DETERMINATION OF FETAL RHESUS D STATUS
BY MATERNAL PLASMA DNA ANALYSIS
Aykut A1,*, Onay H1, Sagol S2, Gunduz C3, Ozkinay F1, Cogulu O1
*Corresponding Author: Ayça Aykut, M.D., Ph.D., Department of Medical Genetics, Ege University Faculty
of Medicine, 35100, Bornova, Izmir, Turkey; Tel.: +90-232-390-3961; Fax: +90-232-390-3971; E-mail:
aycaaykut@hotmail.com
page: 33
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DISCUSSION
It has become a common practice to offer routine
antenatal anti-D prophylaxis, usually at 28-34 weeks
of gestation, or within 72 hours after delivery to prevent
anti-D immunization in RhD-negative pregnant
women. Anti-D prophylaxis is not indicated in about
40.0% of all RhD-negative cases because the fetus
is also RhD-negative. We have shown that real-time
PCR analysis of cffDNA in maternal plasma is a
feasible and reliable technology after analyzing 29
maternal plasma samples which resulted in sensitivity
and specificity rates of 100.0%. These high sensitivity
and specificity rates were also achieved in the
second and third trimesters.
Due to the high complexity of the Rh system
and the possibility of false results, more than one region
of the RHD gene is suggested to be examined
for RhD typing [11-13]. Therefore, two RHD-specific
exons (7 and 10) were amplified, which is importantly
below the mean size of circulating cffDNA in
maternal serum (range 145-201 bp) [14]. The sizes
of the two amplicons are 89 and 73 bp for exons 7
and 10, respectively. In this study, two regions of the
RHD gene was examined for RhD typing and elimination
of false-negative results.
There are several distinctive results concerning
the quantification cycle (Cp) values of RHD-positive
fetuses. It is accepted that RHD-positive fetus gives
Cp values in the range of 35-40, and no Cp values are
observed when the fetus is RhD-negative. Rouillac-
Le Sciellour et al. [15] highlighted the presence of
a silent variant RHD gene such as the RHDY (pseudogene)
in the maternal genome when Cp values are
in the range of 26-30 cycles. They also pointed out
different levels of expression of exon 7 and exon 10
of the RHD gene depending on the gestational age. If
amplification of exon 7 is [+] but exon 10 is [−], it was
suggested that exon 7 PCR was more sensitive than
exon 10 PCR in which the result was considered as
RHD-positive. This is usually indicative for a sample
collected during early pregnancy (less than 10 weeks
of gestation) when the level of fetal DNA is low in
the mother’s plasma. In a completely opposite condition,
if exon 7 is [–] but exon 10 [+] and Cp >35, they
emphasized the presence of a Rh variant that could be
either RhD-negative or weak or partial D type. The
amplification of only exon 7 was suggested to be useful
for the determination of the fetal RHD genotype
[15]. The discrepancies between the results of exons
7 and 10 might be solved by the third RHD-specific
PCR. In our study, Ct values of exons 7 and 10 in
RhD [–] cases did not show amplification, however,
Ct values of both exons were >35 in RhD [+] cases.
Insufficient amplification of exon 10 was detected in
only two cases by the confirmation of PCR analysis;
therefore, amplification solely of exon 7 PCR has
been accepted to be indicative of RhD status.
Towards the end of the third trimester, there is a
rapid increase in the amount of circulating cffDNA
in maternal plasma [16,17]. In this study, exon 7,
exon 10 and SRY DNA copy numbers were significantly
increased in parallel with the increasing gestational
age as pointed out in the literature.
The existence of the SRY and RHD genes definitely
indicate the presence of fetal genetic material
without considering maternal genome in Rh [–]
pregnant women. In SRY [+] and RhD [+] cases, the
copy number of the SRY gene was significantly correlated
with the copy number of exons 7 and 10. In
addition, the correlation between the exons 7 and 10
in RhD [+] female fetuses showed that the method
used in this study is highly reliable to determine fetal
genetic material.
The sensitivity of genotyping fetal DNA from
maternal plasma varies in the literature. Bischoff et
al. [2] observed a sensitivity of 70.0% in 20 sensitized
RhD [–] pregnant women. Fetal RHD genotyping
in another study correctly predicted fetal Rh
status in 92 of 98 (94.0%) cases [3]. By combining
amplification of three exons, the concordance rate of
fetal RHD genotypes in maternal plasma and newborn
with RHD phenotypes at delivery was 100.0%
(99.8% including one unusual false-positive) in the
Belgian group [4]. Since then, numerous groups
have reported similar results for fetal RHD genotyping
in RhD-negative mothers [5-8] as in the presented
study, which indicated a sensitivity and specificity
of 100.0%.
Our results have shown that non invasive fetal
RHD genotyping can be performed rapidly and reliably
using cffDNA in maternal plasma with TaqMan
real-time PCR assay with a sensitivity and specifity
of 100.0%. The weakness of our study is the low
number of tested samples, which could be the reason
for the 100% sensitivity and specificity. A further
prospective study with a larger number of samples
will be performed in the future to confirm the reliability
of this protocol.
Declaration of Interest. The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of this article.
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