and denaturation for 1 min at 95 °C, PCR was performed for 35 cycles (denaturation for 1 min at 95 °C, annealing for 1 min, and extension for 2 min at 72 °C), followed by a final extension for 10 min at 72 °C and cooling. All reactions were performed identically except that the annealing temperature was set at the temperature predicted to be optimal for each target (Oligo 4.0). The gene-specific primers used were as follows:

• DSCR4-F (5'ATC TTG ACG AGA GAT GAT GAA CCC C-3') and DSCR4-R (5'-CTT TAC CTC CAT GGG CTC CAC A-3'); T_annealing = 57 °C
  
• PTTG1IP-F (5'-TGA AGA ACG TCT CCT GTC TT-3') and PTTG1IP-R (5'-ACT ACC GAC ATG GTG ATG AT-3'); T_annealing = 53 °C)
  
• LOC90625-F (5'-ACA CCG CCT CGT GTT GTC TGT TGG-3' and LOC90625-R (5'GGA TCC ATC GCA CCA GGG TCA A-3'); T_annealing = 61 °C
  
• HPL-F (5'-GCA CCA GCT GGC CAT TGA CA-3') and HPL-R (5'-CCG TGC GGT TCC TCA GGA GTA T-3'); T_annealing = 58 °C
  

For expression analysis of early placental tissues and cells, RNA was obtained and isolated as described previously (11). These samples were representative of total chorionic villi, villus fibroblast cells, and extravillus trophoblast cells (SGHPL5) (12). The latter cells were kindly provided by Dr. Judith Cartwright (St. George’s Hospital Medical School, London, UK).

sequence analysis
For sequence analysis of amplified cDNA fragments, PCR products were electrophoresed in agarose for size separation, purified by affinity-based isolation (Qiagen), subjected to cycle sequencing using BigDye terminators, and analyzed using an ABI Prism 3100 Genetic Analyzer.

	Results

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placental expression of chromosome 21-encoded genes
We tested three chromosome 21-encoded genes (8)(10) for their expression and cell type distribution in early placental tissues (Fig. 1 ). For comparison, expression analysis of CSH1 (hPL) was done identically. Of the three genes tested, LOC90625, PTTG1IP, and DSCR4, all located within or near the DSCR on chromosome 21q22, the strongest expression was observed for LOC90625 (Fig. 1A ), with expression in all major cell components of the early human placenta, i.e., trophoblast cells, both villus and extravillus, as well as villus fibroblast cells. Expression of LOC90625 (Fig. 1A ) was similar in intensity to that of CSH1, although expression of the latter was restricted to the trophoblast (Fig. 1D ), whereas LOC90625 was expressed in all placental cells. Expression of PTTG1IP (Fig. 1B ) and DSCR4 (Fig. 1C ) in early placenta was weak compared with that of LOC90625 and CSH1 as analyzed in weeks 8–12 of pregnancy.

View larger version (46K): [in this window] [in a new window]   Figure 1. Expression of chromosome 21-encoded genes in early human placenta. High RNA expression in early placental tissues and cells can be seen for LOC90625 (A) similar in intensity to the chromosome-17-encoded control gene CSH1 (hPL; D). Signals for the other chromosome 21-encoded genes, PTTG1IP (B) and DSCR4 (C), are weak. Lane C, early placental tissue; lane S, extravillus trophoblast cell line; lane V, villus fibroblast cells; lane MW, molecular weight marker (100-bp ladder).

detectability of chromosome 21-encoded rna in maternal plasma
We subsequently tested whether chromosome 21-encoded RNA from LOC90625 could be identified in maternal plasma samples obtained from pregnant women in a similar gestational age window (weeks 9–13). Before this, our RNA isolation procedure was validated using CSH1 (1). In all pregnant females tested (n = 7), CSH1 RNA was detected easily (Fig. 2 ) in 800 µL of maternal plasma between weeks 9 and 13 of gestation, whereas plasma specimens from nonpregnant females (n = 7) were negative. No false-positive or -negative results were obtained. Subsequently, RNA detection was performed identically for the chromosome 21-encoded gene LOC90625. mRNA from this gene was successfully detected in maternal plasma, although with a lower intensity compared with CSH1. When 800 µL of plasma was used, detection was successful in 60% of samples; when 1600 µL of plasma was used, detection of LOC90625 mRNA in first-trimester plasma from pregnant females (n = 8) was 100% (Fig. 3 ). Moreover, sequence analysis of the LOC90625 cDNA amplicons generated in this way confirmed the specificity of the products.

View larger version (55K): [in this window] [in a new window]   Figure 2. Detection of CSH1 mRNA in maternal plasma samples. CSH1 cDNA amplicons generated by RT-PCR from RNA isolated from maternal plasma (weeks 9–13) can be seen in all pregnant samples analyzed (lanes 4–10). All negative controls (n = 8) from nonpregnant females were negative (only one is shown, in lane 11). Positive controls consisting of early placental tissues and cells are shown in lanes 1–3: lane 1, early placental tissue; lane 2, extravillus trophoblast cell line; lane 3, villus fibroblast cells. Lane MW, molecular weight marker (100-bp ladder).

