BLOOD GROUP GENOMICS

RHD and RHCE variant and zygosity genotyping via multiplex

ligation–dependent probe amplification

Lonneke Haer-Wigman, Barbera Veldhuisen, Remco Jonkers, Martin Lodén, Tracey E. Madgett,
Neil D. Avent, Masja de Haas, and C. Ellen van der Schoot

T
he Rh blood group system is one of the most
BACKGROUND: The presence of a D variant may complex and immunogenic blood group
hamper correct serologic D typing, which may result in systems in humans.1,2 Antibodies against the Rh
D immunization. D variants can be determined via RHD antigens can give severe hemolytic transfusion
genotyping. However, a convenient single assay to reactions and/or hemolytic disease of the fetus and/or
identify D variants is still lacking. We developed and newborn.3,4 Therefore, it is crucial to correctly determine
evaluated a multiplex ligation–dependent probe amplifi- the Rh status in blood donors, blood recipients, and preg-
cation (MLPA) assay to determine clinically relevant nant women to prevent immunization against the Rh
RHD and RHCE variant alleles and RHD zygosity. antigens.5-7
STUDY DESIGN AND METHODS: We analyzed 236 The antigens of the Rh blood group system are
cases (73 normal and 163 selected samples) with the encoded by two closely linked genes, RHD and RHCE, that
RH-MLPA assay, which is able to determine 79 RHD encode the homologous RhD and RhCE proteins, respec-
and 17 RHCE variant alleles and RHD zygosity. To tively.8 The RhD protein carries the D antigens and the
confirm the results, mutations were verified by RHD RhCE protein carries the C, c, E, and e antigens.9-13
and/or RHCE exon–specific sequencing and RHD A wide genetic diversity exists in RHD and RHCE,
zygosity was verified by quantitative real-time poly- resulting in more than 250 RHD and RHCE variant alle-
merase chain reaction (PCR) for 18 cases. les.14 These variant alleles have been classified into four
RESULTS: In 99% of the cases, the RH-MLPA assay groups: the partial RHD alleles, alleles encoding the weak
correctly determined whether a person carried only D, and D-elution (Del) phenotypes, the D– null alleles and
wild-type RHD and RHCE alleles (n = 69) or (a) variant the RHCE variant alleles, based on their phenotypic
RHD allele(s) and/or (a) variant RHCE allele(s) expression (see Web resources). Individuals carrying a
(n = 164). In only three cases, including two new RHD partial RHD allele lack one or more D epitopes and are
variant alleles, the variant allele was not identified, due prone to make alloantibodies against the epitopes they
to lack of detecting probes. These were RHD*DCS2, a miss.2,15 Individuals carrying a weak D or Del allele express
new partial RHD allele, RHD*525T (Phe175Leu), and a
new D– null allele, RHD*443G (Thr148Arg). All RHD
(n = 175) and RHCE variant alleles (n = 79) indicated by ABBREVIATIONS: MLPA = multiplex ligation–dependent probe
the RH-MLPA assay were confirmed by sequencing. amplification; RHD-MPX = RHD multiplex; SNP = single-
RHD zygosity was confirmed by quantitative PCR. Two nucleotide polymorphism.
hematopoietic chimeras were recognized.
From the Sanquin Research and Landsteiner Laboratory,
CONCLUSION: The RH-MLPA genotyping assay is a
Academic Medical Centre, University of Amsterdam, and
fast, easy, and reliable method to determine almost all
MRC-Holland, Amsterdam, the Netherlands; and School of
clinically relevant RHD and RHCE variant alleles, RHD
Biomedical and Biological Sciences, Plymouth University,
zygosity, and RHD+/RHD– chimeras in blood donors,
Plymouth, United Kingdom.
blood recipients, and pregnant women.
Address reprint requests to: Lonneke Haer-Wigman,
Immunohematology Experimental, Sanquin Blood Supply,
Plesmanlaan 125, Amsterdam, 1066 CX, The Netherlands;
e-mail: [email protected]
Received for publication March 16, 2012; revision received
July 17, 2012, and accepted August 20, 2012.
doi: 10.1111/j.1537-2995.2012.03919.x
TRANSFUSION 2013;53:1559-1574.

Volume 53, July 2013 TRANSFUSION 1559

HAER-WIGMAN ET AL.

the RhD protein, but in low amounts.2,15 In theory, indi- sis equipment. The aim of this study was to evaluate the
viduals with a weak D or Del variant express all D epitopes; performance of the RH-MLPA genotyping assay in the
hence they will not make anti-D.14,15 There have been, determination of clinically relevant RHD and RHCE
however, reports describing alloanti-D formation in indi- variant alleles and RHD zygosity.
viduals with weak D variants.16-18 Both weak D and Del can
elicit an immune response in D– recipients.19,20 Individu-
als with a D– null allele lack the complete expression of the MATERIALS AND METHODS
RhD protein.14,15 D negativity is most often caused by the Materials
entire deletion of the RHD gene (the RHD*01N.01 allele) in
DNA samples from 73 random blood donors with normal
the Caucasian population or the presence of the RHD*y
Rh serology were included after informed consent was
allele (RHD*Pseudogene) or the hybrid RHD*DIIIa-CE(3-
given (including 11 D– and 62 D+ samples; 44 C+, 60 c+, 27
7)-D allele, formerly called (C)ceS Type 1 in the black
E+, and 68 e+ samples).
African population.21-23
Sanquin Diagnostics is the national reference labora-
Serology with monoclonal anti-D is currently the tory for the analysis of the presence of Rh variants in blood
standard method to determine the Rh status of blood recipients, blood donors, and pregnant women. For this
donors, recipients, and pregnant women.2,15,24 Serologic study 97 cases were selected, which were analyzed
typing will, in most cases, only reveal the presence of an between 2003 and 2011. This selection included preferen-
RhD variant, but cannot exactly determine which variant tially a minimum of three samples for each known RHD
is present.2,5 Since transfusion policy, as well as preventive variant allele and all samples of which the specific RHD
measurements during pregnancy, are based on the exact variant was not yet determined. Twenty DNA samples
nature of the RhD variant, it is important to correctly were provided by other blood group centers and were
determine which RhD variant is present.2,5,6,14 RHD and selected for the presence of a specific RHD variant allele.
RHCE variant alleles can be determined via genotyping of Forty-six cases positive for the RHD*y allele were obtained
the RHD and RHCE genes.5,25,26 Since molecular genotyp- from the screening program of D– pregnant women.
ing methods are increasingly applied for blood donor
typing and for noninvasive fetal RHD genotyping, the
number of cases with discrepant serologic and genotyping
D serology
results that require further molecular analysis is expand-
Serologic D typing was routinely preformed with two
ing.5,25 A method to identify a broad range of clinically
anti-D reagents. A monoclonal anti-D reagent (immuno-
relevant Rh variants (all frequently occurring variants and
globulin [Ig]M clone MS201, Sanquin Reagents, Amster-
all variants where the carrier is able to make alloanti-D) in
dam, the Netherlands) and a monoclonal blend reagent
a convenient and cheap assay is still lacking.14,25
(IgM clone TH28 and IgG clone MS26, Sanquin Reagents)
Only pregnant women who are D– or express an RhD
were used in a method with an immediate spin at room
variant and who are carrying a D+ fetus are at risk for
temperature. All D– samples were also tested with the
anti-D formation and subsequently of anti-D–mediated
monoclonal blend reagent in the indirect antiglobulin
hemolytic disease of the fetus and/or newborn.3,6 Cur-
test. In case of a discrepancy between the results of the
rently, the presence of a D+ fetus can be determined with
two reagents, the D-epitope pattern was determined with
noninvasive fetal RHD typing.27,28 If the father is homozy-
an in-house panel of anti-D reagents and/or the extended
gous for the RHD*01 allele (wild-type RhD) this test can be
partial RhD typing set from Bio-Rad Laboratories B.V.
omitted.6,14 Upon serologic typing of the C, c, E, and e
(Veenendaal, the Netherlands).
antigens of the father, a prediction of the paternal zygosity
can be made based on the most frequently occurring
RHD/RHCE haplotypes.6,29 Because the haplotype fre-
quencies differ between ethnic populations, these predic- DNA isolation
tions are not reliable without knowledge of the ethnic Ethylenediaminetetraacetic acid–anticoagulated blood
background.6,29 The exact paternal zygosity can be det- was collected and genomic DNA was isolated from
ermined by genotype-based methods; however, the white blood cells (WBCs) using a DNA extraction kit
methods used at the moment are cumbersome and can be (QIAamp DNA blood mini kit, Qiagen Benelux, Venlo, the
confounded by genetic alterations, especially in the black Netherlands).
African population.30-33
We therefore developed a multiplex ligation–
dependent probe amplification (MLPA) genotyping assay. MLPA
This single assay detects mutations and copy number An MLPA assay specific for the analysis of RHD and RHCE,
variation of the RHD and RHCE genes and is easy to use, as their variants and zygosity was developed. This RH-MLPA
it only requires a thermocycler and capillary electrophore- assay contains 17 RHD wild type, five RHCE wild type, 21

