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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 375-387
REVIEW ARTICLE
The Rh blood group system: a review
Neil D. Avent and
Marion E. Reid
From the Department of Biological and Biomedical Sciences,
University of the West of England, Bristol, England, and the New York
Blood Center, New York, NY.
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Abstract |
The Rh blood group system is one of the most polymorphic and
immunogenic systems known in humans. In the past decade, intense investigation has yielded considerable knowledge of the molecular background of this system. The genes encoding 2 distinct Rh proteins that carry C or c together with either E or e antigens, and the D
antigen, have been cloned, and the molecular bases of many of the
antigens and of the phenotypes have been determined. A related protein,
the Rh glycoprotein is essential for assembly of the Rh protein complex
in the erythrocyte membrane and for expression of Rh antigens. The
purpose of this review is to provide an overview of several aspects of
the Rh blood group system, including the confusing terminology,
progress in molecular understanding, and how this developing knowledge
can be used in the clinical setting. Extensive documentation is
provided to enable the interested reader to obtain further information.
(Blood. 2000;95:375-387)
© 2000 by The American Society of Hematology.
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Introduction |
The Rh blood group system was first described 60 years
ago. A woman had a severe transfusion reaction when she was transfused with blood from her husband following delivery of a stillborn child
with erythroblastosis fetalis. Her serum agglutinated red blood cells
(RBCs) from her husband and from 80% of Caucasian ABO-compatible
donors.1 The following year, Landsteiner and Wiener2 found that sera from rabbits (and later guinea
pigs) immunized with RBCs from Macaca mulatta (Macacus
rhesus in the original paper) agglutinated 85% of human RBC
samples. Initially, it was thought that the animal and human antibodies
identified a common factor, Rh, on the surface of rhesus and
human RBCs. It was soon realized that this was not the
case.3 Therefore, the original terms (Rh factor and
anti-Rh) coined by Landsteiner and Wiener, although being misnomers,
have continued in common usage. The heteroantibody was renamed anti-LW
(after Landsteiner and Wiener), and the human alloantibody was renamed
anti-D.4
The Rh blood group system is the most polymorphic of the human blood
groups, consisting of at least 45 independent antigens and, next to
ABO, is the most clinically significant in transfusion medicine. The
ability to clone complementary DNA (cDNA) and sequence genes encoding
the Rh proteins has led to an understanding of the molecular bases
associated with some of the Rh antigens. Serologic detection of
polymorphic blood group antigens and of phenotypes provides a valuable
source of appropriate blood samples for study at the molecular
level. This review summarizes our present understanding of the
complexities of Rh blood group expression and how this knowledge
impacts on clinical situations that arise through Rh blood group incompatibility.
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Terminology |
Several nomenclatures have been used to describe antigens, proteins,
and genes in the Rh system. Throughout this review, we will use
traditional terminology recommended by the International Society of
Blood Transfusion (ISBT) committee for terminology of blood group
antigens.5 The numeric portion of the ISBT terminology for
Rh antigens is based on the nomenclature described by Rosenfield et
al.6-9 RH30 and RH50 have been used to
describe genes encoding Rh proteins (Rh30) and Rh glycoprotein (Rh50),
respectively, where the numbers relate to the apparent molecular mass
of the proteins on a SDS-polyacrylamide gel. Because Rh30 and Rh50 also
relate to Goa and FPTT antigens, respectively, we will use
RH as a generic term for genes encoding either the RhD protein
or the RhCcEe (also known as RhCE) protein and use RHAG for the
gene encoding the Rh-associated glycoprotein (RhAG). The common Rh
antigens: D, C or c, and E or e, were originally written in
alphabetical order (CDE) but later, when it was recognized that C and E
antigens are inherited en bloc, the order was changed to DCE. Although d antigen, which was thought to be antithetical to D, does not exist,
the letter "d" is used to indicate the D-negative phenotype. The
most frequently occurring forms of RHCE and RHD encode
8 haplotypes: Dce, dce, DCe, dCe, DcE, dcE, DCE, and dCE, known in
short, respectively, as R0, r, R1, r',
R2, r , Rz, and ry. The
uppercase "R" is used when the D antigen is expressed, lowercase
"r" when it is not. This notation has practical value in
transfusion medicine as a means to communicate the Rh phenotype of a
patient or donor. Rare deletion phentoypes use dashes in the notation
to indicate a lack of antithetical antigens; eg,
Dc . RBCs lack E and e antigens, and D
RBCs lack C, c, E, and e antigens. RBCs with the Rhnull phenotype do not express any of the Rh antigens.
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The Rh complex |
Biochemical studies, protein purification, and amino acid sequencing
of Rh and RhAG are beyond the scope of this article but have been
reviewed elsewhere.10-16
The Rh proteins carry Rh antigens but are only expressed on the
erythrocyte surface if RhAG is also present. The amino acid sequence
homology (approximately 40%) of the Rh and RhAG proteins indicates an
ancestral relationship, and collectively they are referred to as the
"Rh protein family." Hydrophobicity profiles, immunochemical
analyses, and data obtained through site-directed mutagenesis imply
that Rh and RhAG proteins have 12 transmembrane spans with both the
N-terminus and C-terminus oriented to the cytoplasm (Figure
1).17-24
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| Fig 1.
