Fetal RhD genotyping by real time quantitative PCR in maternal

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Fetal RhD genotyping by real time quantitative PCR in maternal
Original article
Ann Biol Clin 2012; 70 (6): 683-8
Fetal RhD genotyping by real time quantitative PCR
in maternal plasma of RhD-negative
pregnant women from the Sahel of Tunisia
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 12/08/2017.
Génotypage RhD fetal par PCR quantitative en temps réel
à partir du plasma maternel des femmes enceintes RhD-négatif
dans le Sahel tunisien
Hajer Moussa1,2
Marthe Tsochandaridis2
Saloua Jemni-Yacoub1
Slama Hmida3
Hédi Khairi4
Jean Gabert2
Annie Levy-Mozziconacci2
1 Unité de recherche UR06SP05,
Centre régional de transfusion
sanguine, Sousse, Tunisie
2 Laboratoire de biochimie et biologie
moléculaire, Unité fonctionnelle
de biologie materno-fœtale,
AP-HM-Hôpital Nord, Aix-Marseille
Université, Marseille, France
<[email protected]>
3 Centre national de transfusion
sanguine, immunohématologie,
Tunis, Tunisie
doi:10.1684/abc.2012.0769
4 Service de gynécologie obstétrique,
Hôpital Farhat Hached, Sousse, Tunisie
Received March 9, 2012,
accepted May 7, 2012
Abstract. Objective: The aim of this study was to evaluate the diagnostic value
of RhD fetal genotyping from the plasma of RhD-negative pregnant women.
Methods: We analysed the plasma samples of 65 pregnant women. DNA quantification was done using real time quantitative PCR (RQ-PCR) in multiplex
targeting multiple RhD exons 5, 7 and 10, with a standardized pool of plasmid calibrators. Results were compared with serological analysis of cord blood
after delivery. Results: Fetal RhD status was predicted with 95.38% accuracy
from maternal plasma of pregnant women in the 11th to 40th weeks of gestation. One false positive but no false negative results were found. Thus the
sensitivity of the assay was 100% and the specificity was 94.44 %. Conclusion: The present data demonstrates that the fetal RhD genotyping approach
could be achieved efficiently with RQ-PCR for RhD-negative tunisian pregnant
women.
Key words: maternal plasma, fetal DNA, cell-free DNA, RhD gene, real-time
quantitative PCR, RhD genotyping
Résumé. Objectif : évaluer la valeur diagnostique de l’approche du génotypage rhésus fœtal à partir du plasma maternel dans une population de femmes
enceintes RhD-négatif. Patientes et méthodes : les prélèvements plasmatiques
des 65 femmes enceintes ont été analysés. L’ADN a été quantifié par PCR quantitative en temps réel (RQ-PCR) par multiplexage ciblant les exons 5, 7 et 10
du gène RhD, et à l’aide d’une gamme de calibration plasmidique standard. Les
résultats ont été comparés avec ceux de l’analyse sérologique du sang du cordon suite à l’accouchement. Résultats : le statut RhD fœtal a été prédit avec une
précision de 95,38 % à partir du plasma maternel des femmes enceintes entre la
11e et la 40e semaine d’aménorrhée. Un faux positif, mais aucun faux négatif
n’a été identifié. La sensibilité et la spécificité du test sont respectivement de
100 % et 94,44 %. Conclusion : ces résultats démontrent que l’approche de
génotypage rhesus fœtal pourrait être efficace par PCR quantitative en temps
réel chez les femmes enceintes tunisiennes de phénotype RhD négatif.
Mots clés : plasma maternel, ADN fœtal, ADN des cellules libres, gène RhD,
PCR quantitative en temps réel, génotypage RhD
Reprints: A. Levy-Mozziconacci
To cite this article: Moussa H, Tsochandaridis M, Jemni-Yacoub S, Hmida S, Khairi H, Gabert J, Levy-Mozziconacci A. Fetal RhD genotyping by real time quantitative
PCR in maternal plasma of RhD-negative pregnant women from the Sahel of Tunisia. Ann Biol Clin 2012; 70(6): 683-8 doi:10.1684/abc.2012.0769
683
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Original article
Rhesus (Rh) blood group incompatibility between the pregnant women and her fetus is a significant problem due to
the possibility of maternal allo-immunisation and consequent haemolytic disease of the newborn (HDN) [1]. In
Tunisia, HDN due to RhD immunisation is currently prevented in the vast majority of cases by administration of
anti-D immunoglobulin to D-negative women within 72 h
of each delivery of a RhD-positive newborn as well as
in abortion cases. A dose of 300 ␮g is commonly used
and this can be increased in case of feto-maternel haemorrhage.
