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1995 85: 829-832
Rh E/e genotyping by allele-specific primer amplification
BH Faas, S Simsek, PM Bleeker, MA Overbeeke, HT Cuijpers, AE von dem Borne and CE van
der Schoot
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Rh E/e Genotyping by Allele-Specific Primer Amplification
By B.H.W. Faas,
S.Simsek, P.M.M. Bleeker, M.A.M. Overbeeke, H.Th.M. Cuijpers, A.E.G. Kr. von dem Borne,
and C.E. van der Schoot
It has beenshown that theRhesus (Rh)blood group antigens
are encodedby two homologous genes: the Rh D gene and
the Rh CcEe gene. The Rh CcEe gene encodesdifferent peptides: the Rh C,c,E, and e polypeptides. Only onenucleotide
difference has beenfound between the alleles encodingthe
Rh E and the Rh e antigen polypeptides. It is a C G transition at nucleotide position 676, which leads to an amino
acid substitution from proline to alanine in the Rh e-carrying
polypeptide. Here we present an allele-specific
primer amplification (ASPA) method t o determine the Rh E and Rh e
genotypes. In onepolymerasechainreaction,
the sense
primer had a 3'-end nucleotide specific for the cytosine at
position 676 of the Rh E allele. i n another reaction, a sense
primer was used with a 3'-end nucleotide specific for the
guanine a t position 676 of the Rh e allele and the Rh D
gene, whereas the antisense primer had a 3'-end
nucleotide
specific for theadenine at position787 of the Rh CcEe gene.
We tested DNA samples from 158 normal donors (including
non-Caucasian donors and
donors with rare Rh phenotypes)
in these assays. Therewas full concordance with the results
of serologic Rh E/e phenotyping. Thus, we may conclude
that the ASPA approachleads t o a simple and reliable
method t o determine the Rh E/e genotype. Thiscan be useful in Rh €le genotyping of fetuses and/or in cases in which
no red blood cells are available for serotyping. Moreover,
our results confirm the proposed association between the
cytosine/guanine polymorphism at position 676 and the Rh
Ele phenotype.
0 1995 by The American Society of Hematology.
T
Here we describe an Rh E/e genotyping method using a
polymerase chain reaction (PCR) with two primer sets, with
the 3'-end nucleotides of one or both primers specific to
amplify the Rh E or Rh e allele [allele-specificprimer amplification (ASPA)]. We performed these two PCRs to determine the Rh E and Rh e genotypes in DNA samples from
158 white and non-white volunteer blood donors that
had been serologically phenotyped for Rh D, Rh We, and
Rh c/c.
-+
HE RHESUS (Rh) blood group system is of clinical
interest, because it is involved in hemolytic disease of
the newborn (HDN), in hemolytic transfusion reactions, and
in autoimmune hemolytic anemia (AIHA). The Rh system
is complex; as many as 46 different antigens have been
serologically defined.'.' Among these antigens are those of
the Rh D, C/c, and We series. The two highly homologous
genes encoding these antigens are localized on chromosome
lp34.3-p36.1 and are inherited t ~ g e t h e rOne
. ~ gene encodes
the Rh D antigen. The Rh D-negative phenotype is caused
by the absence of the entire or at least part of the Rh D
gene:5 rather than by an allele of the gene. The other gene,
the Rh CcEe gene, encodes the polypeptides carrying the Rh
C/c as well as those carrying the RhE/e polymorphisms.
Recently, the cDNA structure of the Rh gene has been elucidated.6.7The Rh CcEe gene encodes different polypeptides,
and there are several alleles. Alternative splicing probably
plays a role in the production of these polypeptides.' In the
limited number of donors tested so far, only one nucleotide
difference has been found between the alleles encoding the
polypeptide carrying the Rh E and the Rh e antigen.'.'' This
difference involves the nucleotide at position 676 of the
coding sequence6,' and leads to a proline to alanine substitution in the allele encoding the Rh e antigen.
Until recently, it was only possible to determine the Rh
phenotype by serologic typing of red blood cells. This serologic approach can be inconclusive, eg, in the case of Rh
phenotyping of fetuses and of patients who have recently
been transfused and who harbor a large quantity of donor
red blood cells. In these cases, Rh genotyping is an option.