Model of topology for RhAG, RhCE, and RhD.
RhAG (Mr 50 000) consists of 409 amino acids and
is encoded by RHAG on chromosome 6p11-p21.1. RhCE and RhD
(Mr 30 000) are predicted to have a similar
topology and are encoded by RHCE and RHD, which are
adjacent on chromosome 1p34-p36. The domain of the RhD protein encoded
by each exon is depicted by numbered boxes, which represent the start
and finish of each exon. Of the D-specific amino acids, 8 are on the
exofacial surface (yellow ovals), and 24 are predicted to reside in the
transmembrane and cytoplasmic domains (black ovals). Red ovals
represent amino acids that are critical for C/c (Ser103Pro) and E/e
(Pro226Ala) antigens; purple ovals represent Ser103 and Ala226 on RhD.
The zigzag lines represent the Cys-Leu-Pro motifs that are probably
involved in the palmitoylation sites. The N-glycan on the first loop of
RhAG is indicated by the branched structure of red circles.
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"Rh accessory proteins" is a collective term for other
glycoproteins that are associated with the Rh protein family as defined by their absence or deficiency from Rhnull RBCs (see below
and Table 1).25 Together, the
association of the Rh protein family and the Rh accessory proteins is
called the "Rh complex."
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Table 1.
Proteins in the Rh Complex in Normal RBC Membranes That
May Be Absent or Reduced in Rhnull RBC Membranes
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Rh protein family
The complex of the Rh protein family is estimated by density
ultracentrifugation to be 170 000 daltons26 and to consist of a tetramer with 2 RhAG molecules and 2 RhCcEe or RhD protein molecules stabilized by both N-terminal and C-terminal domain associations.18,19,26,27 The mode of association of this core complex with Rh-accessory proteins, some of which interact directly with the membrane skeleton, remains undefined.
RhD and RhCcEe proteins.
The RhD protein expresses the D antigen, while the RhCcEe protein
carries either C or c antigens (involving the second extracellular loop) together with E or e antigens (involving the fourth extracellular loop) on the same protein.19,28-30 Characteristics of the
RhD protein (synonyms: Rh30, Rh30B, Rh30D, D30, Rh30
polypeptide [30 kd], RhXIII, Rh13) and of the RhCcEe protein
(synonyms: Rh30, Rh30A, Rh30C [RhCE], Rh30 polypeptide [32 kd],
RhIXb cDNA, [RhcE], Rh21 cDNA [RhcE], R6A32, Rhce,
RhCe, RhcE, RhCE, CcEe) are summarized in Table 1 and depicted in
Figure 1. Analysis of the primary amino acid sequences (inferred from
cDNAs) shows that the first 41 N-terminal amino acids of RhD and RhCE/e
are identical.20,31-33 and that RhD differs from the
common forms of RhCE by only 30 to 35 amino acids along the
entire protein.20,28,31-33,35,36 Despite the high degree of
homology, the various RhCcEe proteins do not express any D
epitopes, and RhD protein does not express C or e antigens.
The Rh proteins are thought to interact with the membrane bilayer by
palmitoylation,26,37 where acylated palmitic acid residues
are attached to cysteine side chains. These cysteine residues are
predicted to be at the boundary of the cytosol and lipid bilayer
(Figure 1). Cys-Leu-Pro motifs, flanked by charged amino acids (2 are
on RhD and 3 are on RhCcEe) are likely candidates although 2 other
cysteine residues (315 and 316) may be alternative sites.20,26 This interaction may explain why alteration of membrane cholesterol concentration affects the accessibility of the D
antigen.38 The ability to label Rh proteins with
3H-palmitate26,37 indicates that a reversible
coenzyme A and adenosine triphosphate (ATP)-dependent
acylation-deacylation cycle occurs in mature RBC membranes, which is of
unknown significance.
Rh-associated glycoprotein.
The characteristics of RhAG (synonyms: Rh50, Rh glycoprotein Rh50A,
D50, MB-2D10 protein, R6A45, GP50, GP50A) are
summarized in Table 1 and depicted in Figure 1. One of the 2 potential
N-glycan sites is glycosylated. A third site is predicted to be
cytoplasmic and, therefore, not accessible for
glycosylation.17,39 The N-glycan carries ABH
antigens,12 but RhAG is not known to possess a
protein-based blood group polymorphism. Based on the predicted amino
acid sequence, RhAG shares 39.2% and 38.5% amino acid sequence identity with, respectively, the Rhce and RhD
proteins.17,20,31-33
Expression of Rh proteins and RhAG during erythropoiesis.
Rh antigens appear early during erythropoietic differentiation. Anti-D
binds to approximately 3% of BFU-E (burst-forming unit, erythroid),
68% of CFU-E (colony-forming unit, erythrocyte), and to all of the
more mature erythroid cells. However, the binding of anti-D to
proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and normoblasts was, respectively, 25%, 50%, 66%, and
75% compared with mature RBCs.33 RhAG protein is
detectable on CD34 progenitors isolated from cord blood, after culture
for 3 to 5 days, while RhCcEe appears after 5 to 7 days, and RhD
appears after 9 to 11 days of culture.40 In the fetus, Rh
antigens are expressed on RBCs from the 6-week conceptus.41
Possible function of Rh protein family.