A systematic antenatal prophylaxis that could allow the
strengthening of prevention for all pregnant women is not
recommended in Tunisia since such proceedings would
expose to a potential shortage in anti-D immunoglobulin and a significant potential cost. Therefore antenatal
prophylaxis is applied only in cases with obstetrical problems (amniocentesis and every cause of feto-maternel
haemorrhage). Despite the previous precautions of Rh
immunoglobulin prophylaxis in RhD-negative pregnant
women, residual cases of Rh immunisations still occurs
[2], unfortunately, the frequency of fetal immunization in
Tunisia has not been determined, studies performed in
France suggest that it would represent 0.9/1000 births [3-5].
The recent discovery of cell free fetal DNA in maternal peripheral blood (serum or plasma) in relatively high
amounts compared with the cellular fraction [6]: 3.4 and
6.2% in early and late pregnancy respectively [5, 7] opened
new possibilities for non-invasive prenatal diagnosis [8].
Using real time PCR technology, it is possible to successfully determine the RhD status of the fetus based
on the assumption that the RhD gene is absent in RhDnegative pregnant women. Although, RhD gene deletion is
the most common cause of the RhD-negative phenotype
in the Caucasian population, RhD-negative phenotype in
the black African population is commonly caused by the
presence of a RHD␺ with the frequency of 67% [9] and
the d(C)ces haplotype with the frequency of 15% [10]. In
Tunisia, a recent study revealed that the RhD-negative phenotype was highly associated with a deletion of the RhD
gene in 99.32% of cases, nevertheless, there was a low
African contribution associated to the presence of RHDψ
(0.23%) and d(C)ces (0.46%) which is considerably low
compared to African black groups [11] As these phenotypes
could generate “false-positive” results in RhD genotyping assays, a genotyping strategy with cell free fetal DNA
should be more specific (amplification of the desired DNA
sequence only) and sensitive (able to detect a very low DNA
copy number) than a strategy developed for use on genomic
DNA.
Therefore, in the current study, we evaluate the diagnosis
value of RhD fetal genotyping using a novel approach that
consist to quantify fetal DNA level in maternal peripheral
684
blood using the real time quantitative PCR targeting multiple exons of RhD gene with a standardized pool of plasmid
calibrators.
Material and methods
Maternal blood samples
10 mL of EDTA anticoagulated blood samples were collected from 65 pregnant women recruited with informed
consent at the Department of obstetrics and gynecology,
Farhat Hached hospital and at the Center of maternal and
child prevention of Sousse. The study was approved by the
committee of ethics and research for the university hospital
Farhat Hached of Sousse. Samples were obtained at a gestational stage ranging from 11 to 40 weeks. The RhD status
of the fetuses was confirmed at the time of birth by standard
serology protocols on cord blood cells. Blood samples were
shipped to the laboratory within 2 hours to 3 days at room
temperature and directly centrifuged two times at 3,000 g
for 10 min. Supernatants were aliquoted and stored at -20◦ C
until further processing.
Plasma DNA extraction
DNA was extracted from 200 ␮L of plasma sample with the
biorobot easyMAG (Biomérieux) using the specific protocol according to the manufacturer’s instructions. For a
control of the fetal DNA isolation, each run included a
plasma pool of RhD-negative women pregnant with a RhDpositive fetus and a plasma pool of D-negative women
pregnant with a RhD-fetus. The isolated DNA was eluted
in 70 ␮L.
RQ-PCR analysis
The DNA was quantified by real time quantitative (RQ)PCR in multiplex targeting RhD exon 5, RhD exon 7, RhD
exon 10 and ß-globin gene.