1560 TRANSFUSION Volume 53, July 2013

 GENOTYPING OF RHD VARIANTS AND ZYGOSITY

RHD mutation, and two RHCE mutation probe combina- hybridization and 2 hours of hands-on time. Between 1
tions (Table 1; hybridizing sequences are shown in and 16 samples can be analyzed simultaneously in a
Supplementary Table S1, available as supporting informa- single run consisting of all three RH-MLPA mixes.
tion in the online version of this paper). So-called wild-
type probe combinations are used to determine the copy
number of wild-type RHD or RHCE sequence, while muta- RHD multiplex PCR
tion probe combinations detect the presence of a mutated
In our institution all samples that show unexpected weak
sequence. Twenty-five probe combinations were conven-
or negative reactions with D-typing reagents are cur-
tionally designed and consisted of two probe pieces and
rently analyzed using a multiplex PCR, simultaneously
for the additional 20 probe combinations a three-piece
detecting RHD Exons 3, 4, 5, 6, 7, and 9 and an internal
design was chosen (Table 1).
control.34 The RHD multiplex (RHD-MPX) PCR was per-
The RH-MLPA assay is able to distinguish between
formed on a DNA engine thermocycler (Dyad, Bio-Rad
51 RHD and 13 RHCE variant alleles. For another 28 RHD
Laboratories B.V.) in a total volume of 50 mL, containing
and four RHCE variant alleles the assay can identify the
10 to 1000 ng DNA, 5 mL of 10¥ FastStart Taq DNA poly-
main type, but cannot discriminate between several sub-
merase buffer without MgCl2 (Roche, Woerden, the Neth-
types, for example, RHD*DOL1 or RHD*DOL3 (Table 2).
erlands), 3 mL of MgCl2 25 mmol/L stock solution
Since probes hybridizing to the same regions will
(Roche), 1 mL of PCR-grade nucleotide mix (Roche), 15 mL
hamper each others’ binding, the probes of the RH-
of primer mix (primer dilution between 0.1 and
MLPA had to be divided into three pools, p401, p402, and
1.2 mmol/L; Eurogentec, Maastricht, the Netherlands).
p403 (Table 1).
PCR conditions were as follows: 5 minutes at 95°C, 33
The MLPA reaction was performed according to the
cycles of 1 minute at 95°C, 1 minute at 56°C, and 1
manufacturers’ protocol (MRC Holland, Amsterdam, the
minute at 72°C, followed by 5 minutes at 72°C. The pres-
Netherlands) on a thermocycler (Biometra T1, Wester-
ence or absence of the RHD exons and internal control
burg BV, Leusden, the Netherlands; or Veriti, Applied Bio-
was analyzed by gel electrophoresis on a 1.5% (wt/vol)
systems, Nieuwerkerk aan de IJssel, the Netherlands).
agarose gel (Invitrogen, Breda, the Netherlands).
Supplementary Fig. S1 (available as supporting informa-
tion in the online version of this paper) shows a sche-
matic overview of the MLPA reaction. In short, 5 mL
containing 100 ng of DNA was denatured and 1.5 mL PCR and sequence reaction
probe mix and 1.5 mL SALSA MLPA dilution buffer were RHD and RHCE exon–specific sequencing was performed
added at 25°C. After hybridization at 60°C for between 16 using RHD- and RHCE-specific primers flanking each
and 20 hours, 1 mL of SALSA ligase-65, 1.5 mL of SALSA exon. The primers were synthesized by Eurogentec and
ligase Buffer A, and 1.5 mL SALSA ligase Buffer B were sequences are shown in Supplementary Table S2 (avail-
added and incubated for 15 minutes at 54°C. A poly- able as supporting information in the online version of
merase chain reaction (PCR) was performed in a total this paper). The PCR was performed on a thermocycler
volume of 50 mL containing 4 mL of SALSA dilution buffer, (Veriti, Applied Biosystems) in a total volume of 20 mL,
2 mL of SALSA enzyme dilution buffer, 2 mL of universal containing 50 to 150 ng DNA, 10 mL of 2¥ PCR master mix
primers, 0.5 mL of SALSA polymerase, and 10 mL of liga- (GeneAmp Fast, Applied Biosystems), 0.5 mmol/L forward
tion sample. PCR conditions were as follows: 5 minutes at and reverse primer. PCR conditions were as follows: 10
95°C, 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C seconds at 95°C, 35 cycles of 10 seconds at 95°C, and a
and 1 minute at 72°C, followed by 20 minutes at 72°C. A specific annealing-elongation temperature and time for
mixture of 1.5 mL of MLPA sample, 8.5 mL of formamide each primer set (listed in Supplementary Table S2), fol-
(Hi-Di, Applied Biosystems), and 0.5 mL of size standard lowed by 1 minute at 72°C. PCR products were purified
(GeneScan, 500-Liz, Applied Biosystems) was analyzed using PCR product cleanup (ExoSAP-IT, GE Healthcare,
on a genetic analyzer (Model 3130, Applied Biosystems). Eindhoven, the Netherlands), according to manufactur-
Data analysis was performed using computer software er’s protocol. The sequence reaction was performed on a
(Genemarker, Version 1.85, Softgenetics, State College, thermocycler (Veriti, Applied Biosystems) in a total
PA). For each run two control samples hemizygously volume of 20 mL, containing 1 mL of purified PCR product,
positive for the RHD*01 allele, as determined via RHD 1 mL of 2.5¥ polymerase mix (BigDye Terminator v1.1,
Exons 5- and 7-specific quantitative real-time PCR were Applied Biosystems), 3.5 mL of 5¥ buffer (BigDye Termina-
used as reference samples to determine zygosity. Supple- tor, Applied Biosystems), and 0.25 mmol/L forward or
mentary Fig. S2 (available as supporting information in reverse primer. Sequence conditions were as follows:
the online version of this paper) shows an example of the 25 cycles of 15 seconds at 95°C, 10 seconds at 50°C, and
results of an MLPA reaction. The total RH-MLPA assay 4 minutes at 60°C. Sequence products were analyzed on
takes approximately 20 hours, including 16 hours of a genetic analyzer (3130, Applied Biosystems).

