Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies
Cell Biology Laboratory, Paediatric Clinic (IH,LV,KV), Clinic of Obstetrics and Gyneacology (JD,RV), and Blood Bank, 2nd Medical Faculty (BB), Charles University, University Hospital Motol, Prague, Czech Republic
Correspondence to: Dr. Ilona Hromadnikova, Clinic of Paediatrics, 2nd Medical Faculty, University Hospital Motol, V Uvalu 84, 150 18 Prague 5, Czech Republic. E-mail: ilona.hromadnikova{at}lfmotol.cuni.cz
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Summary |
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Key Words: fetal DNA maternal plasma real-time PCR RHCE gene RHD gene hemolytic disease of the newborn
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Introduction |
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In this prospective study, we assessed the feasibility of fetal RHD, RHC, and RHE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women homozygous for c and/or e alleles of the RHCE gene using real-time PCR and primers and probes targeted toward the RHD (exon 7 and exon 10) and RHCE (exon 2 and exon 5) genes.
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Materials and Methods |
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Forty-five consecutive RhD-negative pregnant women including those alloimmunized (1x anti-D, 3x anti-D+C, 1x anti-Kell) at risk for HDN at a gestational stage ranging from 11 to 40 weeks were recruited for the study.
The study was approved by the Ethics Committee of our local institution, and informed consent was obtained from all study patients.
DNA Extraction from Plasma Samples
Ten milliliters of maternal peripheral blood from pregnant women was collected into EDTA-containing tubes and processed within a few hours (maximally 24 hr). In detail, blood samples were centrifuged first at 1200 x g (protocol 1) (Hahn et al. 2000; Hromadnikova et al. 2003
) and then at 3000 x g (protocol 2) for 10 min. (Lo et al. 1998b
). Plasma samples were then recentrifuged, and the supernatants were collected and stored at 80C until further processing. DNA was extracted from 400 µl plasma using QIAamp DNA Blood Mini Kit (Qiagen; Hilden, Germany) according to the manufacturer's instructions. To minimize the risk of contamination, DNA was isolated in laminar airflow and aerosol resistant tips were used. DNA was eluted in 50 µl Buffer AE. Five µl of DNA were used as a template for the RHD PCR reaction and 2.5 µl of DNA for the ß-globin (GLO) PCR reaction.
Real-time PCR Analysis
Real-time PCR analysis was performed using ABI PRISM 7700 Sequence Detection System (Applied Biosystems; Branchburg, NJ).
Primer and probe sequences are shown in Table 1. The GLO gene served as a control to confirm the presence and quality of DNA in each sample (Lo et al.1998b; Hromadnikova et al. 2003
). Amplicons for the GLO control gene were detected in all analyzed samples.
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Results |
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Non-invasive prenatal fetal RHD exon 7 and exon 10 genotyping analysis of maternal plasma samples was in complete concordance with the analysis of cord blood in 45 out of 45 RhD- negative pregnant women delivering 24 RhD-positive- and 21 RhD-negative newborns. Non-invasive prenatal fetal RHC exon 2 genotyping was performed as well in 41 out of 41 Rhc homozygote pregnant women delivering 17 RhC-positive- and 24 RhC-negative newborns. Similarly, non-invasive prenatal fetal RHE genotyping was done as well in 45 out of 45 Rhe homozygote pregnant women delivering 7 RhE-positive- and 38 RhE-negative newborns. Results are summarized in Table 2.
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Fetal RHC and RHE genotyping is also important in pregnancies bearing an RhD-negative female fetus because amplification of another paternally inherited allele (RhC or RhE positivity) proves the presence of fetal DNA in maternal circulation, as we showed in the case of patient no. 902.
SRY real-time PCR analysis of maternal plasma was in complete concordance with fetal sex in all 45 RhD-negative pregnant women (Table 2). We confirmed the sensitivity and specificity of the system previously in a cohort of pregnancies at risk for X-linked hemophilia and chromosomal aneuploidies when we determined fetal sex by analysis of DNA in maternal plasma using real-time PCR and the SRY gene as a marker for the detection of the fetal Y chromosome (Hromadnikova et al. 2003).
The amplification efficiencies of the assays specific for SRY, RHD exon 7, RHD exon 10, and RHC were comparable. The amplification occurred on a similar Ct (the difference less than 1.5 Ct). However, a lower efficacy was observed for RHE system. The amplification of fetal DNA occurred 1.0 to 4.0 (mean 2.0) Ct later.
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Discussion |
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We suggest checking one of the RhD-specific nucleotides in RHD exon 7 and at least one second region: exon 4, intron 4 (Cotorruelo et al. 2002) or exon 10 of RHD gene. RHD genotyping based solely on RhD-specific sequences in the 3' untranslated region of RHD exon 10 is not considered a safe procedure (Flegel and Wagner 2002
). When D-positive fetuses are identified and anti-D alloantibodies are already present, it is important to carefully monitor anti-D alloantibody titer variations during the pregnancy so that fetal-blood sampling can be performed in time to determine the level of fetal hemolysis and to start early treatment of sensitized RhD-negative pregnancies (ultraviolet phototherapy or exchange or intrauterine transfusion, if required). On the other hand, detection of a D-negative fetus excludes the risk of HDN caused by anti-D alloantibodies that may be present in the maternal circulation from previous pregnancies for a variety of reasons, including non-administration of Rh immunoglobulin, unrecognized miscarriage, leakage of fetal RBCs into the maternal circulation in late pregnancy, etc. (Avent 1998
; Avent and Reid 2000
). However, the identification of a D-negative fetus should be verified if another fetal marker is detected in maternal plasma samples. SRY-positive- and RHD-, RHC-, and RHE-negative genotyping results on the same maternal plasma sample identify a male fetus with cde paternally inherited haplotype. SRY positivity proves the presence of fetal DNA in a maternal plasma sample and may confirm the specificity of the RHD-, RHC-, and RHE-negative results. Similarly, in a case of an identification of an RhD-negative female fetus, amplification of another paternally inherited allele (RHC or RHE positivity) proves the presence of fetal DNA in the maternal circulation.
In other cases (e.g., a female fetus with ccddee phenotype), the specificity of RhD-negative results remains uncertain, inasmuch as we do not have any other marker to confirm the presence of fetal DNA in maternal circulation.
We recommend performing fetal RHD, RHC, and RHE genotyping together with fetal sex determination in alloimmunized pregnancies at risk of HDN due to the presence of anti-D, anti-D+C, or anti-D+C+E alloantibodies. When maternal anti-D+C are identified in sera from alloimmunized women, it is important to genotype fetuses for RhD, RhC, and RhE status to establish whether they express Dce, DCe, dCe, DcE, DCE, or dCE paternally inherited haplotypes and are at risk for HDN (Avent 1998). While larger and confirmatory studies are required, these data present a compelling argument that this type of testing may be incorporated into our clinical diagnostic algorithm for following pregnancies at risk for hemolytic disease of the newborn.
Anti-E rarely produces clinically significant alloimmunization (Avent 1998; Avent and Reid 2000
).
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Acknowledgments |
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Footnotes |
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Received for publication May 4, 2004; accepted September 29, 2004
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