RQ-PCR analysis was performed using Abi-Prism 7900
Sequence Detection (Applied Biosystems, France). The
RhD exon 10 TaqMan system consisted of forward primer,
5’- CCT CTC ACT GTT GCC TGC ATT-3’; reverse primer
5’- AGT GCC TGC GCG AAC ATT-3’ and a probe 5’
(VIC) TAC GTG AGA AAC GCT CAT GAC AGC AAA
GTC T (TAMRA)-3’ [12]. The RhD exon 7 TaqMan system consisted of forward primer 5’- GGG TGT TGT AAC
CGA GTG CTG-3’; reverse primer, 5’- CCG GCT CCG
ACG GTA TC-3’ and a probe 5’-(FAM) CCC ACA GCT
CCA TCA TGG GCT ACA A (TAMRA)-3’ [12]. The RhD
exon 5 TaqMan system consisted of forward primer 5’CGC CCT CTT CTT GTG GAT G-3’; reverse primer, 5’GAA CAC GGC ATT CTT CCT TTC-3’ and a probe 5’(VIC) TCT GGC CAA GTT TCA ACT CTG CTC TGC
Ann Biol Clin, vol. 70, n◦ 6, novembre-décembre 2012
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Fetal RhD genotyping in maternal plasma of RhD- women
T (TAMRA)-3’ [13]. The ␤-globin TaqMan system consisting of two primers: (forward) 5’-GTG CAC CTG ACT
CCT GAG GAG A-3’; (reverse) 5’- CCT TGA TAC CAA
CCT GCC CAG-3’; and a dual-labelled fluorescent TaqMan probe, 5’-(FAM) AAG GTG AAC GTG GAT GAA
GTT GGT GG (TAMRA)-3’; served as a control to confirm the presence and quality of DNA in each sample [14],
amplicons for the ␤-globin control gene were detected in
all analysed samples.
RQ-PCR was performed in multiplex amplifying RhD exon
10 with ␤-globin gene and RhD exon 5 with RhD exon
7 separately in two tubes. Taq Man amplifications reactions were set up in a reaction volume of 25 ␮L using the
Taq Man Universal PCR master mix (Applied Biosystems).
The RhD and ␤-globin probes (Fam-labeled probe ␤-globin,
VIC-labeled probe RhD exon 10, Fam-labeled probe RhD
exon 7 and VIC labeled probe RhD exon 5 were used at
a concentration of 100nM; the PCR primers were used at
a concentration of 300 nM. All reactions were run with a
template volume of 6 ␮L. Amplifications were carried out
in duplicate in 96 wells reaction plates. The TaqMan PCR
conditions consisted in a first step at 50◦ C for 2 min, followed by denaturation at 95◦ C for 10 min and then 50 cycles
at 95◦ C for 15 s and 60◦ C for 1 min.
␤-globin plasmid calibrator purchased from Ipsogen (Marseille, France) was used to quantify the total amount of
DNA (maternal DNA and fetal DNA) for RhD exons 5,7 and
10 quantification plasmid calibrators were developped and
produced in the laboratory of molecular biology oh Hospital Nord (Marseille, France). The standard curves consisted
on serial dilution of mixed plasmids (␤-globin + RhD exon
10) and (RhD exon 7 +RhD exon 5) with a range of 5 to
50,000 copies/ 6 ␮L. PCR efficiency was evaluated from
these standard curves for each target.
In the RQ-PCR approach, the liberation of a fluorescent
reporter, tagged onto a sequence specific probe, is coupled
to the amplification reaction. The sequence detector monitors the increase in fluorescent signal of each reaction well
and determines the number of amplification cycles required
to reach a fixed threshold signal intensity, termed the threshold cycle (Ct). The threshold chosen for the Ct calculation
was 0.03. Background signal was under the threshold. The
standard curve of plasmid calibrators generated a mean
slope of –3.3±0.3 for both RhD and ␤-globin plasmids.
Amplification results were reported by cycle threshold (Ct ),
that is the calculated cycle number at which the PCR products crossed a detection threshold and then results were
expressed in terms of copies per milliletter (copies/mL) [8].
Interpretation criteria
The amount of maternal DNA was considered suitable for
analysis when ␤-globin copies were comprised between
Ann Biol Clin, vol. 70, n◦ 6, novembre-décembre 2012
1,700/mL and 100,000/mL. A fetus was characterized as
RhD-positive if at least one of two replicates was positive
for all three RhD exons. When there were discrepancies
between the three RhD exons, RQ-PCR assays targeting the
exons with negative results were repeated on two additional
replicates prepared from a new extract of the last preserved
maternal plasma sample. If the discrepancy persisted, the
result was considered inconclusive and the sample was
excluded assuming that problems with sample storage or
extraction of fetal DNA would compromise accuracy and
reproducibility of the analysis test.
RhD phenotyping on cord red blood cells
The RhD phenotype was determined on blood samples collected at birth for Rh immunoprophylaxis. RhD serotyping
was performed with commercial anti-D reagents (Diagast, France) testing the following specificities (P3X61;
P3X21223B10; P3X290; P3X35) using a direct agglutination test. Indirect antiglobulin test was performed
systematically for RhD-negative results.
Contributions
This study was done between two distinct groups; sample
collection, plasma preparation and RhD phenotyping was
performed in blood transfusion center of Sousse in Tunisia.
Fetal DNA extraction and RhD fetal genotyping were performed in the laboratory of biochemistry and molecular
biology in the hospital Nord of Marseille in France where
the method has been developed.