View larger version (46K): [in this window] [in a new window]   Figure 3. Detection of chromosome 21-encoded mRNA of placental origin in maternal plasma samples. LOC90625 cDNA generated by RT-PCR of amplicons isolated from maternal plasma (week 9–13) are identified in all (n = 8) pregnant samples analyzed (lanes 4–12). Note the absence of the nonspecific lower bands with retention of the specific 362-bp band when RNA isolation was performed under vacuum-controlled conditions (lanes 11 and 12). All negative controls from nonpregnant females (n = 7) were negative (only one is shown, in lane 13). Positive controls consisting of early placental tissues and cells are shown in lanes 1–3: lane 1, early placental tissue; lane 2, extravillus trophoblast cell line; lane 3, villus fibroblast cells. Lane MW, molecular weight marker (100-bp ladder).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In this report, we have shown the presence and detectability of chromosome 21-encoded mRNA of placental origin in maternal plasma during weeks 9–13 of pregnancy. Given that the gene involved, LOC90625, has up-regulated expression in trisomic placentas (8) and is located within the DSCR, our data, which extend the pioneering work of Dennis Lo’s group (1), indicate that prenatal screening of Down syndrome becomes a realistic option with this plasma-RNA-based assay. To convert the experimentally validated strategy as described in this study to a clinical diagnostic application, quantitative plasma analysis of a placentally expressed, chromosome 21-encoded mRNA (target gene) must be complemented by correction for experimental and biological variability. This can be done through identical analysis of a second, placentally expressed gene from a different autosome (placenta-specific reference gene). We have shown that LOC90625 is a good candidate for the target gene that meets the criteria presented above: it codes for placentally expressed, chromosome 21-encoded mRNA; it is located within the DSCR; it is overexpressed in trisomy 21 placentas (8); and it is detectable in maternal plasma in the first trimester, as shown in this study.

Although CSH1 (hPL) is placentally expressed and CSH1 mRNA can be detected easily in the maternal plasma [Ref. (1) and this study], we feel that CSH1 is unsuitable for use as a placenta-specific reference gene. CSH1 belongs to a large gene cluster on chromosome 17q24 with a complex genomic organization and transcription pattern. This cluster encompasses five genes (CSH1, CSH2, GH1, GH2, CSHL1) with very similar organization and nearly identical sequences. In addition, each gene codes for at least four alternative transcripts (13). All of these genes except for GH1 are expressed in the placenta, but expression differs according to time (gestational age), cellular origin, and the amounts expressed. The CSH primers used in our study react with eight transcripts, of which seven are produced by the placenta (CSH1A, -B, CSH2-A and -B, and GH2-A, -B, and -C). The same holds for the primers used previously by Ng et al. (1). This feature explains the clear difference we observe between the signal intensities for CSH1 and LOC90625 mRNA as detected in maternal plasma. Unfortunately, the obvious strategy of designing alternative primers reactive only with single CSH transcripts is not possible. Consequently, the mRNA copy number of placental mRNA molecules expected to circulate in maternal plasma during the first trimester is likely to be within the range observed for ß-human chorionic gonadotropin, with an upper limit of 5000 copies/mL of maternal plasma, rather than the threefold higher amounts determined for hPL (1) in the first trimester.

The RNA isolation procedure we used is similar in design to the method used by Ng et al. (1), i.e., it involves denaturation followed by silica-based affinity isolation. However, our procedure is less hazardous and simpler because it lacks Trizol and chloroform treatments. We noticed that the presence of nonspecific RT-PCR signals when low-abundancy targets were analyzed (see right hand panel in Fig. 3 ) was attributable to reactivity with the carrier RNA present in the isolation reagents. However, these nonspecific bands disappeared, whereas the specific bands were retained, when the vacuum manifold system was used. In addition, with the vacuum manifold system, up to 24 samples can be processed simultaneously. Using the same affinity-based isolation principle, the RNA isolation procedure can be automated and modified to a walk-away procedure for use with a robotic workstation (such as the BioRobot MDX).

In conclusion, we demonstrate the presence and detectability of chromosome 21-encoded mRNA (LOC90625) of placental origin in the plasma of pregnant women between weeks 9 and 13 of pregnancy. Because expression of the LOC90625 gene is up-regulated in trisomy 21 placentas, this could permit the development of clinical diagnostic tests based on analysis of plasma RNA for Down syndrome screening during the first trimester.

	Acknowledgments

 
This work was supported by Grant 01245 from the Health Insurance Council. We thank K. Deurloo, M. Engels, F. Gerards, and M. Bekker for their support.

	Footnotes

 
¹ Nonstandard abbreviations: hPL, human placental lactogen; DSCR, Down syndrome critical region; and RT-PCR, reverse transcription-PCR.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (24) Citing Articles via Google Scholar Google Scholar Articles by Oudejans, C. B.M. Articles by van Vugt, J. M.G. Search for Related Content PubMed PubMed Citation Articles by Oudejans, C. B.M. Articles by van Vugt, J. M.G. Related Collections Molecular Diagnostics and Genetics