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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 12-18
PLENARY PAPER
The presence of an RHD pseudogene containing a 37 base
pair duplication and a nonsense mutation in Africans with the Rh
D-negative blood group phenotype
Belinda K. Singleton,
Carole A. Green,
Neil D. Avent,
Peter G. Martin,
Elizabeth Smart,
Abigail Daka,
Edwin G. Narter-Olaga,
Linda M. Hawthorne, and
Geoff Daniels
From the Bristol Institute for Transfusion Sciences, Bristol,
England; the International Blood Group Reference Laboratory, Bristol,
England; the Natal Blood Transfusion Service, Pinetown, South Africa;
the Blood Transfusion Service, Harare, Zimbabwe; the National Blood
Transfusion Service, Accra, Ghana; and the Louisiana State University
Medical Center, Shreveport, LA.
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Abstract |
Antigens of the Rh blood group system are encoded by 2 homologous
genes, RHD and RHCE, that produce 2 red cell membrane
proteins. The D-negative phenotype is considered to result, almost
invariably, from homozygosity for a complete deletion of
RHD. The basis of all PCR tests for predicting fetal D
phenotype from DNA obtained from amniocytes or maternal plasma is
detection of the presence of RHD. These tests are used in order
to ascertain the risk of hemolytic disease of the newborn. We have
identified an RHD pseudogene (RHD  ) in Rh
D-negative Africans. RHD contains a 37 base pair (bp) insert
in exon 4, which may introduce a stop codon at position 210. The insert
is a sequence duplication across the boundary of intron 3 and exon 4. RHD contains another stop codon in exon 6. The frequency of
RHD in black South Africans is approximately 0.0714. Of 82 D-negative black Africans, 66% had
RHD, 15% had the RHD-CE-D hybrid gene associated
with the VS+ V- phenotype, and only 18% completely lacked
RHD. RHD is present in about 24% of D-negative
African Americans and 17% of D-negative South Africans of mixed race.
No RHD transcript could be detected in D-negative individuals
with RHD, probably as a result of nonsense-mediated mRNA
decay. Existing PCR-based methods for predicting D phenotype from DNA
are not suitable for testing Africans or any population containing
a substantial proportion of people with African ethnicity. Consequently, we have developed a new test that detects the 37 bp
insert in exon 4 of RHD. (Blood. 2000; 95:12-18)
© 2000 by The American Society of Hematology.
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Introduction |
Rh is a highly complex red cell blood group system with
52 antigens and numerous phenotypes.1,2 The Rh antigens are
encoded by 2 closely linked homologous genes, each consisting of 10 coding exons. RHCE encodes the Cc and Ee antigens, and
RHD encodes the D antigen. There are also many uncommon fusion
genes, comprising part of RHCE and part of RHD, which
may encode abnormal D and CcEe antigens and 1 or more low- frequency Rh
antigens.3,4
The most important Rh polymorphism from the clinical aspect is the D
polymorphism. About 82%-85% of Caucasians are D-positive, the
remainder lack the D antigen. The D-negative phenotype has a frequency
ranging between 3% and 7% in Africans and <1% in the people from
the Far East.1 D-negative red cells lack the D protein, and
alloanti-D consists of antibodies to a variety of epitopes on the D
polypeptide. Colin et al5 found that the D-negative
phenotype resulted from homozygosity for a complete deletion of
RHD. Rare exceptions to this molecular basis of D-negative, in
which at least some RHD exons are present, have been described. In almost all these exceptions, the aberrant RHD belongs to a haplotype producing C antigen. Examples found in
Caucasians include RHD containing a nonsense
mutation6 or a 4- nucleotide deletion,7 and
RHD-CE-D fusion genes containing RHD exons 1 and 10 or
RHD exons 1-3 and exons 9 and 10, with the remainder of the
exons derived from RHCE.8,9 An RHD-CE-D
fusion gene, in which the 3' end of exon 3 plus exons 4-8 are
derived from RHCE, is sometimes associated with a D-negative
phenotype in people of African origin.10 In 130 D-negative
Japanese, 36 (28%) had an apparently intact RHD, 2 had at
least exon 10 of RHD, and 1 of these also had RHD exon
3.11 Despite these variants, however, it is generally
accepted that lack of RHD is the usual molecular background for
the D-negative phenotype.