The function of the Rh complex remains unclear. Rh proteins have
approximately 20% homology to the methylamine permease (Mep) transporters and ammonium transporters (Amt) in yeast, bacteria, and
simple plants.42 This family of transporters are uniporters that have evolved to concentrate ammonium salts from the surrounding environment. Higher animals use more complex nitrogen sources, and they
eliminate toxic ammonium via the urea cycle and transport it in the
form of glutamine and alanine. The role of the Rh complex as a
dedicated ammonium transporter is unlikely, but the complex could
cotransport ammonium with other cations; however, further study is
needed. Matassi et al43 report that RHAG shares greatest homology to MEP2, which behaves as an ammonium sensor and transporter in yeast.44 Furthermore, the presence of RhAG
homologs in Caenorhabditis elegans and Geodia cydonium infers
they have roles that are not confined to RBCs.
Rh accessory proteins
The blood group antigens associated with the Rh family of
proteins, the gene location, their molecular mass, number of copies per
RBC, and selected accession numbers are summarized in Table 1.
LW glycoprotein.
The LW glycoprotein (synonym: ICAM-4) is a single pass (type I)
membrane protein with homology to intercellular adhesion molecules (ICAMs), which are ligands for  2 integrins. LW has been reported to
be a ligand for the integrin LFA-1 (synonyms:  L2,
CD11a/CD18).45
While the LW glycoprotein is absent from RBCs of LW(ab)
and Rhnull individuals, expression of Rh antigens is normal
on LW(ab) RBCs. LW antigens are more abundant on
D-positive RBCs than on D-negative RBCs from adults, which led
to the initial interpretation that anti-D and anti-LW were the
same.46,47 It is possible that the LW glycoprotein
interacts preferentially with RhD as compared with RhCcEe; however, the
nature of such an interaction awaits definition. Interestingly, LW
antigens are expressed equally well on D-positive and D-negative RBCs
from fetuses and newborns and more strongly than on RBCs from
adults.48,49
Integrin-associated protein.
Isoform 2 of integrin-associated protein (IAP; synonyms: CD47, BRIC 125 glycoprotein, AgOAB, 1D8) is present in the RBC membrane, where it is
predicted to pass through the RBC membrane 5 times and have 6 potential
N-glycan motifs.50,51 IAP carries ABH antigens but no known
protein-based blood group antigen. IAP occurs as different isoforms in
various tissues where it binds to 3 integrins.50,52 The
IAP isoform in RBCs does not bind integrins but does bind
thrombospondin53 and may be involved in calcium transport,
possibly as a gated channel.54 While the amount of IAP is
reduced in RBC membranes from Rhnull and D
people, it is present in normal levels in lymphoblastoid cell lines
from these people.55-57
Glycophorin B.
Glycophorin B (GPB; synonyms: Ss sialoglycoprotein [SGP],  -SGP,
PAS-3) is a type I membrane glycoprotein that has several O-glycans but
no N-glycan. The Rh complex appears to aid, but is not essential for,
the correct insertion of GPB in RBC membranes. In
SsU RBCs that lack GPB, the Rh proteins are
apparently normal, but RhAG has increased glycosylation, suggesting a
slower migration through the endoplasmic reticulum and Golgi
apparatus.39 An interaction of GPB and RhAG may be required
for full expression of the U antigen58,59 and, to a lesser
extent, S and s antigens (Table 2).
Further, the known ability of GPB to form heterodimers with glycophorin
A (GPA) may bridge the Rh complex with the band 3/GPA
complex, forming a large unit in the RBC membrane.
Fy glycoprotein.
A possible association between the Fy glycoprotein (synonyms:
Duffy, DARC) and the Rh complex is indicated by the Fy5 antigen, which
is absent from Fy(ab) and Rhnull
RBCs.60 However, Rhnull RBCs have normal
Fya, Fyb, Fy3, and Fy6 antigens, and
Fy(ab) RBCs have normal Rh antigens. The specific
requirements for expression of the Fy5 antigen remain unknown.
Band 3.