Volume 53, July 2013 TRANSFUSION 1561

1562
TABLE 1. Performance of RHD- and RHCE-specific probes from the RH-MLPA tested in 236 cases
Exon or Primary Secondary Probe Present Signal detected Mutation verfied
Probe name Gene intron ligation site* ligation site† size Use of probe/detecting in mix in tested cases by sequencing
D00_-132A RHD 5′ UTR –132A –173C 217 RHD*01 P401 222
MD01_008G RHD Exon 1 8G 149 RHD*weak D Type 3 P401 3 3
DCE01_048C RHD/RHCE Exon 1 48C 143 RHCE P402 191
MD01_048A RHD Exon 1 48A 144 RHD*48A P403 3 3
MCE01_122G RHCE Exon 1 122G 156 RHCE*CeCW P402 4 4
HAER-WIGMAN ET AL.

DCE02_IVS1-20G RHD/RHCE Intron 1 IVS1–20G 180 RHD*01 and RHCE*C P402 221
MD02_186T RHD Exon 2 186T 225C 241 RHD*DIIIa, RHD*DIII.4, RHD*DIVa.1, P401 14 5
RHD*DIVa.2, RHD*DIIIa-CE(3-7)-D
DCE02_203G RHD/RHCE Exon 2 201G/203G 224 RHD*01 and RHCE*C P403 220
MD02_270A RHD Exon 2 270A 214 RHD*270A P403 0 0
CE02_307C RHCE Exon 2 307C 269G 218 RHCE*c P402 208
MD02_329C RHD Exon 2 329C 158 RHD*DVII.1 P403 6 6
CE02_ins_C RHCE Intron 2 RHCE*C insertion 156 RHCE*C P401 123
D03_380T RHD Exon 3 380T 405C 167 RHD*01 P402 220

TRANSFUSION Volume 53, July 2013

MD03_410T RHD Exon 3 410T 383A 192 RHD*DIIIa, RHD*DIII.4, RHD*DIII.6, RHD*DIVa.2, P401 13 9
RHD*DOL3, RHD*DIIIa-CE(3-7)-D
MD03_446A RHD Exon 3 446A 419T 188 RHD*weak D Type 5 P403 4 3
D03_455A RHD Exon 3 455A IVS3+4T 175 RHD*01 P402 207
MD03_IVS3+1A RHD Intron 3 IVS3+1A 247 RHD*IVS3+1A P401 4 4
MD04_509C RHD Exon 4 509C 163 RHD*DOL1, RHD*DOL2, RHD*DOL3 P402 1 1
D04_514A RHD Exon 4 514A IVS3–5T 187 RHD*01 P401 168
D04_602C RHD Exon 4 602C 224 RHD*01 P402 196
MD04_609A RHD Exon 4 609A 130 RHD*y P401 46 5
D05_667T RHD Exon 5 667T 639C 211 RHD*01 P402 159
CE05_676G RHCE Exon 5 676G 712A 205 RHCE*e P401 230
CE05_676C RHCE Exon 5 676C 712A 211 RHCE*e P401 51
D05_697G RHD Exon 5 697G 192 RHD*01 P403 195
MCE05_733G RHCE Exon 5 733G 787A 204 RHCE*ceVS, RHD*DIIIa-CE(3-7)-D P402 19 10
D05_787G RHD Exon 5 787G IVS5+17C 181 RHD*01 P401 198
D05_800A RHD Exon 5 800A 148 RHD*01 P403 202
MD06_807G RHD Exon 6 807G 167 RHD*807G, RHD*y P403 46 4
MD06_809G RHD Exon 6 809G 192 RHD*weak D Type 1 P402 24 5
MD06_819A RHD Exon 6 819A IVS5-9T 169 RHD*DIIIa, RHD*DIII.6, RHD*weak D Type 4.0, P401 5 3
RHD*weak D Type 4.1, RHD*weak D Type 4.3
MD06_845A RHD Exon 6 845A 164 RHD*weak partial D Type 15 P403 5 4
MD06_872G RHD Exon 6 872G 132 RHD*weak D Type 4.3 P402 0 0
MD06_885T RHD Exon 6 885T 906G 174 RHD*weak partial D Type 11 P401 5 4
D06_932A RHD Exon 6 932A 237 RHD*01 P402 203
D07_941G RHD Exon 7 941G 174 RHD*01 P403 211
D07_989A RHD Exon 7 989A 143 RHD*01 P401 211
MD07_1025C RHD Exon 7 1025C 992A 151 RHD*DIV.3, RHD*DAR1, RHD*DAR2, RHD*weak P402 7 4
D Type 29
D07_1061T RHD Exon 7 1061T 181 RHD*01 P403 206
MD07_1063A RHD Exon 7 1063A IVS7+25T 222 RHD*DNB P401 3 3
MD08_1136T RHD Exon 8 1136T 211 RHD*DAU P403 5 5
MD09_1154C RHD Exon 9 1154C 1999A 236 RHD*weak D Type 2 P401 8 5
D09_1193A RHD Exon 9 1193A IVS9+2T 187 RHD*01 P402 219
MD09_1227A RHD Exon 9 1227A IVS9+46C 204 RHD*1227A P403 3 3
D10_1375C RHD Exon 10 1375C 162 RHD*01 P401 223
* Position as counted from ATG translation start site and nucleotide of the ligation site that is specific for the nucleotide detected by the probe.
† Position as counted from ATG translation start site and nucleotide of a second ligation site which is used to make the probe specific for RHD or RHCE.
 TABLE 2. RHD and RHCE variant alleles that can be determined using the RH-MLPA assay
Loss or gain of wild-type-probe combination(s) in specific variant allele

