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TRANSFUSION MEDICINE
The DAU allele cluster of the RHD gene
Franz F. Wagner, Birgit Ladewig, Katharina S. Angert, Guido A. Heymann, Nicole I. Eicher, and Willy A. Flegel
Variant D occurs frequently in Africans.
However, considerably less RHD alleles
have been described in this population
compared with Europeans. We characterized 5 new RHD alleles, dubbed DAU-0 to
DAU-4, that shared a T379M substitution
and occurred in a cDe haplotype. DAU-1
to DAU-4 were detected in Africans with
partial D phenotypes. They harbored one
and 2 additional missense mutations, re-
spectively, dispersed throughout the RhD
protein. An anti-D immunization was found
in DAU-3. DAU-0 carrying T379M only was
detected by screening European blood
donors and expressed a normal D phenotype. Within the phylogeny of the RHD
alleles, DAU formed an independent allele
cluster, separate from the DIVa, weak D
type 4, and Eurasian D clusters. The characterization of the RH phylogeny pro-
vided a framework for future studies on
RH alleles. The identification of the DAU
alleles increased the number of known
partial D alleles in Africans considerably.
DAU alleles may be a major cause of
antigen D variability and anti-D immunization in patients of African descent. (Blood.
2002;100:306-311)
© 2002 by The American Society of Hematology
Introduction
The D antigen of the RH blood group (ISBT 004.001; RH1;
CD240D; “Rhesus D”) is the most important blood group antigen
determined by a protein, because D-negative individuals are easily
anti-D immunized.1 This antibody remains the leading cause for the
hemolytic disease of the newborn,2,3 and antigen D–compatible
transfusion is standard in modern transfusion therapy.
D-positive individuals harboring a “partial” D antigen may
produce an allo–anti-D, too. Among Europeans, the population
frequency of all known partial D phenotypes combined is less than
1%.4,5 The molecular basis is generally a gene conversion, in which
parts of the RHD gene were substituted by the respective segments
of the RHCE gene, and single missense mutations.6 The molecular
characterization of aberrant RHD alleles was much facilitated in
Europeans by the frequent occurrence of the RHD gene deletion.7,8
Transfusion strategies were devised to ensure D-negative transfusion in carriers of D category VI, which was known to be the
clinically most relevant partial D occurring in Europeans.9
The situation is more intricate in Africans: The occurrence of
aberrant RHD alleles and anti-D immunizations in D-positive individuals is much more frequent than in Europeans.10 The serologic testing is
confounded by frequent “African” alleles that almost defy serologic recognition, like D category III types.11 Molecular analysis
revealed that there are often multiple missense mutations, rather
than single ones.12 The molecular characterization of RHD alleles
was hampered in Africans by the frequent occurrence of RHD␺,13
Ccdes,14 or the concomitant presence of 2 different partial D alleles.
RHD␺ and Ccdes do not express a D antigen, yet they harbor a
grossly intact RHD allele or an RHD-CE-D hybrid allele, respectively, that often interferes with RHD polymerase chain reaction
(PCR) and RHD-specific sequencing.
We described 5 RHD alleles that shared a T379M substitution.
Four of these alleles expressed a partial D phenotype characterized
by the lack of distinct D epitopes or by an anti-D immunization
event. We provide a detailed RHD phylogeny in which the DAU
alleles formed a previously unknown cluster.
From the Abteilung Transfusionsmedizin, Universitätsklinikum Ulm and DRK
Blutspendedienst Baden-Württemberg–Hessen, Institut Ulm, Ulm, Germany;
BiotestAG, Dreieich, Germany;Abteilung Transfusionsmedizin, Universitätsklinikum
Aachen, Germany; Institut für Transfusionsmedizin, Charité, Berlin, Germany;
and Blutspendedienst SRK Bern, Bern, Switzerland.
by the Deutsche Gesellschaft für Transfusionsmedizin und Immunhämatologie
(project DGTI/fle/00-01).