Band 3 (synonyms: AE1, anion exchanger, solute carrier family 4 anion
exchanger member 1) is a glycosylated protein that is predicted to pass
through the RBC membrane 12 or 14 times and is the major anion
transporter.61,62 Unlike the proteins described above, band
3 is apparently normal in Rhnull RBCs; however, based on
hemagglutination studies, antigens on Rh proteins and on band 3 are
decreased in South-East Asian ovalocytic RBCs.63 The
molecular defect associated with South-East Asian ovalocytic RBCs
results from a deletion of a segment of DNA encoding 9 amino acids
located at the boundary of the cytoplasmic N-terminal domain and
membrane domain of band 3.64-67 Recent evidence that the
expression of endogenous and retrovirally expressed Rh antigens were
enhanced following transduction of K562 cells with band 3 suggests that
band 3 and Rh proteins associate in erythroid cells.68
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Structure of RH and RHAG genes |
The genes encoding RhD and RhCcEe are highly homologous, while the
gene encoding RhAG is almost 40% homologous. The 3 genes are each
composed of 10 exons; RHCE and RHD in tandem encompass 69 kilobases (kb) of DNA (Figure 2), while
RHAG encompasses 32 kb. The RhD protein is encoded by
RHD (synonyms: RH30, RH30B, RH30D, RHXIII, RH13); the
RhCcEe protein is encoded by RHCE (synonyms: RH30, RH30A,
RH30C (RHCE), RHIXB, RH21); and the RHAG
glycoprotein is encoded by RHAG (synonyms: RH50,
RH50A).
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| Fig 2.
RHCE-RHD gene organization.
Organization of exons (E) and introns of RHCE and RHD
is shown. Exon sizes are indicated above the line as number of
nucleotides, and intron sizes are indicated below the line. A
c-specific short tandem repeat (STR) is located in intron 2 of
RHCE and another in intron 6 of RHD. The information
used to compile this figure came from the database accession numbers
given in the figure.
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The intron-exon boundaries of the RHCE gene21 and
the complete nucleotide sequences of some RHCE and RHD
introns have been described.69-74 Selected GenBank
accession numbers for cDNA are listed in Table 1, and those for introns
are given in Figure 2. The intron-exon structure of the RHAG
gene also has been defined and is remarkably similar to RHCE
and RHD.22,75-77 Several mutations in RHAG
have been described that cause the regulator type of Rh deficiency
syndrome (see below).
Evolution of the RH gene family
It was thought that Rh proteins were erythroid-specific and confined
to higher vertebrates. However, the discovery of sequence-related RHAG homologs in invertebrates suggests otherwise. These
homologs have been found as 2 different RHAG-like genes in
Caenorhabditis elegans (a nematode; GenBank accession U64 847
and Z74 026)78 and as at least 1 in Geodia
cydonium (a marine sponge; GenBank accession
Y12 397).79 These genes are predicted to encode proteins with remarkably high (respectively, 46%, 39%, and 47%) amino acid identity to human RhAG. The highest homology is within the
transmembrane domains, suggesting a conserved functional role for the
RhAG protein family. Recent work has also demonstrated the presence of
RHAG counterparts in mouse (GenBank accession AF065 395;
AF057 524-27, AF012 430), macaque (AF058 917) and RH
orthologs in chimpanzee (L37 048-50), gorilla (L37 052, L37 053),
orangutan (AF012 425), gibbon (L37 051), baboon (AF012 426), macaque
(L37 054 570 343), New World monkeys (AF012 427-9, AF021 845) and
cow (U59 270).77,80,81
As the invertebrate homologs more strongly resemble human RHAG
than human RH, it is likely that an ancient gene duplication event, estimated to have occurred 250 million to 346 million years ago,
caused divergence of RH from RHAG.75
Subsequent to the gene duplication, RH and RHAG
underwent different evolutionary pathways.43 A second gene
duplication event, being the origin of the human RHCE and
RHD genes, occurred much later in a primate ancestor 5 million
to 12 million years ago. Based on the evolutionary rates of
RHAG and RH genes in different species, it appears that RHAG evolved some 2.6 times slower than RH,
suggesting that RhAG has a more important functional role than Rh
proteins.22,73,77,82
The order of the Rh genes on chromosome 1 is probably
RHCE-RHD.83 (After submission of this
manuscript, a paper was published that questions the order of the Rh
genes on chromosome 1. Sequencing the intergenic region of the two RH
genes suggests that the order may in fact be
RHD-RHCE.211) The primordial human Rh haplotype is
believed to be Dce, and the other 7 common Rh haplotypes most likely
each arose from this gene complex by a single genetic event. The
predominant Caucasian D-negative haplotype (dce) probably arose by a
deletion of the RHD gene84 from the
RHce/RHD gene complex, whereas the DCe haplotype (the most
common D-positive haplotype in Caucasians) arose by gene
conversion with exon 1 and 2 from RHD replacing the same exons
of RHce. The remaining haplotypes arose through point mutations
(eg, the E/e polymorphism) or rare recombination events of the various
haplotypes.83
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Molecular basis of Rh antigens |
Since the first descriptions of Rh
cDNAs,20,31-33 much effort has been expended in
differentiating the molecular bases underlying the antigens of the Rh
system. The different genetic mechanisms that give rise to the major
clinically relevant Rh antigens are described within this section.
These include gene deletion (D-negative phenotype); gene
conversion (C/c polymorphism); antithetical missense mutations (E/e);
and other missense mutations (VS and V). The RH genes appear to
be a source of massive diversity, and combinations of these different
genetic rearrangements abound among all racial groups. We have selected
examples of Rh polymorphisms that are of clinical significance and have
been defined at the molecular level. Figures 3-6 detail the molecular
basis of published examples of Rh variants. Enthusiastic readers
requiring more data regarding Rh variants should consult
references.16,25,85,86
D antigen
The D antigen is a collection of conformation-dependent
epitopes along the entire RhD protein. While in most D-negative
Caucasians there is a deletion of RHD, in other populations
(notably Japanese and African blacks) the D-negative phenotype is
associated with a grossly normal RHD, and the reason for the
lack of expression of the D antigen is not known (except in Africans;
see later). Figure 3 depicts the molecular
basis of some D-negative phenotypes.70,74,84,87-89
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| Fig 3.