P402 D03_380T
P402 D05_667T

P402 D03_455A
P401 D04_514A
P403 D05_800A
P402 D06_932A
P401 D07_989A

P402 D04_602C
P403 D05_697G
P401 D05_787G
P403 D07_941G

P401 D00_-132A
P402 D09_1193A

P403 D07_1061C
P401 D10_1357C

P402 CE02_307C
P401 CE05_676C
P401 CE05_676G

P401 CE02_ins_C
P402 DCE02_-20C

P402 DCE01_048C
P403 DCE02_203G
Gain of mutation-probe combination(s)
Variant allele RHCE RHD in specific variant allele
RHD*DII/RHD*DIV.4 -
RHD*DIIIa - - - MD02_186T MD03_410T MD06_819A
RHD*DIIIb + - - - - - MD03_410T MD06_819A
RHD*DIII.3 - -
RHD*DIII.4/RHD*DIVa.2 - MD02_186T MD03_410T
RHD*DIII.6 - - - MD03_410T MD06_819A
RHD*DIVa.1 - MD02_186T
RHD*DIV.3 - - - - - MD07_1025C
RHD*DIV.5 - - - -
RHD*DIV.6 - -
RHD*DV.1 - -
RHD*DV.2 + - - - -
RHD*DV.3/RHD*DBS2 + - -
RHD*DV.4/RHD*DV.5/ -
RHD*DV.9
RHD*DV.6/RHD*DV.8 + - -
RHD*DV.7 + - - -
RHD*DVI.1 + - - - - - -
RHD*DVI.2 + - - - - - - -
RHD*DVI.3 + - - - - - - - - -
RHD*DVI.4 + - - - - - - - -
RHD*DVII.1 MD02_329C
RHD*DVII.2 + MD02_329C
RHD*DAR1 - - MD07_1025C
RHD*DAR2 - - - MD07_1025C
RHD*weak partial D type 4.0/ RHD*weak - - MD06_819A
partial D type 4.1
RHD*weak partial D type 4.3 - - MD06_819A MD06_872G
RHD*DAU0/ MD08_1136T
RHD*DAU1/
RHD*DAU2/
RHD*DAU3/
RHD*DAU6/
RHD*DAU7
RHD*DAU4 - MD08_1136T

Volume 53, July 2013

RHD*DAU5 - - MD08_1136T
RHD*DOL1/RHD*DOL2 - MD04_509C
RHD*DOL3 - MD03_410T MD04_509C
RHD*DBS1 + - - - -
RHD*DBT1 - - - - - - - -
RHD*DBT2 - - - - - - - - -
RHD*DCS1/RHD*DTO -
RHD*DFR1/RHD*DFR3 -

TRANSFUSION
RHD*DFR2 - -
RHD*DNB - MD07_1063A

1563
GENOTYPING OF RHD VARIANTS AND ZYGOSITY
1564
TABLE 2. Continued
Loss or gain of wild-type-probe combination(s) in specific variant allele
HAER-WIGMAN ET AL.

P402 D03_380T
P402 D05_667T

P402 D03_455A
P401 D04_514A
P403 D05_800A
P402 D06_932A
P401 D07_989A

P402 D04_602C
P403 D05_697G
P401 D05_787G
P403 D07_941G

P401 D00_-132A
P402 D09_1193A

P403 D07_1061C
P401 D10_1357C

P402 CE02_307C
P401 CE05_676C
P401 CE05_676G

P401 CE02_ins_C
P402 DCE02_-20C

P402 DCE01_048C
P403 DCE02_203G
Gain of mutation-probe combination(s)
Variant allele RHCE RHD in specific variant allele
RHD*weak partial D type 11 MD06_885T
RHD*weak partial D type 15 MD06_845A
RHD*weak D type 1 MD06_809G

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RHD*weak D type 2 MD09_1154C
RHD*weak D type 3 MD01_008G
RHD*weak D type 5 MD03_446A
RHD*weak D type 14/RHD*weak D type -
40/RHD*weak D type 51
RHD*weak D type 29 - - MD07_1025C
RHD*weak D type 41 -
RHD*1227A MD09_1227A
RHD*IVS3+1A MD_IVS3+1A
RHD*01N.01 - - - - - - - - - - - - - - - - -
RHD*y - - MD04_609A MD06_807G
RHD*CE(1-9)-D + + - - - - - - - - - - - - - - - -
RHD*CE(2-7)-D + - - - - - - - - - - - - - -
RHD*CE(2-9)-D + + - - - - - - - - - - - - - - -
RHD*DIIIa-CE(3-7)-D - - - - - - - - - - - MD02_186T MD03_410T MCE05_733G
RHD*CE(3-7)-D + - - - - - - - - - - - -
RHD*CE(3-9)-D + - - - - - - - - - - - - -
RHD*CE(4-7)-D + - - - - - - - - - -
RHD*48A MD01_048A
RHD*270A MD02_270A
RHD*807G MD06_807G
RHD*941T -
RHCE*ceAR/RHCE*ceEK + - + +
RHCE*ceBI/RHCE*ceSM + -
RHCE*ceCF + + MCE05_733G
RHCE*ceHAR ⱖ1 - + + + +
RHCE*ceMO + +
RHCE*ceVS MCE05_733G
RHCE*ce-D(9)-ce +
RHCE*CeCW MCE01_122G
RHCE*CeFV - + +
RHCE*CeRN1 + +
RHCE*CeRN2 + + +
RHCE*CeVA ⱖ1 - + + + +
RHCE*Ce800A +
RHCE*cEFM + - +
RHCE*cE602C +
 GENOTYPING OF RHD VARIANTS AND ZYGOSITY