Submitted January 31, 2002; accepted March 1, 2002. Prepublished online as
Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2002-01-0320.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the DRK-Blutspendedienst Baden-Württemberg–Hessen,
Mannheim; by the University of Ulm (Forschungsförderungsprojekt P. 531); and
306
Materials and methods
Blood samples
There were 6 ethylenediaminetetraacetic acid (EDTA)–anticoagulated
blood samples referred to our laboratory for problems with D typing or
anti-D production in a D-positive individual (sample RIR no. 38 of the
Rhesus Immunization Registry, accessible at http://www.uni-ulm.de/
⬃wflegel/RIR). In addition, EDTA-anticoagulated blood samples were
collected from blood donors of ccDee phenotype in Baden-Württemberg,
Germany. DNA was isolated using QiaAmp blood kit (Qiagen, Hilden,
Germany) or by a modified salting-out procedure.15 In addition, 2 DNA
samples with rare RhCE phenotypes typically occurring in Africans
(95-012: hrs- and 95-013: Rh: -34) were obtained from the serum, cells, and
fluid exchange (SCARF).
Sequencing of the 10 RHD exons from genomic DNA
Nucleotide sequencing of the 10 RHD exons was performed as previously
described.16-18 The standard PCR for RHD exon 2 amplification failed with
some alleles belonging to the DIVa cluster (eg, in sample 95-013); in such
samples RH exon 2 was amplified using primers re12c and re2317 and
sequenced in a RHD-specific manner using primer re17. Primer sequences
were re12c, attagccgggcacggtggtg; and re17, ctcgtctgcttcctcctcg.
Reprints: Willy A. Flegel, Abteilung Transfusionsmedizin, Universitätsklinikum
Ulm, and DRK Blutspendedienst Baden-Württemberg–Hessen, Institut Ulm,
Helmholtzstrasse 10, D-89081 Ulm, Germany; e-mail: [email protected].
© 2002 by The American Society of Hematology
BLOOD, 1 JULY 2002 䡠 VOLUME 100, NUMBER 1
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BLOOD, 1 JULY 2002 䡠 VOLUME 100, NUMBER 1
DAU ALLELE CLUSTER
Polymerase chain reaction with sequence-specific priming
A PCR with sequence-specific priming (PCR-SSP) was devised to detect or
to confirm the 1136 C ⬎ T substitution in the DAU alleles and triggered to
work under similar PCR conditions as a PCR-SSP system previously
developed for RHD typing.18,19 The positive control was a 434–base-pair
(bp) PCR fragment of the human growth hormone gene. Specific primers
dau1b and daub as well as control primers were used at concentrations of 1
␮M. Amplifications were carried out with Taq (Qiagen) in a final volume of
10 ␮L. Primer sequences were dau1b, ttggccatcgtgatagctcacat; and daub,
ggagatggggcacatagacatc.
Population screen for DAU
To screen for DAU alleles among Europeans, 194 random ccDee donations
were screened for the 1136 C ⬎ T substitution by PCR-SSP. The presence
of a DAU allele was confirmed by sequencing of RHD exon 8. The DAU
type was determined by sequencing the 10 RHD exons from genomic DNA.
DAU allele frequencies were calculated based on phenotype and haplotype
frequencies previously determined for the local donor population.9
Antigen density and Rhesus index
Flow cytometric determination of antigen density and Rhesus index was
performed as described previously.16,20 The secondary antibody was goat
anti–human IgG, Fab-fragment, fluorescein isothiocyanate (FITC)–
conjugated (Jackson Immunoresearch, supplied by Dianova, Hamburg,
Germany). For the DAU-3 sample, which had a positive direct agglutination test, the background fluorescence was determined by incubating the
sample with secondary antibody only. Monoclonal anti-D antibodies were
provided by the 3rd International Workshop on Monoclonal Antibodies
against Human Red Blood Cells and Related Antigens.21 The following IgG
anti-D antibodies were used as primary antibodies: D-89/47 (workshop no.