Results
Accuracy of fetal RhD genotyping
from maternal plasma
The RhD status of the fetus in 65 pregnancies was analyzed
by RQ-PCR using fetal DNA derived from maternal plasma.
Fetal RhD status was predicted in 63 pregnant women, 2
plasma samples were undetermined because of an excess
of maternal DNA indicated by a high concentration of
␤-globin copies (> 100,000 copies/mL).
Non-invasive prenatal fetal RhD exon 10, exon 5 and exon
7 genotyping analysis of maternal plasma samples was in
complete concordance with the analysis of cord blood in 62
RhD-negative pregnant women delivering 45 RhD-positive
and 17 RhD-negative newborns.
One maternal plasma derived DNA, was predicted to be
RhD-postive but at birth the baby was serologically RhDnegative, this discordance being most likely explained by
the presence of an RhD polymorphism (weak D or partial
D). Unfortunately, no cord blood was available to perform
685
Original article
an RhD genotyping and to validate this hypothesis. However, the positive and the negative predictive value of the
fetal RhD status were 97.83% and 100% respectively.
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The quantity of total DNA and fetal DNA
in maternal plasma
The quantity of maternal DNA and fetal DNA in maternal
plasma were investigated in 63 pregnancies. The amplification efficiencies of the assays specific for RhD exons 5,
7 and 10 were comparable, as indicated by similar slope
values.
Plasma samples from pregnant women carrying a RhDpositive fetus gave Ct values in the range 33-40 according
to the gestationnel age of pregnancy, whereas no Ct values
were observed when the fetus was RhD-negative.
When positive signals were obtained, the Ct values were
used to predict the quantity of fetal and total plasma DNA
from a standard curve calculated from known quantities of
imput DNA. Our data, therefore, report the mean number of
copies of fetal DNA detected in maternal plasma, 17 maternal plasma samples were uninformative because no fetal
DNA (RhD gene) was detected. The fetus was subsquently,
confirmed to be RhD-negative in these pregnancies. The
quantity of total DNA (␤-globin) in maternal plasma was
found to vary considerably (2667.21 copies/mL to 95405.16
copies/mL; mean: 13652.26 copies/mL). The mean number of copies of fetal DNA present in maternal plasma was
found to increase with each trimester (table 1).
Representative amplification data of the RQ-PCR of 2
plasma samples: one predicted to be RhD-positive and
another one predicted to be RhD-negative were shown in
figure 1.
Discussion
Non-invasive prenatal diagnosis is one of the many major
goals in human genetics. The discovery of cell free fetal
DNA in maternal plasma in 1997 [6] opened new possibilities for non-invasive prenatal diagnosis. Circulating fetal
DNA has been shown to increase in concentrations with
gestational age and to be cleared rapidly following delivery
[6, 11]. With the use of RQ-PCR methodology, circulating
fetal DNA has been detected robustly in the plasma of pregnant women, even early in the first trimester of pregnancy
[14].
In the present study, we performed a fetal RhD genotyping
using maternal plasma from 65 pregnant women in weeks
11-40 of pregnancy. Postpartum serological RhD typing of
the babies showed an overall discrepancy between genomic
and serological typing of 1.53% related to one false positive
result encountered in this study. In this case, RhD exon 5, 7
and 10 were detected in maternal plasma with ratio (number of fetal DNA copies/number of maternal DNA copies)
below 6% which represent obviously fetal DNA. This pattern suggests the presence of paternally inherited weak or
partial D allele taking account the high polymorphism of
the Rh system. Such RhD variant was previously found in
Tunisian blood donors in an independent study [11]. Contamination is not unlikely as the negative control was always
negative. Unfortunately, this suspected result could not be
ascertained since no infant blood was available, but this
false positive is surely lesser worry than false negatives
since the consequence of a false positive RhD determination
for the fetus, would result only in unnecessary prophylaxis.
Two pregnant women could not be evaluated for prenatal
RhD typing, in the current assay, because of an excess of
maternal DNA detected in the plasma which compromising the sensitivity of the test to detect fetal DNA. Excess
maternal DNA could arise from cellular lysis before the
plasma is removed. This was probably due to variation in
time spent in transit (2 hours to 3 days). Furthermore, it
has been reported that significant random variation can be
found in the amount of total DNA, thus samples of maternal
blood processed a long transit time (more than 2 days) after
maternal venipuncture would yield a high amount of free
maternal DNA [15].