The D antigen is of clinical importance because anti-D
antibodies are capable of causing severe hemolytic disease
of the fetus and newborn (HDN), resulting in hydrops fetalis and fetal
death. In those severely affected infants who are born alive, jaundice may lead to kernicterus.12 The prevalence of RhD HDN has
been greatly reduced since the 1960s by the introduction of anti-D immunoglobulin prophylaxis, which prevents D-negative mothers from
being immunized by fetal D-positive red cells as a result of
feto-maternal hemorrhage at parturition. Some D-negative women, however, still produce anti-D for a variety of reasons including undetected antepartum feto-maternal hemorrhage and failure to administer anti-D, and any subsequent D-positive fetus is at risk from
HDN. It is now common practice for anti-D immunoglobulin to be
administered antenatally as well as postnatally in an attempt to reduce
the risk of D immunization.
Discovery of the molecular basis of the D polymorphism created the
possibility of predicting fetal D phenotype from fetal DNA derived from
amniocytes obtained by amniocentesis. D phenotyping of the fetus of a
D-negative woman with anti-D provides valuable information for
predicting the risk of HDN and for subsequent management of the
pregnancy. The first tests to be devised involved a polymerase chain
reaction (PCR). The tests either determined the presence or absence of
RHD-specific sequences in exon 10 or exon 7 or recognized the
size differences between introns 4 of RHD and
RHCE.13-16 These techniques were found to be
unreliable.16 The absence of certain RHD regions in
hybrid genes encoding partial D antigens may predict a D-negative
phenotype, and the presence of some RHD regions in genes
encoding no D antigen may predict a D-positive phenotype. In order to
avoid these complications, methods where more than 1 region of
RHD is detected in a single PCR were
introduced.6,11,17
Amniocentesis is a highly invasive procedure and not without risk to
the fetus. It may also enhance the risk of maternal immunization. Methods have been described for predicting D phenotype from fetal DNA
in maternal plasma. It has been proposed that these techniques could be
used for testing all D-negative pregnant women to ascertain whether
they require administration of antenatal anti-D
immunoglobulin.18,19 Testing would reduce wastage of this
valuable resource by avoiding anti-D immunoglobulin administration to
women with a D-negative fetus.
All techniques for predicting D phenotype from DNA or mRNA are based on
the dogma that, with only very rare exceptions, the D-negative
phenotype results from the absence of all or most of the RHD
gene. Most tests, however, have been performed on people of European
origin, and the molecular basis of D polymorphism has not been
adequately investigated in other races, particularly in people of
African origin. We have tested for the presence of the RHD gene
in Africans and African Americans and found that the majority of
D-negative Africans and about a quarter of D-negative African Americans
have an inactive RHD gene with a 37 base pair (bp) insert and a
nonsense mutation; the remainder have either the RHD-CE-D gene
or a complete deletion of the RHD gene.
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Materials and methods |
Blood samples
D-negative blood samples were obtained from black African blood
donors from South Africa, Zimbabwe, and Ghana; from South African
donors of mixed race, referred to as "Coloured" in South Africa;
and from African American donors, selected on the basis of appearance,
from Shreveport, Louisiana. Blood samples from D-positive black South
Africans were derived from another study.20 All blood
samples were collected into a citrate-phosphate-dextrose (CPD)
anticoagulant. Blood samples used for screening purposes were the
by-product of blood donation and were collected according to the
approved protocol of the transfusion center involved. Whole units of
blood, for transcription analysis, were taken with informed consent.