III-1-29); HG/92 (III-30); D-90/7 (III-31); D-90/17 (III-32); D-90/12
(III-33); 17010C9 (III-36); AUB-2F7/Fiss (III-41); LOR11-2D9 (III-43);
LOR12-E2 (III-44); LOR17-6C7 (III-45); LOR17-8D3 (III-46); LOR2821D3 (III-47); LOR28-7E6 (III-48); LOR29-F7 (III-49); LORA (III-50);
LORE (III-51); NAU3-2E8 (III-53); NAU6-4D5 (III-55); NOI (III-56);
SAL17-4E8 (III-58); SAL20-12D5 (III-59); SALSA-12 (III-60); 822
(III-68); BTSN4 (III-71); BTSN6 (III-72); BTSN10 (III-73); LHM76/58
(III-74); LHM76/55 (III-75); LHM76/59 (III-76); LHM77/64 (III-77);
LHM59/19 (III-78); LHM50/2B (III-80); LHM169/80 (III-81); LHM169/81
(III-82); C205-29 (III-88); CLAS1-126 (III-89); F5S (III-90); H2D5D2F5
(III-93); RAB.B15 (III-94); BIRMA-DG3 (III-95); BIRMA-D6 (III-96);
BIRMA-D56 (III-97); P3G6 (III-101); P3AF6 (III-102); BRAD3 (III-105);
L87.1G7 (III-108); MS26 (III-112); D10 (III-114); HIRO-3 (III-117);
HIRO-4 (III-118); ID6-H8 (III-119); HIRO-7 (III-120); HIRO-8 (III-121);
HIRO-2 (III-122); D6DO2 (III-123); and MCAD-6 (III-124).
Epitope patterns
Agglutination was tested in a gel matrix test (LISS-Coombs 37°C,
DiaMed-ID Micro Typing System; DiaMed, Cressier sur Morat, Switzer-
307
land) using the following antibodies in addition to those tested in flow
cytometry: B9A4B2 (workshop no. III-1-28); HeM-92 (III-34); 175-2
(III-35); NaTH28-3C11 (III-37); NaTH87-4A5 (III-38); NaTH53-2A7
(III-39); CAZ7-4C5 (III-42); MAR-1F8 (III-52); NAU6-1G6 (III-54);
NOU (III-57); VOL-3F6 (III-61); ZIG-189 (III-62); 819 (III-69); LHM70/45
(III-79); LHM174/102 (III-83); LHM50/3.5 (III-84); LHM59/25 (III-85);
LHM59/20 (III-86); T3D2F7 (III-87); P3187 (III-98); P3F17 (III-99);
P3F20 (III-100); MS201 (III-113); HIRO-1 (III-115); HIRO-6 (III-116);
HS114 (III-134); and BS87 (III-180).
Routine D typing
Reactions of the DAU phenotypes in routine D-typing conditions were
established using commercial anti-D BS226 (Biotest, Dreieich, Germany),
BS232 (Biotest), RUM1 (Immucor, Norcross, GA), and D14E11 (Immucor) in tube technique. In addition, P3⫻61 (Diagast, Loos, France) was
tested in a gel matrix test.
Phylogeny of RHD alleles
A possible phylogenetic tree for RHD alleles was developed which was
based on the RHD coding sequence and the presence of a C or E allele.
Nucleotide substitutions, gene conversions, recombinations, and mutations
in the accompanying RHCE alleles were counted as equivalent single
events. The tree was devised manually to minimize the required number of
events. Within RHCE, only standard C alleles caused by the gene
conversion around exon 222 were counted as C positive. Because of
insufficient data, further intricacies of RHCE alleles like the 16 Trp/Cys,
245 Leu/Val, and 336 Gly/Cys polymorphisms were disregarded. Sequences from chimpanzee (Pan troglodytes Rh-like protein IIR, nucleic
acid accession number L3705023) were used for external rooting. The RHD
alleles shown in the phylogeny tree were published previously13,14,16-18,24-31
or described in this study for DAU-0 to DAU-4 and DIII type 5.
Nomenclature
The name DAU derived from “D of African origin” (in German: D
afrikanischen Ursprungs) and is pronounced like in “now.”
Results
DAU alleles
There were 5 RHD alleles identified (Table 1). These alleles constituted
a cluster, because they shared a 1136C ⬎ T single nucleotide
polymorphism (SNP) causing a T379M substitution. T379M only
was found in DAU-0 which represented the primordial allele of the
DAU allele cluster. The other 4 DAU alleles harbored one or 2
additional substitutions dispersed in the various segments of the
protein (Figure 1).