Therefore, despite the previous cases, a reliable fetal RhD
genotype determination was highly accurate in the present
study. The positive and the negative predictive value of
the fetal RhD status were 97.83% and 100% respectively,
results were in complete concordance with that obtained
by serologic study of the newborn for 62 pregnant women
(95.38%). Accordingly, 45 samples were correctly genotyped as RhD positive and 17 as RhD negative, in these last
Table 1. The estimated quantity of fetal DNA in maternal plasma.
Gestation
(weeks)
≤14
15-28
29-40
Fetal DNA
(copies number/mL maternal plasma)*
Mean
Range
143
80.88-225.43
371
152.49-755
1363
180.27-3494.66
Number
6
17
22
*The number of copies of fetal DNA in maternal plasma was estimated from the mean number of copies of RhD exon 5, 7 and 10.
686
Ann Biol Clin, vol. 70, n◦ 6, novembre-décembre 2012
Fetal RhD genotyping in maternal plasma of RhD- women
A
B
Amplification Plot
Amplification Plot
Amplification Plot
1.000 E+1
β-Globin
1.000
Amplification Plot
1.000 E+1
β-Globin
1.000
RHD exon 5
RHD exon 10
1.000 E-1
1.000 E-1
∆Rn
∆Rn
RHD exon 7
1.000 E-2
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1.000 E-2
1.000 E-3
1.000 E-3
1.000 E-4
1.000 E-4
1.000 E-5
1.000 E-5
0
5
10
15
20
25
30
35
40
45
50
0
5
10
15
20
Plot: ∆Rn vs. Cycle
Threshold:
0.030
25
30
35
40
45
50
Cycle
Cycle
Detector: All
Detector: All
Plot: ∆Rn vs. Cycle
Threshold:
0.030
Figure 1. Representative real-time PCR amplification plots showing the presence of fetal and maternal DNA in maternal plasma of RhDnegative women pregnant with a RhD-positive fetus (A) and only the presence of maternal DNA in maternal plasma of RhD-negative
women pregnant with a RhD-negative fetus (B). Real-time multiplex PCR was performed in duplicate.
cases, no prenatal anti-D prophylaxis is needed and injection of anti-D immunoglobulin can be avoided. However,
for the other cases, the pregnant women are subsquently
monitored.
Most importantly, no false negative result was observed in
this study. Thus the sensitivity of the assay was 100% and
the specificity was 94.44%.
These high percentage is most likely the result of the use
of an efficient protocol for the RhD genotyping: the quantification of fetal DNA was done using a RQ-PCR that
incorporate: 1) a pool of plasmid calibrators estimating
the PCR efficiency; 2) a ␤-globin gene serving as a control gene that confirm the presence and quality of DNA
in each sample and 3) a positive and negative controls.
Thus, the quantitative nature of the test will assures that
a maternal signal is not mistaken for a fetal signal. Moreover, this analysis test is efficient since a multiple RhD exons
were targeted by RQ-PCR which allows to overcome false
positive results and, thus, improve accuracy of fetal RhD
genotyping.
The amount of free fetal DNA is obviously a limiting factor for fetal RhD genotyping in maternal blood. It is well
documented that the amount of fetal DNA increases during
pregnancy [8], as was observed in our study. However, there
is a considerable variation in the amount of circulating fetal
DNA from pregnancy to pregnancy with the progress of
gestational age as indicated in table 1. Our current findings
would suggest that accurate RhD genotyping is possible in
the first trimester, since the earliest pregnancy in which the
presence of fetal DNA was confirmed in our study was 11
weeks. It would be advisable to repeat the test at later stages
of gestation to insure fetal genotyping results.
Traditional management for RhD-negative women at risk
for RhD alloimmunization involves determining the RhD
status of the fetus through chorionic villus sampling from
Ann Biol Clin, vol. 70, n◦ 6, novembre-décembre 2012
amniotic fluid, and administrating the prophylactic human
derived RhD immunoglobulin during pregnancy and after
delivery. Both practices carry their own risks and associated
costs [16].
Therefore, the feasibility of mass testing for the fetal RhD
genotype with maternal plasma is highly desirable for ethical and economical reasons. Indeed, it avoids unnecessary
administration of anti-D immunoglobulin, to approximately
40 percent of RhD-negative women bearing RhD-negative
fetus [17].
Non-invasive fetal RhD genotyping by targeting multiple
exons with real time PCR is highly sensitive and accurate.
It’s the best method for assessing RhD fetal status in RhDnegative mothers and represents the first step in identifying
fetus at risk for alloimmunization. In conclusion, it will be
of interest to plan the practice of this analysis in the routine testing of all RhD-negative pregnant women, this will
present surely a significant achievement in the application
of research to clinical practice.
Conflicts of interest: none.
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