RHD Exons 5- and 7-specific quantitative RHD Exon 2 also detect RHCE*C, because the RHD and
real-time PCR RHCE*C sequence of Exon 2 is completely identical.
Zygosity was determined with an RHD Exons 5- and RHCE*C genotyping is based on the detection of
7-specific quantitative real-time PCR. Primers were devel- the RHCE*C specific 109-nucleotide insertion in Intron
oped to detect ALB35 (quantification of input DNA), SRY,36 2 and 203G in Exon 2. RHCE*c genotyping is based on the
and RHD Exons 527 and 7.37 The RHD Exons 5 and 7 detection of 307C. RHCE*E and RHCE*e genotyping is
primers and probes were synthesized by TIB MOLBIOL based on the detection of 676C and 676G, respectively. To
(Berlin, Germany) and the primers for SRY and the ALB ensure RHCE specificity, the latter two probe combina-
were synthesized by Invitrogen. The real-time PCR was tions consist of three probe pieces, creating a second
performed on a sequence detection system (ABI PRISM ligation site for the 712A RHCE-specific nucleotide. In all
7000, Applied Biosystems) in a total volume of 25 mL, con- D+ cases (n = 62) all RHD wild-type probe combination
taining 25 ng of DNA, 12.5 mL of universal primer PCR signals were present, while in the D– cases (n = 11) all RHD
master mix (Taqman, Applied Biosystems), 0.1 mmol/L of wild-type probe combination signals were absent. The
the RHD Exon 5 and SRY probe or RHD Exon 7 and ALB RH-MLPA RHCE*ce, RHCE*Ce, RHCE*cE, and RHCE*CE
probe and 0.3 and 0.9 mmol/L of the RHD Exon 5 and SRY genotyping results were completely concordant with
forward and reverse primers, respectively, or 0.3 mmol/L of serology. All possible common RHD/RHCE haplotypes
the RHD Exon 7 and ALB forward and reverse primers. were determined at least once (data not shown). In four
Real-time PCR conditions were as follows: 2 minutes at cases one or more mutation probe combination signals
50°C, 10 minutes at 95°C, and 50 cycles of 15 seconds at were detected: two cases showed the presence of the
95°C and 1 minute at 60°C. Data analysis was performed RHD*DVII.1 allele next to an RHD*01 allele (wild-type
using computer software (Microsoft Office Excel 2003, RhD); similarly one case had an RHD*y allele next to an
Microsoft, Amsterdam, the Netherlands). RHD*01 allele and one case carried an RHCE*CeCW allele.
Sequencing of the involved exons in these cases con-
firmed the presence of the variant alleles detected by the
Short-tandem-repeat multiplex PCR RH-MLPA.
The presence of chimerism was determined with genomic
DNA isolated from WBCs using short-tandem-repeat mul- Mutation probe combinations
tiplex (Powerplex 16 HS system, Promega, Leiden, the The mutation probe combinations of the RH-MLPA were
Netherlands). The reaction was performed according to selected to recognize, in combination with the wild-type
the manufacturer’s protocol. probe combinations, the majority of clinically relevant
RHD variant alleles and most frequently occurring RHCE
variant alleles in the Caucasian and black African popula-
RESULTS tions. The performance of the mutation probe combina-
tions was evaluated with DNA from 97 cases, for whom
Performance of RH-MLPA assay with plasmid DNA
serology indicated the presence of an Rh variant and with
First, all RH-MLPA wild-type (n = 22) and mutation probe
DNA from 66 cases, for whom the presence and/or
combinations (n = 23) were analyzed using a cloned DNA
expressing of an Rh variant was already determined. With
plasmid sample, containing the genomic sequence of all
this set of samples 19 RHD mutation probe combination
probes, as a template. For all probes correct signals were
signals and two RHCE mutation probe combination
detected (data not shown) and subsequently the perfor-
signals were obtained at least once. To confirm the pres-
mance of the RH-MLPA was evaluated with a set of
ence of a mutation indicated by one of the mutation probe
genomic DNA samples.
combinations, the exons containing the respective muta-
tions were sequenced for at least three independent
samples (if available). All sequencing results were concor-
Performance of RH-MLPA assay with
dant with the results of the RH-MLPA (Table 1). For two
genomic DNA
RHD mutation probe combinations MD02_270A specific
Wild-type probe combinations for the RHD*270A allele and MD06_872G specific for the
Next, the performance of the RH-MLPA assay was ana- RHD*weak partial D Type 4.3 allele no genomic DNA was
lyzed with DNA from 73 healthy donors with normal Rh available.
serology, representing all common Rh phenotypes. The The RHCE mutation probe combination MCE05_
RHD wild-type probe combinations are able to detect the 733G was designed to determine whether an individual is
copy number of all RHD exons, except that of Exon 8. No positive (733G) or negative (733C) for the VS antigen. Of
probe combination for RHD Exon 8 was included, since note, individuals carrying an RHCE*ceAR allele have the
the sequence of RHD and RHCE Exon 8 is completely 733G mutation, but are negative for the VS antigen.38 We
identical. The probe combinations detecting wild-type designed the MCE05_733G with a second ligation site

Volume 53, July 2013 TRANSFUSION 1565

HAER-WIGMAN ET AL.

specific for the 787A RHCE wild-type single-nucleotide (Thr148Arg) mutation. The latter was determined as D–
polymorphism (SNP), which is present in all VS+ alleles via the absorption-elution technique using a polyclonal
and absent in the RHCE*ceAR allele. Six of the nine cases anti-D (anti-D bromelain, Sanquin Reagentia). This
carrying a RHCE*ceAR allele were VS negative and indeed patient was included in our series, because her current D
these samples showed correctly the absence of signal for serology (D–) was discordant with a previously reported
MCE05_733G. In the three other cases the second RHCE D+ phenotype by the blood bank of Azerbaijan. Pro-
allele was a VS+ allele and as expected the MCE05_733G bably the donor was regarded as D+ because of the CE
signal was obtained. phenotype (RhCcee). The new RHD*525T (Phe175Leu)
variant allele results in absence of expression of Epitope
1.2 (determined with monoclonal antibodies [MoAb]
Determination of RHD and RHCE variant alleles LHM174/102 and LHM70/45 of the extended partial
using the RH-MLPA assay RhD typing set of Bio-Rad Laboratories) and Epitope 2.2
In 160 of the above-described 163 individuals the (determined with MoAb 5C839). The LHM169/81 MoAb
RH-MLPA indicated the presence of one or more variant (extended partial RhD typing set), which detects Epitope
alleles. In these 163 cases a total of 172 RHD and 78 RHCE 1.1 showed the same strength of reactivity for both the
variant alleles or variant allele groups were determined as D+ control and the RBCs expressing the RHD*525T
shown in Table 3. (Phe175Leu) allele.40 Similarly, all other D epitopes tested
The assigned RHD and RHCE variant allele(s) were in were normally present. The patient was typed positive for
all cases compatible with the initial serologic results the C, c, and e antigens. In two other cases no RHD variant
obtained for the cases. In addition, in 99 of the 163 allele was detected, but an aberrant RHD copy number of
selected samples, we performed an RHD-MPX PCR, in 0.3 and 0.5, respectively, was detected. These samples are
which RHD Exons 3, 4, 5, 6, 7, and 9 are RHD specifically further discussed below, see “RHD and RHCE zygosity”
amplified. The results of the RHD-MPX combined with the results.
results of the serology were concordant with the RHD vari- For three variant alleles a loss of signal of a wild-type
ants concluded from the RH-MLPA, except for 11 cases. probe combination was obtained, whereas presence of
In these cases, carrying the RHD*DV.5, RHD*DAU2, the RHD-specific nucleotide was shown by sequencing. In
RHD*DAU3, RHD*weak partial D Type 11, or RHD* weak three cases carrying the RHD*DNB or the new partial
partial D Type 15 allele, the RHD-MPX results showed no RHD allele (150T>C, 178A>C, 201G>A, 203G>A, 307T>C,
aberrant pattern, suggesting the presence of weak D vari- 1063G>A), the D07_1061T signal was absent. Apparently,
ants, while the RH-MLPA indicated the presence of partial the 1063A mutation present in these alleles impaired the
RhD variants. Sequencing of the exons containing the complete hybridization and ligation of D07_1061T. In the
mutations showed that in these cases the RH-MLPA 46 cases carrying the RHD*y allele, the D04_514A signal
assigned the correct RHD variant alleles (Table 3). In one was absent. The 37-bp duplication in intron 3 (IVS3-19)
case the RH-MLPA indicated the presence of a new partial present in the RHD*y allele prevents the hybridization of
RHD allele, containing the mutations of both RHD*DIIIb the D04_514A probe combination, since the insertion
and RHD*DNB on one allele (150T>C, 178A>C, 201G>A, results in a shortening of the hybridization site to only 13
203G>A, 307T>C, and 1063G>A). Zygosity analysis showed nucleotides instead of the normal 34 nucleotides. All 46
the presence of one RHD allele and sequencing confirmed cases carrying the RHD*y allele were also associated with
the presence of the mutations. Unfortunately, no red an unexpected gain of the D09_1193A signal. The RHD
blood cells (RBCs) were available for further serologic and RHCE Exon 9 were therefore PCR amplified in three
typing. In 23 cases the RH-MLPA detected the presence of cases that were homozygous for the RHD*y allele. In these
a variant allele, but could not discriminate between differ- cases RHCE Exon 9 could not be amplified, while RHCE
ent subtypes (e.g., RHD*DAU alleles and RHD*weak Exons 8 and 10 were normally amplified and sequencing
partial 4.0 or RHD*weak partial 4.1 alleles) and the of these exons and surrounding introns showed RHCE
samples were sequenced to assign the specific RHD or wild-type sequence. We therefore hypothesize that in this
RHCE variant subtype (Table 3). RHCE variant allele, linked to the RHD*y allele, RHCE
In three cases in which serology indicated the pres- Exon 9 is replaced by RHD Exon 9 resulting in a RHCE*ce-
ence of an Rh variant, the RH-MLPA indicated the pres- D(9)-ce allele. This hybrid RHCE allele is normally
ence of a single normal RHD*01 allele. Sequencing of all expressed since these three homozygous cases were all
RHD exons revealed that one case concerned a hemizy- serologically positive for the c and e antigens and also no
gously present RHD*DCS2 allele (676G>C), for which no abnormal reaction patterns have been described for D–
RHD-specific probe combination is included. In the two black Africans. As no product was amplified in the RHCE
other cases two new RHD variant alleles were detected. Exon 9 PCR, the break points of the mutated RHCE allele
One new RHD variant allele has the 525C>T (Phe175Leu) are located outside the range of at least one of our RHCE
mutation and the other RHD variant allele has the 443C>G Exon 9 primers. To confirm the presence of this hybrid