Table 1. RHD alleles described in this study
Trivial name
Allele
Nucleotide change
Effect on protein
sequence
Exon
involved
Number of
probands
Ethnic
origin
DAU-0
RHD (T379M)
1136 C ⬎ T
Thr to Met at 379
8
3
European*
DAU-1
RHD (S230I,T379M)
689 G ⬎ T
Ser to Ile at 230
5
3
African
1136 C ⬎ T
Thr to Met at 379
8
209 G ⬎ A
Arg to Gln at 70
2
1
African†
998 G ⬎ A
Ser to Asn at 333
7
1
African
1
African†
DAU-2
DAU-3
DAU-4
RHD (R70Q,S333N,T379M)
RHD (V279M,T379M)
RHD (E233K,T379M)
1136 C ⬎ T
Thr to Met at 379
8
835 G ⬎ A
Val to Met at 279
6
1136 C ⬎ T
Thr to Met at 379
8
697 G ⬎ A
Glu to Lys at 233
5
1136 C ⬎ T
Thr to Met at 379
8
*Detected in the local donor population of southwestern Germany; in addition, sequencing of the 95-012 DNA sample revealed heterozygosities for 509 T/C, 667 T/G, and
1136 C/T, suggestive of a DOL/DAU-0 genotype.
†Samples referred without explicit information on ethnic origin; African descent inferred from African names.
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BLOOD, 1 JULY 2002 䡠 VOLUME 100, NUMBER 1
WAGNER et al
Routine D-typing issues. Applying routine methods for D
typing,34 DAU-0 typed D positive, whereas DAU-1, DAU-2, and
DAU-4 were not agglutinated by most commercial monoclonal
IgM anti-D antibodies (Table 4). Hence, DAU-1, DAU-2, and
DAU-4 would usually be typed as D-negative, triggering Dnegative transfusions, which is the clinically favored management.
DIII type 5
Sequencing of DNA sample 95-013 revealed 6 nucleotide substitutions, 186G ⬎ T, 410C ⬎ T, 455A ⬎ C, 692C ⬎ G, 667T ⬎ G,
819G ⬎ A. This result predicted the homozygous or hemizygous
presence of an RHD (L62F, A137V, N152T, T201R, F223V) allele
that was dubbed DIII type 5, because of its similarity to the
molecular structure described for mditDIIIa.12
Figure 1. Schematic representation of the RhD proteins observed in the 5 DAU
phenotypes. All DAU types share a T379M substitution (black disk) that is located in
the twelfth transmembrane protein segment. DAU-1 to DAU-4 have additional
substitutions: the S230I substitution of DAU-1 and the E233Q substitution of DAU-4
are both located in exofacial loop 4; the R70Q and S333N substitutions of DAU-2
position near the border of intramembrane and intracellular protein segments; the
V279M substitution of DAU-3 locates at an intramembraneous protein segment
proximate to exofacial loop 5.
Population frequencies
DAU-1 to DAU-4 samples were referred to our laboratory because
of typing problems or, in the case of DAU-3 (sample RIR no. 38),
of an anti-D immunization. All probands carrying these alleles
were, if known, of African descent (Table 1). To determine the
possible presence of such alleles in Europeans, we screened for the
common 1136C ⬎ T SNP by PCR-SSP (Figure 2). Among 194
random samples of ccDee phenotype, 3 samples (1.5%) were
positive for the 1136C ⬎ T SNP. These samples, however, lacked
any additional SNP in the coding sequence and represented DAU-0.
The haplotype frequency of the cDe haplotype with DAU-0 was
1:3159 (95% confidence interval: 1:1170-1:11 587). The frequency
of the DAU-0 phenotype in the German population was calculated
to be 1:3843. All 4 other DAU alleles were infrequent in whites
(cumulative frequency ⬍ 1:3164, upper limit of 95% confidence
interval according to the Poisson distribution).
Phenotypes of DAU alleles
Antigen density and Rhesus index. As previously noted for weak
D samples,16 there was no simple relation of the type of substitution
(Figure 1) to the antigen density or to the Rhesus index (Table 2).
Both the antigen density and the Rhesus index of the DAU-0
phenotype were about normal, rendering it indistinguishable from
the normal antigen D–positive phenotype. However, the extracellular substitutions in the DAU-1 and DAU-4 phenotypes correlated
well with their much diminished Rhesus index, which is typical for
partial D. The DAU-2 with its low antigen density was reminiscent
of weak D because its 2 unique substitutions were located at the
inner boundary of the red cell membrane. The Rhesus index of
DAU-3 indicated its propensity to anti-D immunization, while its
antigen D density at the lower end of the normal range would
render DAU-3 carriers being transfused with D-positive blood units.
Epitope patterns. The D epitope patterns of the DAU phenotypes were distinct (Table 3). Despite DAU-1 having a much higher
antigen density than DAU-2, more anti-D antibodies agglutinated
DAU-2 than DAU-1 red blood cells. The profile of DAU-4 was
almost identical to that reported for DHK,32 alias DYO,33 which
shared the E233K substitution. DAU-0 had a normal D-positive
epitope pattern.