Serological studies
All red cell samples were tested for D, C, c, E, e, G, VS, and Rh32
antigens. VS+ samples were also tested for V antigen. Serological tests
were performed by the method most suited to the particular reagent:
direct hemagglutination, with native or papain-treated cells, or
indirect hemagglutination with antihuman IgG (BPL, Elstree,
England). All D-negative red cells were tested by an antiglobulin test with 2 potent alloanti-D that are known to
react with red cells of all partial D phenotypes. An acid-elution method (Elu-kit II, Gamma Biologicals, Houston, TX) was used for adsorption/elution tests with anti-D.
Molecular analyses on genomic DNA
Genomic DNA was isolated from mononuclear cells by a standard
phenol-chloroform-isoamyl alcohol procedure or from whole blood with a
DNA purification kit (Promega, Madison, WI). PCR primers are listed in
Table 1. The method used for RHD
screening, the multiplex PCR described by Avent et al,6
detected the presence of RHD exon 10 and intron 4 and RHCE intron 4, as an internal control.
The method for screening for the 37 bp insert of RHD exon 4 was
a PCR with a sense primer (RHDIN3F) in intron 3 and an
RHD-specific antisense primer (RHD4R) in intron 4 (Table 1).
Electrophoresis of amplification products in 1.5% agarose gel revealed
a 381 bp product from wild type RHD and a 418 bp product from
RHD with the 37 bp insert. As an internal control, primers
RHEX2IN1F and RH2R amplified a 509 bp product across exon 2 of
RHD and RHCE for all samples. Screening for the
RHD exon 6 T807G nonsense mutation involved PCR with RH6F
(sense primer) and a sequence-specific antisense primer,
RHDE × 6R (Table 1).
The sequences of RHD exons 1, 3-7, and 10 were determined by
direct sequencing of PCR products. Many of the primers used for exon
amplification were described by Wagner et al21 (Table 1). Following agarose-gel electrophoresis, PCR products were cut out of the
gel, purified (QIAEX II kit; QIAGEN, Dorking, England), and sequenced (373A DNA sequencer; Applied Biosystems, Warrington, England).
Screening for the presence of RHD exons 3, 4, 5, 6, 7, and 9 was performed in separate PCRs, with pairs of primers (Table 1) in
which at least 1 had an RHD-specific sequence by the conditions employed. The test for an RHD-CE hybrid exon 3, characteristic of the D-negative, (C)ces
(r'S) complex, utilizes an
RHD-specific primer to the 5' end of exon 3 and an
RHCE-specific primer to the 3' end.20 A test
for identifying the C733G polymorphism in RHCE exon 5, which
encodes a Leu245Val substitution in the RhCcEe protein responsible for
the absence or presence of VS antigen, involved an
RHCE-specific sense primer in intron 4 and an allele-specific
antisense primer in exon 5 of RHCE.20
Transcript analysis
Erythroblasts were cultured from a D-negative black South African
with the RHD 37 bp insert. Mononuclear cells were separated from whole blood in CPD (Histopaque-1077 gradient; Sigma-Aldrich, Poole, England), and contaminating red cells were lysed in ammonium chloride. CD34+ cells were isolated (Mini-MACS columns;
Miltenyi Biotech, Bisley, England) by 2 cycles of positive
selection with anti-CD34 and immunomagnetic beads. The isolated cells
were then cultured in the presence of fetal-calf serum (N.V. HyClone,
Belgium), recombinant human (rH) erythropoietin
(Boehringer Mannheim, Frankfurt, Germany), rH
granulocyte-macrophage colony stimulating factor (R&D Systems, Abingdon, England), and rH interleukin-3 (IL-3) (R&D
Systems), as described by Malik et al.22
After 11 days, cells were harvested and analyzed for the presence of
glycophorin A (GPA) and Rh protein. This was accomplished using flow
cytometry with monoclonal antibodies BRIC 256 and BRIC 69 (IBGRL
Research Products, Bristol, England). BRIC 256 detects an epitope on
the extracellular domain of GPA, and BRIC 69 detects an extracellular
epitope common to the RHCcEe and RhD polypeptides. About 80% of the
cells expressed both Rh protein and GPA.