1566 TRANSFUSION Volume 53, July 2013

 TABLE 3. RHD and RHCE variant alleles detected by the RH-MLPA assay in 163 DNA samples
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
Partial RHD alleles RHD*DIIIa RHD*DIIIa RHCE*ceVS 1
RHD*DIIIb New RHD variant 1
(150T>C, 178A>C,
201G>A, 203G>A,
307T>C, 1063G>A)
RHD*DIII.3 RHD*DIII.3 1
DIVa RHD*DIII.4 or RHD*DIVa.2 3
RHD*DIVa.2
RHD*DIV.4 RHD*DII or RHD*DIV.4 1
RHD*DIV.4
DVI t1 RHD*DVI.1 2
DVI t2 RHD*DVI.2 7
DVII RHD*DVII.1 4
DAR§ RHD*DAR1 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK
RHD*DAR1 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK and
RHCE*ceVS
RHD*DAR1 RHCE*ceAR or RHCE*ceAR 1
(homozygous) RHCE*ceEK (homozygous)
(homozygous)
RHD*DAR1 and RHCE*ceAR or RHCE*ceAR 1
RHD*DIIIa-CE(3-7)-D RHCE*ceEK
RHD*DAR2 RHCE*ceAR or RHCE*ceAR 2
RHCE*ceEK
RHD*weak D Type 29 1
RHD*weak partial D RHD*weak partial D 3
Type 11 Type 11
RHD*DBT2 RHD*DBT2 1
RHD*DCS1 RHD*DCS1 1
DFR RHD*DFR1 1
RHD*DFR2 2
RHD*DOL1 RHD*DOL1 or RHCE*ceBI or RHD*DOL1 RHCE*ceBI 1
RHD*DOL2 RHCE*ceSM
RHD*DNB RHD*DNB 2
Partial D variant RHD*01 RHD*DCS1 1

Volume 53, July 2013

New variant 1
RHD*525T
RHD*DV.7 3
RHD*DAU5 1
RHD*weak partial D RHCE*ceVS RHD*weak partial D 1
Type 4.0 or Type 4.0
RHD*weak partial D

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Type 4.1

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GENOTYPING OF RHD VARIANTS AND ZYGOSITY
1568
TABLE 3. Continued
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
RHD*weak partial D RHD*weak partial D 2
Type 4.0 or Type 4.0
HAER-WIGMAN ET AL.

RHD*weak partial D
Type 4.1 and
RHD*DIIIa-CE(3-7)-D
RHD*weak partial D RHCE*ceVS RHD*weak partial D 1
Type 4.0 or Type 4.0
RHD*weak partial D
Type 4.1 and
RHD*DIIIa-CE(3-7)-D
Weak D variant RHD*DV.4 or RHD*DV.5 1

TRANSFUSION Volume 53, July 2013

RHD*DV.5 or
RHD*DV.9
RHD*DAU0 or RHD*DAU2 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7
RHD*DAU3 1
RHD*DAU0 or RHCE*ce-D(9)-ce RHD*DAU3 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7 and
RHD*y
RHD*weak partial D 2
Type 11
RHD*weak partial D 5
Type15
Weak RHD and Del Weak D Type 2 RHD*weak D Type 2 7
alleles RHD*weak D Type2 1
and
RHD*DIIIa-CE(3-7)-D
RHD*weak D Type3 RHD*weak D Type3 1
RHD*weak D Type5 RHD*weak D Type5 1
Weak D variant RHD*weak D Type1 22
RHD*weak D Type1 1
and RHD*DVI.2
RHD*weak D Type1 1
and
RHD*DIIIa-CE(3-7)-D
RHD*weak D Type3 2
RHD*weak D Type5 3
 TABLE 3. Continued
Selected donors RH-MLPA conclusion Additional genotyping‡
Serology and Variant allele detected
RHD allele group RHD-MPX PCR* Sequence† RHD RHCE RHD RHCE in tested cases
RHD*1227A RHD*1227A 3
RHD*IVS3+1A RHD*IVS3+1A 2
Del variant RHD*IVS3+1A 2
D negative null alleles RHD*y RHD*y RHCE*ce-.D(9)-ce 30
RHD*y RHCE*ce-D(9)-ce and 4
RHCE*ceVS
RHD*y RHCE*ce-D(9)-ce and RHCE*ceAR 1
RHCE*ceAR or
RHCE*ceEK
RHD*y (homozygous) RHCE*ce-D(9)-ce 3
(homozygous)
RHD*y and RHCE*ce-D(9)-ce 5
RHD*DIIIa-CE(3-7)-D.
RHD*y and RHCE*ce-D(9)-ce and 1
RHD*DIIIa-CE(3-7)-D RHCE*ceCF
RHD*48A RHD*48A 3
D–¶ RHD*01 New variant 1
RHD*443G
RHD chimera Chimera Chimera 50% RHD 1
Weak D variant Chimera 30% RHD 1
RHCE variant alleles RHCE*CeCW RHCE*CeCW 3
ceHAR RHCE*ceHAR 1
RhCE variant RHD*DAU0 or RHCE*cE602C RHD*DAU0 1
RHD*DAU1 or
RHD*DAU2 or
RHD*DAU3 or
RHD*DAU6 or
RHD*DAU7
RHCE*CeRN1 1
* RHD and RHCE variant allele determination after serology and/or RHD-MPX PCR results.
† RHD and RHCE variant alleles that were chosen for their specific genotype.

Volume 53, July 2013

‡ Sequencing was performed when the RH-MLPA was not able to determine the specific subtype of the variant allele.
§ Serology and the RHD-MPX PCR are not able to distinguish between the DAR1, DAR2, and Weak D Type 29 variant alleles.
¶ This patient was included in our series, because her current D serology (D–) was discordant with a previously reported D+ phenotype by the blood bank of Azerbaijan.

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GENOTYPING OF RHD VARIANTS AND ZYGOSITY
HAER-WIGMAN ET AL.