Phylogeny of RHD alleles in humans
Based on the molecular structure of the DAU alleles, the phylogenetic models35,36 of the RHD alleles were extended (Figure 3). The
DAU alleles, which were characterized by a cDe haplotype and a
T379M substitution, formed a separate cluster of RHD alleles.
There were 2 other allele clusters also associated with the cDe
haplotype: the weak D type 4 cluster was characterized by a
common F223V substitution. The DIVa cluster comprising DIII
type 4, DIVa, Ccdes, and DIII type 5 was characterized by a
common N152T substitution. In addition, DIII type 4 and type 5 as
well as all DIVa24 and Ccdes14,37 samples investigated by us carried
L62F and A137V. The remaining RHD alleles could be derived
from the RHD allele prevalent in Eurasians by a single event (point
mutation, gene conversion, or deletion) and formed the Eurasian
D cluster.
Discussion
We found 5 RHD alleles that shared a T379M missense mutation
and formed a previously unknown cluster. There were 4 of these
alleles that had one or 2 additional mutations and were observed in
individuals of African descent. The fifth allele, RHD (T379M), was
detected in Europeans by screening blood donors. DAU alleles may
be a major cause of antigen D variability and anti-D immunization
in patients of African descent.
Anti-D immunization in transfusion recipients and pregnant
women harboring “African” partial D is a continuing problem. For
example, 11% of anti-D in pregnancies in the Cape Town area,
South Africa, occurred in D-positive women.10 Current D-typing
strategies are tuned to detect partial D phenotypes that are typical
Figure 2. Specific detection of the T379M substitution by PCR-SSP. A PCR-SSP
was devised that amplified a specific 140-bp product from the aberrant RHD exon 8 in
all DAU alleles tested (lane 1: DAU-0; lane 2: DAU-1; lane 3: DAU-2; lane 4: DAU-3;
lane 5: DAU-4). In a normal D-positive sample (lane 6), only the 434-bp control
product deriving from the human growth hormone gene is amplified.
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BLOOD, 1 JULY 2002 䡠 VOLUME 100, NUMBER 1
DAU ALLELE CLUSTER
309
Table 2. Antigen D density and Rhesus D similarity index
Percentiles of D epitopes detected
Antibodies
RH phenotype
Antigen
density
DAU-0
15 285
0.74
12 412
15 285
16 883
56
56
76
DAU-1
2 113
0.06
260
1 152
4 091
55
41
10
373
0.17
276
373
1 626
55
55
2
10 879
0.11
2 542
9 289
23 850
44
39
54
DAU-2‡
DAU-3
DAU-4
ccDee sample
Rhesus
index
10%
50%
90%
Tested
⬎ Cutoff*
% of
reference†
1 909
0.01
55
358
5 595
56
25
9
20 166
0.74
17 188
20 166
23 302
55
55
NA§
*Number of monoclonal anti-D antibodies detecting at least 1/10 of the 90th percentile of the epitope density detected by all anti-D antibodies.
†Antigen D density as percentage of control cells with ccDee phenotype.
‡The determination of Rhesus index and antigen density may be confounded by the very low overall epitope densities.
§NA indicates not applicable because sample is reference control.
for white populations.9,34 Although carriers of partial D are more
frequent in African populations,25 more than 25 partial D alleles are
predominantly observed in Europeans and to date only 5 partial D
alleles were typical for Africans.12,16,24-26 Thus, the identification of
the DAU cluster increased the number of “African” partial D
considerably. Because anti-D immunization may occur in carriers
of DAU alleles, our molecular characterization is instrumental for
evaluating the clinical relevance in transfusion recipients.