We isolated mRNA from 106 cells (The PolyATtract System
1000 kit; Promega). First-strand cDNA for PCR amplification was
prepared with oligo (dT16) as the primer and avian
myeloblastosis virus reverse transcriptase (Pharmacia, Milton Keynes,
England).23 cDNA was amplified with the
following primers: 5'-CTGAGGATGGTCATCAGTAA-3' (nt 457-476, exon 3 of RHCE and RHD) and
5'-GAGATCAGCCCAGCCAC-3' (nt 914-898, exon 6 of RHCE
and RHD). The 458 bp product was isolated by gel purification
and sequenced. In addition, an RHD-specific PCR was performed
with a sense primer in exon 3 (RHDEX3F, Table 1) and an antisense
primer in exon 7 (5'-ACCCAGCAAGCTGAAGTTGTAGCC-3') to give a
645 bp product with RHD cDNA. As a control, an
RHCE-specific PCR was carried out with a sense primer in exon 3 (5'-TCGGTGCTGATCTCAGCGGG-3') and an antisense primer in
exons 8 and 7 (5'-CAATCATGCCATTGCCGTTCCA-3') to give a 715 bp product with RHCE cDNA.
Reticulocyte RNA was extracted from a D-negative black South African by
isolating CD71+ cells from 10 mL of phosphate buffered
saline-washed peripheral blood using anti-CD71 magnetic beads (Dynal,
Wirral, England). Total RNA was extracted with an RNA
extraction kit (SV total RNA, Promega), and first-strand cDNA was then
synthesized. RHD transcripts were analyzed by the amplification
of 2 overlapping cDNA fragments, 1 encompassing exons 1-7 and the
other exons 7-10. PCR primers were exons 1-7, 5'-TCCCCATCATAGTCCCTCTG-3' and RHDIN7R (Table 1), and
exons 7-10, 5'-TGGTGCTTGATACCGCGGAG-3' and
5'-AGTGCATAATAAATGGTGAG-3'. PCR conditions were as
follows: denaturation at 94°C for 1 minute; annealing at
55°C for 1 minute, and extension at 72°C for 2 minutes (ExpandTM RTPCR kit; Boehringer, Lewes, Sussex,
England) for 30 cycles with 2.5 mmol/L Mg2+ (final
concentration) supplied with the kit. A final 30-minute incubation
at 72°C was performed to maximize the d(A) tailing of the PCR products, which were cloned into the pCRII vector
(Invitrogen, Groningen, The Netherlands) and sequenced on both strands.
A new multiplex PCR-based screening test for fetal D typing
A 10-primer multiplex PCR-based method has been developed for
determining the presence of RHD exon 7 and intron 4, the 37 bp
insert in RHD exon 4, and the RHCE C and
c alleles. The primers and their concentrations are shown in
Table 2. Thirty cycles of PCR were
performed at 94°C for 1 minute, 65°C
for 1 minute, and 72°C for 3 minutes 30 seconds.
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Results |
Identification of an RHD pseudogene in D-negative
Africans
An RHD gene was found in some D-negative Africans.
Sequencing of exon 4 of this apparently inactive RHD revealed a
37 bp insert (Figure 1). This insert
appears to introduce a reading frameshift and a potential
translation-termination codon at codon 210. The 37 bp insert is a
duplication of a sequence spanning the intron 3-exon 4 boundary
(Figure 1). Sequencing of exons 1, 3-7, and 10 of the inactive
RHD revealed additional changes from the common RHD
sequence. In exon 4, there is a G to A point mutation at nucleotide 609 (original position). Normally, this would not result in an amino acid
change, but the 37 bp insertion and subsequent frameshift gives rise to
an aspartic acid to asparagine substitution at codon 203 (original
reading frame) or codon 216 (new reading frame) (Figure 1). In exon 5, 3 missense mutations (G654C, T667G, C674T) were detected, encoding
Met218Ile, Phe223Val, and Ser225Phe substitutions. In exon 6, a T807G
transversion changes the codon for Tyr269 (TAT) to a
translation-termination codon (TAG). PCR with a sequence-specific primer revealed that all individuals with the RHD 37 bp insert also had the T807G nonsense mutation. The inactive RHD gene
will be referred to here as RHD.