Rearrangements at the Rh locus giving rise to D-negative
and Rh deletion haplotypes.
The structures of the RH locus (located at 1p34-36) that has
been defined in various D-negative phenotypes and rare Rh antigen
deletion phenotypes are depicted. Each RH gene is represented
as 10 boxes, each box representing an exon, where RHCE is shown
as gray, RHD as black. Crosshatched boxes depict silent RHD
alleles (eg, RHD Q41 X ). The positions of
microinsertions or deletions of DNA that cause or are indicative of
D-negative phenotypes are shown as triangles. Because exon 8 of
RHCE and RHD are of identical sequence and their
origins are not possible to define, they are shaded according to the
gene loci position. The significance of these rearrangements, and their
impact in particular on molecular genotyping, is discussed within the
text. Sources for the information in this figure: DCW (AM)184; DCW (Glo)185;
D (LM)186; D
(Gou)186; D (SH)72;
D and Evans+ D (JD)187; Evans+
D (AT)69; Evans+ D
(Dav)186; Dc (Bol)186; Dc
(LZ)188; Ce70,74,149; r"G
(SF)105,140; (Ce)Ce184; (C)ceS VS+
(Donor 1077) Vr'S 29;98;104; Amorph
Rhnull (BK/DR)176,189; Amorph
Rhnull (DAA)177; CML.179
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People whose RBCs have an altered form of RhD protein (partial D) may
make alloanti-D. Such RBCs, depending on which D epitopes are altered,
are agglutinated by a proportion of anti-D reagents. Figure
4 summarizes the molecular changes that are
associated with partial D antigens.
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| Fig 4.
Molecular bases of partial D phenotypes.
The different alleles of RHD that cause partial D phenotypes
are depicted here graphically. The genetic structure of each partial D
RHD 10-exon gene is shown, as are associated low-incidence
antigen(s) and the estimated gene frequency. RHD (ie, wild
type) exons are shown as black boxes; where they have been replaced by
RHCE equivalents is shown as white boxes. Missense mutations
are indicated within the exon where they occur. We have used the
original Roman numeral notation (ie, DII to
DVII) and the more recent 3-letter notation (eg, DFR, DBT)
for the different D categories. Where partial D phenotypes have
identical (or very similar) serologic profiles but different genetic
backgrounds, we have adapted the classification originally described by
Mouro et al190 to describe different DVI
phenotypes (types I and II). Thus, we depict DIV types I to
IV, DV types I to VI; DVI types I to III, and
DFR types I and II. We use DVa to indicate the presence of
the DW antigen and DV to represent samples that
have a similar molecular background but that either do not express the
DW antigen or have not been tested for this antigen.
Few = 1 to 10 examples. Many = 11 or more examples as
indicated by serological testing. DVII is common (1 in 900)
in the German population.191 Under "Ethnic Origin,"
B = black, C = Caucasian, and J = Japanese. The information used
for the point mutations used in this figure are as follows: D+G
106; DNU and DII 192;
DHMi 92; DVII 193;
DVa 71,194 DFW195;
DHR.196 The information used for the rearrangements in this
figure was obtained from the following: DIIIa
197; DIIIb 106; DIIIc
198; DIVa type I 194; DIVb
type II194; DIVb type III92;
DIVb type IV195; DVa type
I194; DVa type II194;
DV type III102; DVa type
IV156; DV type V156; DV
type VI156; DVI type I199,200;
DVI type II190; DVI type
III71; DFR type I194; DFR type
II201; DBT type I202; DBT type
II203; ARRO-1204;
DCS205.
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Analysis of genes encoding the weak D phenotype (previously known as
DU) showed a normal RHD sequence but a severely
reduced expression of RHD messenger RNA (mRNA), suggesting a
defect at the level of transcription or pre-mRNA
processing.70,90 More recently, RHD transcripts
from people whose RBCs express a weak form of the D antigen were found
to have missense mutation(s) within the predicted transmembrane or
cytoplasmic domains of RhD (Figure 5).91,92 RBCs with some weak D
antigens may not be agglutinated by all monoclonal anti-D. People whose
RBCs express this type of weak D antigen do not make anti-D.
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| Fig 5.
Molecular basis of weak D phenotypes.
This figure depicts missense mutations in the RHD gene
associated with weak D phenotypes.92,153 The locations of
these mutations on the predicted topology of the RhD protein are
depicted as checkered ovals; the D-specific amino acids are shown as
open ovals. Most of the missense mutations are located within
nonconserved membrane spans (gray) and cytoplasmic regions. Regions of
conserved Rh protein family sequence are indicated as black
rectangles.