Methods to determine the Rh D genotype on genomic DNA
have been d e ~ c r i b e d . ~ ~As
" "HDN,
~
AIHA, and transfusion
reactions are not only due to anti-Rh D antibodies but also
sometimes to anti-Rh We or anti-Rh C/c antibodies, it is
important also to be able to determine the Rh We and Rh
C/c genotype in such cases. Recently, Hyland et a l l 4 applied
restriction fragment length polymorphism (RFLP) patterns
on Southern blots for Rh genotyping. However, they found
a 1 0 0 % correlation for 102 randomly selected blood donors
for the Rh C, Rh e, and Rh D phenotypes, but only 94.8%
for the Rh c and 94.3% for the Rh E phenotypes.
Blood, Vol 85, No 3 (February 1). 1995: pp 829-832
MATERIALS AND METHODS
Red blood cells from 158 mainly white donors were serologically
typed for the Rh C/c,=e,and
D phenotypes(Table 1). Murine
monoclonal antibodies (Pelikloon anti"
D, anti-Rh E, anti-Rh e,
anti-Rh C, and anti-Rh c; all IgM, CLB, Amsterdam, The Netherlands) as well as polyclonal human antibodies (anti-Rh D, anti-Rh
E, anti-Rh e, anti-Rh C, and anti-Rh c for the bromelin technique;
CLB) were used. High-molecular weight DNA was extracted from
the leukocytes of these donors by standard methods described by
Ciulla et d.'5
To performanRh E-specificASPA,thefollowingtwosets
of
primers were used: set A, primer R661c
(sense primer: S'CCAAGTGTCAACTCTC3', position 661 to 676 of the coding
andprimer R768 (antisenseprimer:S'TGACCCTGAGATGGCTGT3'. position 768 to 751); and set B: primer R487(sense primer:
S'ACAGACTACCACATGAAC3', position487 to 504) and primer
R568 (antisense primer: S'GCT'rTGGCAGGCACCAGGCCAC3',
From the Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service and the Luboratory for Experimental and Clinical Immunology, University of Amsterdam; and the Department of
Haematology, Academic Medical Center, Amsterdam, The Netherlands.
Submitted May 23, 1994; accepted October 3, 1994.
Address reprint requests to C.E. van der Schoot, MD, PhD, Central Laboratory of the Netherlands Red Cross Blood Transfusion
Service, 1066 CX Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this facr.
0 1995 by The American Society of Hematology.
0006-4971/95/8503-0$3.00/0
829
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FAAS ET AL
830
Table l.Serologically Predicted Phenotypes and E-Specific and eSpecific ASPA-Determined Genotypes of DNA Samples
Predicted
Serologic Type'
CDelCDe [81
CDe/ce [81
CDe/cDE [91
cDE1ce 161
cDe/ce [ 1I
cDE/cDE
ce/ce [l41
Celce
cE/ce
CDeIcDE (Rhi')
CDEICDE
CEICE
CDEICDe
CDE/cDE
ENo. of Donors
Specific
Tested
ASPA ASPA
23
(5nonwhite)
33
(3nonwhite)
17
11
11
eSpecific
Genotype
+
+
+
+
+
(5 nonwhite)
8
32
10
(1 nonwhite)
6
(1 nonwhite)
~
+
+
+
2
+
1
1
-
between 46°C and 62"C, bands were always obtained. However, at lower temperatures more aspecific bands were seen.
A higher concentration of MgCIZ resulted in stronger bands.
The optimal conditionsfor theE-specific ASPAwere an
annealing temperature of 62°C and 2.5 mmol/L MgCI2. The
combination of primer setsB and C only gave goodproducts
at annealing temperatures of 46°C and 49°C. At higher temperatures, the e-specific band was lost, whereas the control
band was still visible. Changing the MgClz concentrations
2.0 mmol/L
resulted in theloss of the e-specificbandat
MgC1,. Thus, the e-specific ASPA is optimal at 49°C annealing temperature, using 1.5 mmol/L MgCI2.
Set A amplifies a 108-base pair region specific for the Rh
E allele of the Rh CcEe gene (exon S ) because of the use
of the 3'-end nucleotide of primer R661c, which is specific
for nucleotide 676 of the Rh E allele. Set B amplifies a 94-
~
+
2
1
~
Numbers in brackets indicate donors for whom the predicted phenotypes are not confirmed by family analysis.
position 568 to 547). To perform an Rh e-specific ASPA, set B
was used in combination with set C: primer R661g (sense primer:
S'CCAAGTGTCAACTCTG3', position 661 to 676) and primer
R801 (antisense primer: 5'CATGCTGATCTTCCT3', position 801
to 787). All primers were synthesized on a DNA synthesizer (Applied Biosystems model 392, Palo Alto, CA), and the primers R661c,
R661g, and R801 were purified with oligonucleotide purification
cartridges (Applied Biosystems, Foster City, CA).