Fig. 1. RH-MLPA results obtained with a mixture of hemizygous RHD*01 D+ DNA from a donor pheno- and genotyped as (CcDdEe)
mixed with homozygous D– RHD*01N.01 DNA typed as (ccddee). DNA was added in a dilution range from 64% to 0.25% RHD*01 in
RHD*01N. The ratio of the RHD wild-type probe combinations is calculated using the hemizygous RHD*01 (CcDdEe) sample as a
reference sample. For exons of which more than one set of RHD wild-type probe combinations is detected, the mean of the probes
is given. Error bars indicate the standard deviation. Two RHD wild-type probe combinations detected the presence of 0.25%
RHD*01. The presence of 2% RHD*01 is reliably detected by the RH-MLPA and the presence of 4% RHD*01 is detected by all RHD
wild-type probe combinations.

allele in cases which have only one RHD*y allele and RHD genes were deleted (dd). To confirm the zygosity
hence are heterozygous for the RHCE*ce-D(9)-ce allele, we determined by the RH-MLPA, 18 cases (seven DD and
developed an MLPA probe combination for the RHCE- eleven Dd) were compared with zygosity typing using
specific 1193T SNP. Seven cases hemizygous for the RHD Exons 5- and 7-specific quantitative real-time PCR.
RHD*y allele and one case heterozygous for this allele The results were concordant between both assays.
were tested with the CE09_1193T probe combination. In For two cases the RH-MLPA detected an abnormal
all tested samples the CE09_1193T showed a copy number RHD copy number of 0.5 and 0.3 for all RHD-specific
of 1, indicating that these persons were indeed heterozy- probe combinations, indicating the presence of, respec-
gous positive for the RHCE*ce-D(9)-ce allele (data not tively, 50 and 30% hemizygously RHD*01 (RhD+). D serol-
shown). ogy showed a mixed field reaction in the case with an
RHD-copy number of 0.5 and the case with a copy number
of 0.3 was serologically determined as “weak D.” Further
RHD and RHCE zygosity genetic analysis using short-tandem-repeat multiplex
RHD and RHCE zygosity were determined for all 236 DNA PCR showed that these samples were from individuals
samples by RH-MLPA. Of these cases, 163 cases were with a hematopoietic chimerism. To evaluate the sensitiv-
found to be hemizygous for the RHD gene (Dd), 58 cases ity of the RH-MLPA to detect hematopoietic chimerism,
homozygous for the RHD gene (DD), and in 12 cases both DNA from a person hemizygous for RHD*01 (CcDdEe) was

1570 TRANSFUSION Volume 53, July 2013

 GENOTYPING OF RHD VARIANTS AND ZYGOSITY

mixed with DNA from a person homozygous for previously serologically typed for Rh and in some cases
RHD*01N.01 (ccddee). As shown in Fig. 1 the presence of already typed with an RHD-MPX PCR or by RHD exon–
0.25% hemizygous RHD*01 DNA is detected by two RHD specific sequencing. In 160 cases (98%) the RH-MLPA
wild-type probe combinations and the presence of 4% assigned correctly if a wild-type and/or variant allele(s)
hemizygous RHD*01 DNA is detected by all RHD wild- was (were) present. In one case sequencing revealed the
type probe combinations. When 2% or more hemizygous presence of a partial RHD allele (RHD*DCS2), for which no
RHD*01 DNA is present the RH-MLPA can reliably detect a probe was included in the RH-MLPA since it is extremely
hematopoietic chimerism and correct quantification of rare.41 It is still possible to add an extra mutation probe for
the amount of the RhD chimerism is possible when 8% or detection of the RHD*DCS.2 allele. In the other two cases,
more hemizygous RHD*01 DNA is present. two new variant RHD alleles were found: a D– null allele
In 207 of the 236 cases (88%) an RHCE copy number RHD*443G (Thr148Arg) and a partial RHD allele
of 2 was obtained for all RHCE-specific probe combina- RHD*525T (Phe175Leu). We therefore conclude that the
tions. In the other cases, a copy number of 2 was RH-MLPA can be used to determine an RHD and/or RHCE
obtained for all RHCE-specific probe combinations variant in the vast majority of samples analyzed in a ref-
except for the CE05_676C and CE05_676G probe combi- erence laboratory and only in few samples will follow-up
nations that detect RHCE*E and RHCE*e, respectively. analysis be needed to show the presence of either a very
The abnormal RHCE*E/RHCE*e Exon 5 copy number of 0, rare or a new variant allele. In all 254 variant alleles
1, or 3 could be explained in all cases. All cases with a detected by the RH-MLPA the correct variant or variant
RHCE*ceAR, RHCE*ceBI, or RHCE*ceHAR allele have an group was assigned. In one case the RH-MLPA could even
RHCE*E/RHCE*e Exon 5 copy number of 1 (heterozygous determine a new partial RHD allele (150T>C, 178A>C,
variant allele) or 0 (homozygous variant allele), since 201G>A, 203G>A, 307T>C, and 1063G>A). In 26 of the 254
they all contain the 712G mutation, which impairs liga- variant alleles (10%) detected by the RH-MLPA, additional
tion of the second ligation site specific for the 712A sequencing was necessary to determine the specific sub-
RHCE wild-type SNP of CE05_676C or CE05_676G. In 20 group (Table 3). It is only in the RHD*DAU variant group
cases an elevated RHCE*E/RHCE*e Exon 5 copy number (n = 4) where determination of the subtype is clinically
of 3 was obtained. All these cases carried next to their relevant, because the RHD*DAU0 allele, with normal RhD
RHCE alleles an RHD-CE-D hybrid allele that contained a expression, should be discriminated from the other
complete RHCE Exon 5. Therefore, the CE05_676G RHD*DAU alleles.
(including the RHD*DV.7, RHD*DVI.2, and RHD*DIIIa- A new RHD*443G (Thr148Arg) allele was detected in
CE(3-7)-D allele) or CE05_676C (RHD*DVI.1) was able to this study. This seems to be a D– null allele, caused by a
bind to the hybrid allele, causing an elevated RHCE*E/ mutation resulting in the substitution of a polar amino
RHCE*e Exon 5 copy number. acid (threonine) with a positively charged amino acid
(arginine) in the fifth transmembrane region of the RhD
protein. To confirm this unexpected profound effect an
DISCUSSION
RhD expression model should be performed.42
In this study we showed that the RH-MLPA genotyping For the first time it was recognized that the RHD*y
assay is a reliable method to predict the Rh-phenotype allele is linked (in all 46 persons tested) to a hybrid
and to determine the majority of RHD and RHCE variant RHCE*ce variant allele containing RHD Exon 9 (RHCE*ce-
alleles as well as RHD zygosity. The MLPA technique is D(9)-ce). This could be determined with the RH-MLPA,
easy to use, as it only requires a thermocycler and capillary since it determines the RHD copy number of Exon 9. This
electrophoresis equipment. hybrid allele contains two mutations 1170C>T and
First, the specificity of the RHD and RHCE wild- 1193T>A, encoding for one amino acid change Glu398Val
type and RHD and RHCE mutation probe combinations at the intracellular C-terminal tail of the Rhce protein.
was determined with plasmid DNA and subsequently Serologic D and CE typing in persons homozygous posi-
with genomic DNA. All RHD and RHCE wild-type and tive for the RHD*y RHCE*ce-D(9)-ce haplotype showed
mutation probe combinations detected the wild-type or the normal presence of the c and e antigens and the
mutated sequence according to their design (Table 1). We absence of all D epitopes. The amino acid change is
showed two exceptions of wild-type probe combinations present in the intracellular tail of the protein and thus far
in which the loss of signal was not caused by mutation at no effect on the expression of the RHCE epitopes has been
the ligation site, but due to a mutation close by the liga- shown.21
tion site: absence of D07_1061T in variant alleles with The RH-MLPA assay is very accurate in determining
1063G>A mutation and absence of D04_514A in RHD*y gene copy number variation. In the RH-MLPA the RHD
alleles. copy number is based on the signals derived from 17 RHD
To evaluate the accuracy of the RH-MLPA assay its wild-type probe combinations. This makes the RH-MLPA
performance was tested with a set of 163 cases that were more suitable for RHD and RHCE zygosity determination