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CcEe antigens
The RhC/c and RhE/e polymorphisms are caused by nucleotide
substitutions in RHCE.28,93 While 6 nucleotide
substitutions causing 4 amino acid changes (Cys16Trp; Ile60Leu;
Ser68Asn; Ser103Pro) are associated with the C to c polymorphism
(Figure 6), only the Ser103Pro polymorphism
strictly correlates with C/c antigenicity.94 However,
Pro102 appears to be a critical part of the c antigen.95,96 The presence of 2 adjacent proline residues (102 and 103) would be
expected to form a relatively rigid structure that is resistant to
changes in nearby amino acid residues and may explain the relatively low number of c variants as compared with other Rh antigens. It has
been generally accepted that a single nucleotide substitution is
sufficient for expression of the E to e polymorphism (Pro226Ala). However, variants of the e antigen have been described,97
showing that the requirements for expression of the e antigen are not fully understood. For example, the presence of Val at residue 245 instead of Leu,29,98,99 a deletion of Arg at amino acid residue 229,100 or the presence of Cys (instead of Trp) at
amino acid residue 16 101 affects the expression of the e
antigen. The molecular basis of partial E antigens (categories I, II,
and III, and DV type III) has been determined and are shown
in Figures 4 and 6.102,103
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| Fig 6.
Changes in RHCE.
Amino acids encoded by RHCE are shown by gray boxes, and those
encoded by exons from RHD are shown by black boxes. The amino
acids associated with E/e and C/c antigens28,93 are shown
at the top, and single amino acid changes associated with variant forms
of RhCE are shown in the middle. The bottom portion of the figure shows
rearrangements of the RHCE and associated antigens.
Polymorphism that does not have a 100% correlation with expression of
c and C antigens. The information depicted in this figure
was obtained from the following sources: Point
Mutations35,98,99,104,185,206; Rearrangements DHAR
207; rG208;
 N90; E
Cat II and III185; and Variant
e100,101.
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VS and V antigens
The imultaneous presence of 2 low-incidence antigens (VS
and V) occurs with a single amino acid substitution (Leu245Val) that is
predicted to be within a transmembrane domain (Figure
6).104 The V antigen (in the presence of VS) is not
expressed when another transmembrane amino acid substitution is present
at residue 336 (Gly Cys) (Figure 3).98,104 The
membrane location of residues 245 and 336 illustrate that Rh antigen
expression is affected significantly by nonexofacial amino acids and
suggests that the prediction of some Rh epitope expression cannot be
based solely on externalized residues.
G antigen
RhD and RhC proteins carry the G antigen, which is
associated with residues in the second extracellular loop encoded by
exon 2.105,106 In DVIcE (DVI type
I) RBCs, which are predicted to have a hybrid RhD (exons 1-3)-RHCE
(exons 4 and 5)-RhD (exons 6-10) protein, the G antigen was not
detected by 1 of 2 monoclonal anti-G.107 Thus, it would appear that the G antigen is conformation-dependent and not solely dependent on the second external domain of RhC(e/E) or RhD proteins.
Rh variants
Rh-variant phenotypes arise through at least 4 mechanisms: (1) rearrangements of the tandemly arranged
RHCE and/or RHD (Figures 3, 4, and 6); (2) point
mutation(s) in either gene causing amino acid change(s), with
subsequent loss of some epitopes and/or expression of a low-incidence
antigen; (3) nonsense mutations, and (4) deletion of nucleotides
causing a frameshift and premature stop codon. There is some
evidence that there are recombination hot spots due to Alu IV
elements in the RH genes.72,108
Rearranged RHCE genes, associated with D and
D, ablate expression of C, c, E, and e antigens, while the D
antigen expression is exalted to the extent that immunoglobulin (Ig) G
anti-D can agglutinate the RBCs in saline.109 It is now
clear that this increased expression is due to a large insert of
RHD into RHCE in tandem with a RHD gene (Figure
3). In DCW and Dc phenotypes, the region of
the RHCE gene encoding the E/e antigen is replaced by an RhD
equivalent with loss of E/e antigenicity (Figure 3). While these appear
as RHCE deletion phenotypes at the protein level, they are encoded by
rearranged RHCE and thus are RHCE-depleted.
Low-incidence antigens associated with partial D antigens.
Low-incidence antigens associated with some partial D phenotypes are
due to novel structures on the RBC surface and are useful markers for
the identification of the partial D (Figure 4).110 A few
low-incidence antigens are associated with more than 1 molecular background, eg, the FPTT (Rh50) antigen is expressed on DFR,
RoHar, and DIVa(C)
phenotype RBCs; the Rh32 antigen is expressed on DBT and 
The Evans antigen is expressed on D, and a weak form of Evans
is present on DIVb RBCs. RBCs expressing Rh23 or Rh32
possess an antigen (Rh23/32) present on both phenotypes.111
In these cases, it is likely that external surfaces of the altered
proteins have localized similarities.