The PCR was performed on 0.7 pg of genomic DNA template in
a total volume of 50 pL. The reaction mixture contained 75 ng of
each primer, 0.2 mrnol/L of each dNTP (Pharmacia, Uppsala, Sweden), 2 U of Taq DNA polymerase in the appropriate buffer (Promega, Madison, WI), 2.5 mmol/L MgC12 for the E-specific ASPA
and 1.5 mmol/L MgClz for the e-specific ASPA. A negative control
sample without DNA was always included. Thirty-five cycles of
amplification were performed in a thermal cycler (Perkin Elmer
Cetus model 480, Norwalk, CT), with denaturation for 1 minute at
95"C, annealing for 1.5 minutes at 62°C for primer sets A and B
and 1.5 minutes at49°C for primer sets B and C, and extension
for 2.5 minutes at 72°C. The products were separated on a 10%
polyacrylamide gel and visualized with ethidium-bromide staining.
\,
~
),
i
RESULTS
Primers R661cfR768 (setA),R487fR568(set
B), and
R661gR801 (set C) were designed to amplify
specific regions of the Rh CcEe and Rh D genes. Some experiments
were performed to arrive at the optimal conditions to use
the primer sets. PCRs were performed with annealing temperatures of 46"C, 49"C, 5 5 T , and 62°C. MgC12 concentrations were varied using the optimal annealing temperature
(1.0 mmol/L, 1.5 rnmolk, 2.0 mmol/L, and 2.5 mmoVL;
results not shown). The combination of primer sets A and
B worked well in a large range of annealing temperatures;
Fig 1. Schematic diagram of the ASPA for Rh E and Rh e genotyp
ins. In each reaction, two primer sets are used: a control set that
amplies a 94-bp region common to both the Rh CcEe gene and the
Rh D gene and a primer set specific for the Rh E or Rh e allele of the
Rh CcEegene. IA) Schematic result of the Rh E-specificASPA, in
which primer set A (R661cIR768) is used to amplify an Rh E-specific
region of 108 bp. (B) Schematic result of the Rh e-specific ASPA, in
which primer set C (R66lglR801) is used to amplify an Rh e-specific
region of 141 bp (x = E or e).
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Rh €/e GENOTYPING
83 1
n
1
141 b p
108 b p
94 bp
-
2
3
4
5
6
M
-
-
2 0 0 bp
100 b p
Fig 2. Rh Ele genotyping by ASPA analysis. Rh E- and Rh e-specific PCRs were performed using Rh E or Rh e allele-specific primers [set A
(108 bp) and C (141 bp), respectively]. Lanes 1 and 2 DNA sample from a homozygousccDEE donor, amplified with primer set C (lane 1) and
primer Set A (lane 2); lanes 3 and 4 DNA sample from a heterozygous CcDEe donor, amplified with primer set C (lane 3) and primer set A
(lane 4); lanes 5 and 6 DNA sample from a homozygousccdee donor, amplified with primer set C (lane 5) and primer set A (lane 6); lane M:
marker DNA. In all lanes, primer set B, which amplifies a product of 94 bp, was used as a positive control.
bpregion in exon 4 common to the Rh D gene and the
Rh CcEe gene. The 108-bp band and the 94-bp band were
coamplified in one PCR. In Rh E-negative DNA samples
(ee). only the control fragment of 94 bp is found, whereas
in Rh E-positive DNA samples (Ee or EE), the control fragment as well as the Rh E-specific 108-bp fragment are amplified (Fig IA).
Set C amplifies a 141-bp region of the Rh CcEe gene
specific to the Rh e allele. Primer R661g has a 3'-end nucleotide (nucleotide 676) that enables the primer to bind to the
Rh D gene as well as to the Rh e allele of the Rh CcEe
gene, but not to the Rh E allele. Primer R801 has a 3'-end
nucleotide (at position 787) that is specific for the Rh CcEe
gene but not the Rh D gene. When primer sets B and C are
used in the ASPA assay, only the 94-bp control fragment is
amplified in Rh e-negative DNA samples, while in Rh epositive DNA samples two bands are produced: the 94-bp
control bandand the 141-bp Rh e-specific fragment (Fig
1 B).
All of the l58 samples were serologically phenotyped for
Rh C/c, We, and D (Table l). Using the Rh E- and Rh especific ASPA, we typed all of the donors for their Rh E or
R h e genotype. The results of the E-specific and e-specific
ASPA and the conclusions with regard to the genotype are
listed in Table 1. There was complete agreement with the
results of the serologic Rh We phenotyping. All of the samples that were serologically typed as EE or Ee produced two
bands when using primer sets A and B in the ASPA reaction,
whereas all of the samples that were serologically typed as
ee onlyproduced the lower band in this reaction (Fig 2).