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| Fig 1.
Nucleotide and encoded amino acid sequence for the
3' end of intron 3 and the 5' end of exon 4 of RHD
and RHD.
The region of the 37 bp duplication is underlined (single and double).
The putative exon sequence derived from intron 3 is double-underlined.
* Indicates a translation-termination codon.
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Two samples from D+ C- c+ E- e+ Africans were sequenced as controls.
Both had the wild type sequence in exon 4, except 1 had a C602G
transversion encoding Thr201Arg. One had the wild type sequence in exon
5, the other had the Phe223Val change. Neither of the African control
samples had the T807G nonsense mutation, but 1 had a G819A silent
mutation (codon for Ala273). Thr201Arg, Phe223Val, and G819A are
associated with the weak D phenotype called weak D type 4 by Wagner et
al.21
Analsis of Rh mRNA transcripts from Africans with
RHD
mRNA transcripts were isolated from D-negative donors with
RHD using either whole blood or erythroblasts cultured from
CD34+ cells. An RHD-specific PCR gave a 645 bp
product with cDNA prepared from a D-positive control, but the
PCR did not give a product with cDNA prepared from a
D-negative with RHD. An RHCE-specific PCR gave 715 bp products with all samples. We did not find a clone with an
RHD insert, and an RHD sequence was not
detected. These results suggest that there is no transcript from
RHD.
Screening D-negative donors for RHD exon 10 and
intron 4 and for the RHD 37 bp
insert
All D-negative donors were tested by the multiplex PCR method
designed to determine the presence of RHD exon 10 and intron 4. Intron 4 of RHCE was always detected as a control for
successful amplification. Three patterns of reaction were
apparent: presence of both RHD regions; absence
of both RHD regions; and presence of RHD exon 10, but
absence of RHD intron 4. Frequencies of these patterns are
shown in Table 3 for each of the
populations tested. Of the 82 D-negative black Africans tested (from
South Africa, Zimbabwe, and Ghana), 67% had both regions of
RHD, 15% had RHD exon 10, and only 18% lacked
RHD. The frequencies of the 3 genotypes were different in
African Americans and mixed-race South Africans, with a substantially
higher proportion having neither region of RHD (Table 3).
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Table 3.
Results of testing for presence or absence of
RHD intron 4 and exon 10 and for the RHD exon 4 37 bp
insert
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Of 75 D-negative samples from people of African origin with RHD
(including African Americans and mixed-race South Africans), 74 had
the exon 4 37 bp insert (Table 3, Figure
2). None of these 74 had normal RHD
exon 4. These individuals must be homozygous for RHD,
hemizygous for RHD, or heterozygous for RHD and
the grossly abnormal RHD-CE-Ds gene
associated with VS+ V- phenotype. Further investigation of the
Ghanaian sample with RHD exon 4 (without a 37 bp
insert), intron 4, and exon 10 has not been possible.
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| Fig 2.
Agarose gel showing results of PCR screening test for the
37 bp RHD exon 4 insert with samples from black South
Africans.
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PCR-based tests on selected donors with sequence-specific primers
revealed that those donors with RHD exon 10 and intron 4 also
had RHD exons 3, 4, 5, 6, 7, and 9, suggesting the presence of
grossly intact RHD. Red cells of all donors with RHD
exon 10, but without RHD intron 4, were C+ and VS+; almost all
were also V-. In addition to RHD exon 10, donors of this type
had RHD exon 9 and a hybrid exon 3 comprising a 5' end
derived from RHD and a 3' end derived from
RHCE. This suggests that these donors have the RHD-CE-D
gene associated with the (C)ces
(r'S) complex.10 This
gene will be referred to as RHD-CE-Ds.