Table 3. Reactivity patterns of antibodies with DAU samples
Pattern*
Anti-D tested†
DAU-phenotype‡
1-9
1-37
DAU-0
DAU-1
DAU-2
DAU-4
1
1
82, 123
IgG
IgM
⫹
⫺
⫹
⫺
1
2
79
⫹
⫺
⫹
⫺
83
⫹
⫺
⫺
⫺
2
3
44, 51
⫹
⫹
⫹
⫺
2
4
48
⫹
⫺
⫺
⫺
3
5
43, 49, 75
⫹
⫹
⫹
⫹
4
6
45
⫹
⫹
⫹
⫹
5
7
⫹
⫺
⫺
⫺
5
10
88, 89
⫹
⫹
⫹
⫹
41
⫹
⫺
⫹
⫺
⫹
⫺
⫺
⫺
42, 116
52
5
11
69
⫹
⫺
⫺
⫺
6/7
12
102
⫹
⫺
⫹
⫹
46
⫹
⫺
⫹
⫺
113
⫹
⫺
⫺
⫹
35
⫹
⫺
⫺
⫺
6/7
13
29, 36, 47, 90, 93, 105
⫹
⫹
⫹
⫹
6/7
15/16
31, 32, 71, 80,
⫹
⫹
⫹
⫹
56, 58, 81, 95, 97, 114
⫹
⫹
⫹
⫺
108
⫹
⫺
⫹
⫹
59
⫹
⫺
⫹
⫺
37
⫹
⫺
⫺
⫹
28, 34, 39, 98, 99, 115, 134
⫹
⫺
⫺
⫺
30
⫹
⫹
⫹
⫹
119
⫹
⫹
⫺
⫹
⫹
⫺
⫺
⫺
33
⫹
⫹
⫹
⫹
94
⫹
⫹
⫹
⫺
122
⫹
⫺
⫹
⫺
61
⫹
⫺
⫺
⫺
6/7
6/7
17
18
84, 85, 86, 87, 100
6/7
20/21
8
22
74, 78
⫹
⫹
⫹
⫹
9
23
77, 96, 101, 112, 118, 120, 121
⫹
⫹
⫹
⫹
97
⫹
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫹
⫺
⫺
⫺
N/A
31
N/A
32
N/A
33
N/A
34
50
⫹
⫺
⫹
⫺
N/A
35
60
⫹
⫺
⫹
⫺
N/A
36
55, 72, 76
⫹
⫹
⫹
⫹
53
⫹
⫺
⫹
⫺
68, 73, 117, 124, 180
⫹
⫹
⫹
⫹
N/A
37
54, 57
62
38
⫹ indicates a positive result, ⫺ a negative result.
*Pattern as described previously by Lomas et al42 (1-9) and Scott11 (1-37), who did not differentiate epitopes 15 from 16 and epitopes 20 from 2111Tab1.
†Antibody numbers as defined in “Materials and methods.” Reactivity was tested in a gel matrix test.
‡DAU-3 was not tested because of a positive direct antiglobulin test (DAT).
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Table 4. DAU phenotypes in routine D typing
IgM anti-D monoclonal antibody
BS226
BS232
RUM1
D14E11
P3X61
DAU-0
Phenotype
Positive
Positive
Positive
Positive
Positive
DAU-1
Negative
Negative
Negative
Negative
Negative
DAU-2
Negative
Negative
Negative
Negative
Negative
DAU-4
Positive
Positive
Negative
Negative
Negative
DAU-3 could not be tested because of a positive DAT.
All of the more than 50 known aberrant RHD alleles expressed
variant D antigens.6,36 Unexpectedly, DAU-0 encoded a normal
phenotype despite its intramembraneous T379M substitution.
DAU-0 may be the first example of a host of alleles harboring an
amino acid substitution that does not affect their antigen D. The
other 4 DAU phenotypes, however, had a low Rhesus index and
qualify as partial D. The partial D phenotype was most obvious for
DAU-3, in which an anti-D immunization was documented. If such
immunizations were frequently occurring in populations with
African admixture, the specific detection of the involved DAU
alleles might be warranted.
A phylogeny model for the RH haplotypes was originally
presented by Carritt et al35 in 1997 which explained the mechanisms shaping RH heterogeneity in Eurasian populations. More
recently, these haplotypes were recognized to represent just one
branch separated from 2 different clusters of RHD alleles that are
primarily observed in African populations.36 Alleles of the DAU
cluster added to this diversity and represented a third “African”
cluster (Figure 3). Each of these 3 “African” allele clusters was
characterized by a specific amino acid substitution relative to the
“Eurasian” RHD allele: (1) T379M in the DAU cluster, (2) F223V
in the weak D type 4 cluster, which included RHD⌿, DOL, and
many alleles sharing F223V and T201R, and (3) N152T in the DIVa
cluster, which included DIII type 4, DIVa, and Ccdes.