This pattern was foundnot only in the combination of E
with c, but also of E with C (see the rare CDE/CDE, CdEI
CdE and CDE/CDe phenotypes). All samples that were serologically typed as Ee or ee produced two bands when primer
sets B and C were used in theASPA: the Rh e-specific
band of 141 bp and the control band. All samples that were
serologically typed as EE only produced the control band
(Fig 2).
DISCUSSION
Until recently, it was only possible to determine the Rh
C/c, Rh E/e, and Rh D phenotypes serologically; but because
the messenger RNA sequences of the Rh CcEe and Rh D
genes have become known," several methods have been de-
scribed to determine the Rh D genotype at the genomic DNA
IeveI.""3
In the present study, weusedtwoASPA
reactions to
determine the Rh Ele genotype on genomic DNA. To rule
out false-negative reactions caused by insufficient quality of
the reagents or failure of the reaction itself, we included an
internal control, primer set B, that always gives an amplification product irrespective of the phenotype. We performed
the reactions onDNA samples from 158 volunteer blood
donors with diverse serologically determined Rh CcDEe
phenotypes. The results of our Rh E and Rh e genotyping
were in full concordance with the results of the serologic
phenotyping. By genotyping DNA samples from donors with
rare alleles (CDE and CdE), it was shown that only the cE
combination, which is by far the most common, but also the
CE combination could be amplified.
Recently, Hyland et all4 used Msp I RFLP digestion patterns of the 3' noncoding regions of the genes to determine
Rh Ele genotypes. For e they showed a 1 0 0 % concordance
between the results of serologic phenotyping and genotyping
based on RFLP patterns, but for E the concordance was only
94.3%. The discrepancies they found between the results of
serologic phenotyping and molecular genotyping appeared
to be associated with the cE allele in D-negative subjects.
The six cE alleles in four D-negative donors whose DNA
was tested were all genotyped as ce. No discrepancies were
seen when the cE allele occurred in a haplotype with the D
gene. From these results, the investigators concluded that
the cE allele is different in the presence or absence of the D
gene. However, because they were only analyzing noncoding
regions, these results only show linkage between the Msp I
RFLP and the Rh Ele phenotype. Moreover, no DNA from
either D-positive or D-negative subjects with the CE allele
was typed. In theASPA method, we tested DNAof six
donors containing the cE haplotype and never encountered
a discrepancy. Furthermore, we also tested some DNA samples from rare phenotypes. These results were also in full
concordance. For RFLP pattern analysis, a large quantity of
DNA is needed, whereas for this PCR-based method, a small
amount of genomic DNA is sufficient. This small amountpurifiedfrom less than 0.5 mLof wholeblood or from
amniotic cells-makes genotyping samples from fetuses and
from patients with severe transfusion problems feasible.
Although the results of our Rh Ele genotyping were in
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FAAS ET AL
832
full concordance with the results of the Rh Ele phenotyping,
one must be aware of the fact that discrepancies might occur
in rare cases, such as cases in which there is transmission
of silent alleles at the Rh locus. In these cases (eg, the
Rh,,,, and the D- phenotypes), the Rh CcEe gene is present
without anygenomic rearrangements or mutations within the
coding region, but no Clc or Ele antigens can be detected
on the erythrocytes.I6
Our results obtained from this large group of donors confirm the proposed association between the cytosine or guanine polymorphism at position 676 of the Rh CcEe gene and
the Rh E or Rh e phenotype, respectively. These findings
strongly suggest that proline and alanine are involved in the
specific epitopes that are recognized by anti-Rh E and antiRh e antibodies, respectively.
In conclusion, we present a simple and reliable PCR-based
method to determine the Rh Ele polymorphism on genomic
DNA. This can be useful to determine the Rh E/e genotype
in fetuses inan early phase of pregnancy and in recently
transfused patients with large amounts of circulating donor
cells.
ACKNOWLEDGMENT
We thank the staff of the Department of Blood Group Serology
for help serotyping the blood samples and Dr C.P. Engelfriet for
comments on the manuscript.
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