RhD epitope mapping
Partial D antigens were classically identified by testing the RBCs
with well-characterized polyclonal anti-D made by other people with
partial D phenotypes and, also, by testing the patient's anti-D
against RBCs with known partial D antigens. Human monoclonal antibodies
are now being used to classify partial D antigens in terms of expressed
epitopes. The original model consisted of 8- and 9-epitope D
(epD)112,113 but has been expanded to consist of 16,110 30,114 and 37 epitopes.115 When using monoclonal anti-D to define D
epitopes, it is important to perform the testing at the correct pH,
temperature, ionic strength, and antibody concentration; to use RBCs
that have been stored appropriately; and to include controls.110,114 Most D epitopes are conformation-dependent
and may be influenced by other proteins and lipids in the RBC membrane. Indeed, only 1 monoclonal anti-D has been described that reacts strongly by immunoblotting, implying that the epitope it recognizes may
be linear.116
Predictions as to the location of various D epitopes have been based on
which epitopes are absent from RBCs with a partial D for which the
molecular basis is known.117,118 However, the absence of a
D epitope may not always be a direct result of the change in molecular
structure, and the presence of Rh proteins encoded by cis and
trans genes can effect the binding of certain monoclonal
anti-D. For example, R0Har and DVa
do not have any RHD exons in common, but they have overlapping reactivity with monoclonal antibody anti-D, demonstrating
the difficulty of correctly defining the molecular basis of D epitopes. A model proposed by Chang and Siegel119 suggests that
anti-D are essentially similar in that they react with the basic
footprint of the D protein. In this model, a change in the
footprint, induced by an amino acid substitution or a hybrid protein,
is predicted to interfere with binding of anti-D. The involvement of
certain residues for binding of monoclonal anti-D has been investigated by site-directed mutagenesis (SDM), which showed that incorporation of
3 D-specific amino acids (Asp350, Gly353, and Ala354) into an
RhcE construct generated some epD3 and epD9
expression,120,121 and incorporation of 9 exofacial D
residues generated epitopes that were recognized by 40 of 50 monoclonal anti-D.122 These data argue that at least some D
epitopes are spatially distinct. However, SDM studies have not yet
addressed the impact of amino acids located within the lipid bilayer or
on the cytoplasmic side of the RBC membrane. Accurate determination of
the contact points of interaction(s) between antigen and antibody
awaits crystallographic data.
| |
Clinical aspects |
Clinical complications result from RBC destruction due to the
interaction of an alloantibody with RBCs carrying the corresponding antigen. The D antigen is highly immunogenic and induces an immune response in 80% of D-negative persons when transfused with 200 mL of
D-positive blood.123 For this reason, in most countries D
typing is performed routinely on every blood donor and transfusion recipient so that D-negative patients receive D-negative RBC products. Consequently, clinical complications due to mismatched transfusions are
infrequent. In contrast, despite the use of immunosuppressive therapy
with anti-D immunoglobulin prophylaxis, D alloimmunization in pregnancy
still occurs.
Alloantibodies
Alloantibodies that recognize Rh antigens are usually IgG and react
by the indirect antiglobulin test. This is a test in which RBCs are incubated in serum, washed to remove free immunoglobulin, and
then exposed to an antiglobulin reagent that is formulated to detect
the cell-bound IgG. The end point of the test is hemagglutination. Alloantibodies in the Rh blood group system can cause destruction of
transfused RBCs and of fetal RBCs in hemolytic disease of the newborn
(HDN). People whose RBCs have a rare deleted Rh phenotype (Rhnull, D) readily make alloantibodies.
People with the Rhnull phenotype of amorph or regulator
type can make anti-Rh29 (an antibody to "total" Rh), anti-Rh17
(an antibody to the RhCc/Ee protein), anti-D, anti-C, or a mixture of
specificities. Transfusion of a patient with anti-Rh29 is a problem
because only Rhnull RBCs will be compatible: People with
the Rhnull phenotype are not only rare, but they have a
compensated hemolytic anemia and are therefore unlikely to meet
predonation criteria.124 People with either the
D, D, DCW, or Dc
phenotype make anti-Rh17. A patient with anti-Rh17 also represents a
transfusion conundrum because only RBCs with a deleted phenotype will
be compatible.
Autoantibodies
An autoantibody is one that reacts with an antigen on the antibody
maker's own RBCs. Autoantibodies that react optimally at 37°C are
present in the serum of about 80% of patients with warm autoimmune
hemolytic anemia.125 Although most of these autoantibodies appear to be "nonspecific," many have specificity to an Rh
antigen, notably to e. Rarely is the specificity clear-cut, but the
autoantibody commonly reacts more weakly with antigen-negative RBCs
than with antigen-positive RBCs; however, in these cases, transfused
antigen-negative RBCs only rarely survive better than antigen-positive
RBCs.123 Autoantibodies in serum from patients with warm
autoimmune hemolytic anemia may be nonreactive only with
Rhnull and D RBCs (autoanti-Rh17), or only
with Rhnull RBCs (autoanti-Rh29). In such cases,
antigen-negative blood will not be available, and transfusion with
antigen-positive RBCs should not be withheld if the patient has
life-threatening anemia.125,126 In most cases, the
autoantibody is equally reactive with all RBCs tested whether from
donors or antibody detection/identification kits. Thus, in the clinical
setting, it is important to perform tests to ensure that the patient's
serum does not have potentially clinically significant alloantibodies
underlying the autoantibodies before transfusing incompatible RBCs.