Selected donors with neither exon 10 nor intron 4 of RHD also lacked RHD exons 3, 4, 5, 6, 7, and 9, suggesting homozygosity for an RHD deletion.
Fifty-two of the 74 D-negative red cell samples with RHD (and
with the 37 bp insert) in the 5 populations were D- C- c+ E- e+ VS- (probable genotype ce/ce). Of the remaining
22, 11 were D- C- c+ E- e+ VS+ V+, and 11 were D- C+ c+ E- e+
VS+ V-. These 22 were all heterozygous for C733G in exon 5 of RHCE and so had probable genotypes of
ce/ces and
ce/(C)ces, respectively.20
Screening for RHD in random black South
African blood donors
Ninety-eight random samples from black South Africans, consisting of
95 D-positive samples and 3 D-negative samples, were tested for
RHD. Of the 98, 14 had RHD. This gives an
approximate RHD gene frequency of 0.0714. This figure is
based on the assumption that 1 of the D-negative donors is
hemizygous, not homozygous, for RHD. The other randomly
selected D-negative donor with RHD is VS+ V- with the
RHD-CE-Ds gene, and the donor is assumed to
be heterozygous for RHD.
Serological confirmation that the donors tested were
D-negative
The donors used in this study were random D-negatives, as determined
by the routine methods of the relevant transfusion services. In
addition, their red cells gave negative results, by an antiglobulin test, with 2 alloanti-D reagents that react with all known partial D
antigens and weak D antigens (with the exception of the extremely weak form of D known as Del). Several red cell samples from
donors with RHD were tested with alloanti-D by an adsorption
and elution technique that would be expected to detect a very weak D
antigen. No anti-D could be detected in the eluate.
Of 5 African women who had produced anti-D, 4 had RHD, and
the other had no RHD. This provides further evidence that these Africans with RHD are truly D-negative. One of the anti-D
antibodies from a D-negative Ghanaian woman with RHD caused
HDN, which required exchange transfusion.
A pregnant black English woman with anti-D had RHD. Testing
of DNA derived from amniocytes obtained by amniocentesis showed that
her fetus had RHD plus RHD with a normal exon 4. The
fetus is, therefore, almost certainly D-positive and at risk from HDN.
A new PCR-based test for predicting D phenotype from genomic
DNA
We have developed a new multiplex PCR-based test for detecting
RHD, RHD, and the C and c alleles of
RHCE. Examples of the results are shown in Figure
3. RHD and RHD give rise
to 498 bp and 535 bp products, respectively, from RHD intron 4 exon 4. Because of the small difference in size between these 2 products, primers specific for the 37 bp insert in RHD were
incorporated, and they gave rise to a 250 bp product when
RHD was present. Both genes produce a 95 bp product from
RHD exon 7. In addition, primers recognizing a
C-specific sequence in RHCE intron 2 amplify a 320 bp
product when a C allele is present; primers recognizing a
c-specific sequence in RHCE exon 2 amplify a 177 bp
product when a c allele is present.
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| Fig 3.
Polyacrylamide gel showing results with multiplex PCR
method for predicting D and C/c phenotype and for detecting the
presence of RHD.
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Discussion |
The D-negative phenotype of the Rh blood group system is generally
considered to result, almost invariably, from homozygosity for a
deletion of the D gene, RHD. Although this is true in
Caucasians, it is certainly not the case in Africans. We have found
that about two-thirds of D-negative Africans have an inactive
RHD gene. This pseudogene (RHD) has a 37 bp insert
in exon 4, which may introduce a reading frameshift and premature
termination of translation and a translation stop codon in exon 6. Of
the remaining one-third of African D-negative donors, about half appear
to be homozygous for an RHD deletion and about half have the
RHD-CE-Ds hybrid gene characteristic of the
(C)ces haplotype that produces c, VS, and
abnormal C and e, but no D. In D-negative African Americans and South
African people of mixed race (Coloured), the same 3 genetic backgrounds
are present, but 24% of African Americans and 17% of South African
donors of mixed race have RHD, and 54% of African Americans
and 81% of South African donors of mixed race have no RHD.