Recently, Rh-related proteins, including RhAG, have been
shown to transport ammonia.38 It is tempting to speculate that
amino acid substitutions located in transmembraneous Rh protein
segments, like T379M in DAU, F223V and T201R in weak D type
4, and L245V and G336C in Ccdes, may affect the function of the
Rh protein. Even a substitution that does not alter the D antigen,
like T379M in DAU-0, may still be functionally effective. Malaria
and other blood-borne diseases endemic in Africa may favor
functional and antigenic variability, as exemplified by glucose-6phosphate dehydrogenase deficiency39 and lack of Duffy protein expression in red cells,40 respectively. Similar processes
might confer evolutionary advantages to carriers of aberrant
RH alleles.
The RHD alleles of the 3 “African” clusters generally occurred
in a cDe haplotype, which indicated that the cE and Ce alleles
of RHCE evolved in the “Eurasian” branch after its divergence
from the other branches. However, haplotypes of the “Eurasian”
cluster represented a sizeable fraction of haplotypes extant in
Africans. For example, the frequency of antigen E–encoding
“Eurasian” haplotypes is 9.01% among Barotse in Zambia.41 In
contrast, even the most frequent alleles of the “African” clusters are
very rare among Europeans. This was shown for DAU-0 in the
present study (population frequency of 1:3159) and previously
Figure 3. Phylogeny of RHD in humans. A phylogenetic tree of RHD is shown for most “African” alleles and
representative “Eurasian” alleles. There are 4 main clusters that may be discerned. The DIVa cluster encompasses the DIVa, DIII type 4, and Ccdes alleles. Most
samples harboring these alleles share 3 characteristic
amino acids (62F, 137V, 152T) that are ancestral, because they are also observed in chimpanzee RH (Pan
troglodytes Rh-like protein IIR). The weak D type 4
cluster encompasses DAR, DOL, and RHD⌿, too. For
this cluster, the RHD (F223V) allele is postulated36 but
has yet to be shown extant. DIII type 5, a new RHD allele
resembling DIIIa, evolved by a recombination between
alleles of the DIVa and the weak D type 4 clusters. For the
DAU cluster, its primitive type DAU-0 has been found and
was shown to be the most frequent DAU allele in
Europeans. All enumerated alleles occurred in a cDe
haplotype and were predominantly observed in Africans.
In contrast, most other RHD alleles were typical for
Eurasians, derived from standard Eurasian RHD by a
single event, occurred in a CDe or cDE haplotype, and
formed the Eurasian D cluster. The tree was mainly
based on RHD allelic variability, and dismisses the
largely unknown RHCE variability beyond the C and E
polymorphism. For each evolutionary step, the event
is indicated; the depicted distances of the alleles
are arbitrary.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 JULY 2002 䡠 VOLUME 100, NUMBER 1
DAU ALLELE CLUSTER
determined for weak D type 4 (1:15 000).17 The knowledge of RH
phylogeny is of practical importance because it defines the
framework for determining the clinically relevant RH alleles in any
population.
In populations without African admixture, including whites,
Asians, Arabs, and probably American Indians, partial D phenotypes are likely to be rare and to derive from the limited and
serologically well-characterized set of alleles of the Eurasian D
cluster. For these populations, the current D-typing strategies
applied in Europe9 appear to be appropriate and sufficient. Typing
strategies for African populations and those with African admixture
may take account of the various frequently occurring alleles of the
“African” clusters. Several of these alleles characterized by multiple dispersed amino acid substitutions are difficult to discern by
311
serologic means and may in the future warrant genotyping approaches for detection in patients and donors.
Acknowledgments
We are greatly indebted to all contributors of the 3rd International
Workshop on Monoclonal Antibodies against Human Red Blood
Cells and Related Antigens in Nantes, France, 1996, who provided
most other monoclonal anti-D antibodies. We thank John J. Moulds
and Joann M. Moulds, Philadelphia, PA, for rare DNA samples
from the SCARF Exchange program. We acknowledge the expert
technical assistance of Marianne Lotsch, Anita Hacker, Sabine
Kaiser, and Sabine Zahn.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2002 100: 306-311
doi:10.1182/blood-2002-01-0320 originally published online
April 17, 2002
The DAU allele cluster of the RHDgene
Franz F. Wagner, Birgit Ladewig, Katharina S. Angert, Guido A. Heymann, Nicole I. Eicher and Willy
A. Flegel
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