Detection and identification of such antibodies is required to prevent
transfusion reactions but is beyond the scope of this review. For more
information, see a current textbook on laboratory aspects of
transfusion medicine.125-127
Partial and weak D phenotypes
As described earlier, people whose RBCs have a weak D phenotype
(quantitative D variant) do not make anti-D, whereas people whose RBCs
have a partial D phenotype (qualitative D variant with or without
weakening of the D antigen) can make alloanti-D. This presents a
different problem depending on whether the person is a donor or a
patient. For donors, detection of weak and partial D antigens would
eliminate the possibility of immunization should such blood be
transfused to a true D-negative patient. However, historical data show
that weakly expressed D antigens are most unlikely to be immunogenic.
For transfusion recipients and pregnant women, it is common practice to
use a procedure that will classify RBCs with a weak D antigen or some
partial D antigens as D-negative. Thus, blood donated from such a
person should be labeled as D-positive (Rh-positive), but the same
person should be listed as D-negative (Rh-negative) when they are
recipients in need of transfusion. The transfusion recipient will
receive D-negative RBC products, and the pregnant woman will receive
prophylactic Rh immunoglobulin, thereby preventing alloimmunization.
Although a pregnant woman with the DVI partial phenotype
may make alloanti-D, this has rarely caused a clinical problem to a
D-positive fetus.128 In the autologous transfusion setting
(in which the person is both the donor and patient), the above policy
can cause confusion because partial D RBCs may be typed as D-positive
at the donor center but D-negative at the hospital. In practice, it is
difficult to distinguish RBCs with the DVI phenotype from
other weak D; however, this now can be accomplished by immunoblotting
with the unique anti-D, LOR-15C9.129
Rh and hemolytic disease of the newborn
HDN is caused by maternal IgG antibody crossing the placenta,
binding to the fetal antigen-positive RBCs, and initiating their destruction, thereby causing anemia. Prior to the use of prophylactic Rh immunoglobulin, anti-D frequently caused fetal brain damage due to
increased levels of bilirubin (kernicterus) and even death (erythroblastosis fetalis). Despite the widespread use of prophylactic Rh immunoglobulin, a significant number of women still become alloimmunized during pregnancy for a variety of reasons, including nonadministration of Rh immunoglobulin, unrecognized miscarriage, leakage of fetal RBCs into the maternal circulation late in pregnancy, and exposure to maternal D-positive RBCs while in utero (grandmother effect).130
The D antigen accounts for about 50% of cases of maternal
alloimmunization; the remainder is due mainly to incompatibility to K,
c, C/G, E, and Fya antigens and to low incidence antigens
in Rh, MNS, and Diego blood group systems.131-133
Therefore, feto-maternal Rh incompatibility still represents the major
cause of HDN. Ultraviolet phototherapy and, occasionally, exchange
transfusion or even intrauterine transfusion may be required. Invasive
procedures are used as a "last option" in monitoring and treating
HDN, because they may cause further leakage of fetal RBCs into the
maternal circulation. Measures such as determination of the optical
density of amniotic fluid and functional assays (ADCC, MMA,
chemiluminescence) have been used to monitor at-risk pregnancies and to
identify cases requiring treatment (for review, see
Zupanska134). With current molecular technology, it is
possible to perform analyses on fetally derived DNA to predict the
blood type of a fetus.
Interestingly, a fetus that is ABO incompatible with the maternal
anti-A/B is less likely to have HDN due to anti-D, presumably due to
rapid removal of the ABO-incompatible RBCs by the naturally occurring
anti-A/B. Also, because the number of copies of the D antigen per RBC
is higher in the R2 haplotype (range, 14 000 to
16 000) than in the R1 haplotype (range, 9000 to 14 600), fetuses whose RBCs are R2 have
more severe anemia than their R1
counterparts.123 There is also evidence that male fetuses
have more severe HDN than female fetuses.135
Rh immunoglobulin prophylaxis in the prevention of HDN.
The immunologic mechanism responsible for preventing production of
maternal anti-D following administration of prophylactic Rh
immunoglobulin may be due, at least in part, to antigen blocking and
central inhibition of the immune response by negative feedback in the
spleen (for review, see Bowman130). In some instances, recommendations have been made to administer anti-D to partial D-phenotype mothers (eg, DVI and DBT phenotypes) following
the birth of D-positive babies.110,136 In Europe, anti-D
reagents are selected to deliberately type DVI mothers as
D-negative and, thus, ensure that such mothers would automatically
receive prophylactic Rh immunoglobulin therapy following pregnancy.
Prophylactic Rh immunoglobulin preparations for this purpose are
usually for intramuscular injection. However, products approved also
for intravascular injection are used for the treatment of idiopathic
thrombocytopenia.137,138
Legislative restrictions for immunization of D-negative volunteers with
accredited D-positive RBCs are partly responsible for the declining
source of polyclonal anti-D for prophylaxis. Thus, clinica |
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Abstract
Introduction