Volume 53, July 2013 TRANSFUSION 1571

HAER-WIGMAN ET AL.

than real-time quantitative PCR30 or amplification of the pregnant women is still growing, since molecular geno-
hybrid Rh box.31-33 Clinically, it is important to determine typing methods are increasingly applied and therefore
RHD zygosity in fathers, to assess the need for fetal RHD the number of cases with discrepant serologic and geno-
typing in RhD alloimmunized D– women. Furthermore, typing results that require further molecular analysis will
since the RH-MLPA is highly accurate in determining exon increase.
copy number, this is the first high-throughput assay that is
able to recognize hybrid RHD/RHCE alleles next to a
normal RHD gene. ACKNOWLEDGMENTS
Next to the “normal” zygosity scores of 0, 1, and 2, the
We thank Peter Ligthart (Sanquin Diagnostic Services, Amster-
RH-MLPA is also able to reliably determine the presence of
dam, the Netherlands) for his technical assistance, Karel de Groot
hematopoietic chimerism of just 2% RHD*01 (RhD+) in an
(MRC Holland, Amsterdam, the Netherlands) for assistance in
RHD*01N.01 (RhD–) background. It is important to detect
designing the MLPA probe mixes, Christof Weinstock (Institute
RHD+/RHD– chimeras, because it has been shown that
for Clinical Transfusion Medicine and Immunogenetics Ulm,
transfusion of RBCs from a donor with a hematopoietic
Ulm, Germany), Inge von Zabern (Institute for Clinical Transfu-
chimerism of 6% RHD+ DNA was able to immunize two
sion Medicine and Immunogenetics Ulm, Ulm, Germany), Geoff
D– recipients.43 The RH-MLPA can also reliably genotype
Daniels (Bristol Institute for Transfusion Sciences, National
patients who received multiple nonleukoreduced RBC
Health Service Blood and Transplant, Bristol, UK), Martin Pisacka
units. After massive transfusions a (weak) signal derived
(Institute of Haematology and Blood Transfusion, Prague, Czech
from donor WBCs might be detected with the RH-MLPA.44
Republic), and Yanli Ji (Guangzhou Blood Center, Guangzhou
Because the MLPA is a quantitative method, it will be
Guangdong, China) for providing DNA samples with specific
readily recognized if a signal is obtained from a minor
RHD genotypes.
population of transfused WBCs.
Some variants were correlated with a loss or gain of
the RHCE*E/RHCE*e Exon 5 copy number. In the
WEB RESOURCES
RHCE*ceAR, RHCE*ceEK, RHCE*ceBI, and RHCE*ceHAR
alleles the correct binding of the CE05_676G and ISBT Working Party on Red Cell Immunogenetics and
CE05_676C probe combination is inhibited and there- Blood Group Terminology http://www.isbtweb.org/
fore it is not possible to determine whether these working-parties/red-cell-immunogenetics-and-blood-
variant alleles contain the RHCE*E or RHCE*e variation. group-terminology/blood-group-terminology/blood-
Since the RHCE*ceAR, RHCE*ceEK, RHCE*ceBI, and group-allele-terminology/. Accessed 05/10/2012.
RHCE*ceHAR alleles result in partial e antigen expression,
an individual carrying a RHCE*ceAR, RHCE*ceEK,
RHCE*ceBI, or RHCE*ceHAR allele should always be deter- CONFLICT OF INTEREST
mined as RHCE*e+, hence Rhe+.45-47 For the RHD*DVI
NDA is a member of the transfusion Medicine Advisory Board
and RHD*DIIIa-CE(3-7)-D hybrid alleles in which the
for Grifols, SA. All other authors declare that they have no
RH-MLPA suggest an RHCE*E/RHCE*e Exon 5 copy
conflicts of interest relevant to the manuscript submitted to
number of three it is known that the hybrid alleles result
TRANSFUSION.
in the expression of the e antigen (RHD*DVI.1 and
RHD*DIIIa-CE(3-7)-D allele) or the E antigen (RHD*DVI.1
allele).48 For these cases the presence of the RHCE*e
REFERENCES
and/or RHCE*E SNP present in the hybrid allele should be
taken into account when determining the RhEe status. For 1. Daniels G, Reid ME. Blood groups: the past 50 years. Trans-
the precise determination of variant RHCE alleles and the fusion 2010;50:281-9.
identification of rare RHCE variant alleles an additional 2. Westhoff CM. Rh complexities: serology and DNA genotyp-
MLPA has to be developed, but as listed in Table 2 the ing. Transfusion 2007;47 Suppl:17S-22S.
most frequently occurring RHCE variant alleles are recog- 3. Urbaniak SJ, Greiss MA. RhD haemolytic disease of the
nized with the current RH-MLPA. fetus and the newborn. Blood Rev 2000;14:44-61.
In conclusion, the RH-MLPA genotyping assay cor- 4. Daniels G, Poole J, de Silva M, Callaghan T, MacLennan S,
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residual disease in acute lymphoblastic leukemia using 44. Rozman P, Dovc T, Gassner C. Differentiation of autolo-
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Rh antigen D disclosed by testing category DII cells with notype results from substitution of exon 5 of the RHCE
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40. Scott M. Section 1A: Rh serology. Coordinator’s report. Haematol 1996;92:751-7.
Transfus Clin Biol 2002;9:23-9. 48. Avent ND, Finning KM, Liu W, Scott ML. Molecular biology
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J, Pisacka M. DCS-1, DCS-2, and DFV share amino acid
substitutions at the extracellular RhD protein vestibule. SUPPORTING INFORMATION
Transfusion 2008;48:25-33.
Additional Supporting Information may be found in the
42. Smythe JS, Avent ND, Judson PA, Parsons SF, Martin PG,
online version of this article:
Anstee DJ. Expression of RHD and RHCE gene products
using retroviral transduction of K562 cells establishes the Fig. S1. Schematic overview of the MLPA reaction.
molecular basis of Rh blood group antigens. Blood 1996; Fig. S2. Example of analysis of mix P401.
87:2968-73. Table S1. Hybridization sequences of the probes of the
43. Wagner FF, Frohmajer A, Flegel WA. RHD positive RH-MLPA.
haplotypes in D negative Europeans. BMC Genet 2001;2: Table S2. Primer sequences for amplification and
10. sequencing of exons 1-10 of the RHD and RHCE gene.

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