The most common D-negative Rh haplotype in Africans is RHD
with the ce allele of RHCE, although RHD
might also be occasionally associated with
ces. The 37 bp insert in exon 4 of
RHD is a duplication of a sequence spanning the boundary of
intron 3 and exon 4. This insert may introduce a reading frame shift
and a translation stop codon at position 210. However, the duplication
introduces another potential splice site at the 3' end of the
inserted intronic sequence in exon 4 (shown double-underlined in Figure
1). If splicing occurred at this second splice site, the sequence of
exon 4 would not be changed and could be translated normally. No RhD
protein would be expected, however, because another stop codon is
present in exon 6 of the gene. RHD mRNA was not detected in
D-negative individuals with RHD, despite the presence of
RHCE transcripts. mRNA transcripts containing premature
termination codons are often rapidly degraded as a result of
nonsense-mediated mRNA decay.24
The initial report of the molecular basis for the Rh D-negative
phenotype5 was followed by the publication of a number of
PCR-based tests for predicting D phenotype from genomic
DNA.25 Such techniques have proved valuable for predicting
the D phenotype of fetuses of women with anti-D antibodies in order to
assist in assessing the likelihood of HDN and in managing the
pregnancy. Fetal DNA is usually derived from amniocytes obtained by
amniocentesis or from chorionic villus samples. Antenatal
administration of prophylactic anti-D immunoglobulin at around 28 weeks
of gestation to all pregnant D-negative women is an enormous drain on
stocks of anti-D immunoglobulin. About 40% of the fetuses of
D-negative Caucasian women would be expected to be D-negative, yet
these women still receive antenatal immunoglobulin prophylaxis. Less invasive PCR-based tests using maternal plasma are currently being developed, and they may make it possible to test the fetal D phenotype of all D-negative pregnant women.18,19,26
All existing methods for predicting D phenotype from genomic DNA or
mRNA are based on detecting the presence or absence of various regions
of RHD and are unsuitable for testing African populations or
any populations containing a substantial proportion of people with
African ethnicity. We have devised a new test that detects the presence
of RHD. This test incorporates a primer that specifically
detects the presence of the 37 bp insert in RHD. In
addition, the presence or absence of exon 7 and intron 4 of RHD
and RHD are detected. This test would give either an unusual
result or a D-positive result with genes encoding partial D antigens.
The multiplex PCR test also includes primers that determine the
presence of C and c alleles of RHCE. This will
be particularly informative because the D-negative phenotype is not usually associated with C expression. Most inactive RHD genes, with the exception of RHD, are in cis
with a C allele of RHCE. Typing the fetus for the C
allele is also valuable because anti-G may be responsible for
HDN.27
The Rh proteins exist in the red cell membrane as part of a complex of
proteins and glycoproteins. In most people this complex comprises
RhCcEe polypeptide, RhD polypeptide, and the Rh-associated glycoprotein. This complex may also be associated, in the membrane, with LW glycoprotein and CD47 and possibly with Duffy glycoprotein and
glycophorin B.28 In D-negatives, the RhD
polypeptide is not present in the Rh membrane complex. There are 3 common genetic mechanisms responsible for the D-negative
phenotype: deletion of RHD, a pseudogene RHD containing
a 37 bp insert and 1 or 2 stop codons, and a hybrid
RHD-CE-Ds gene that probably produces an
abnormal C antigen but does not produce a D antigen (Figure
4). Functions of the Rh proteins
are not known, so it is difficult to speculate on the
evolutionary significance of the D-negative phenotype and,
therefore, why 3 different mechanisms have evolved to create this
phenotype. D-negative fetuses have an advantage in D-negative
mothers as they are not destroyed by maternal anti-D. Consequently, HDN
may provide a selection pressure in favor of inactive RHD,
which has driven the evolution of the D polymorphism with different
genetic backgrounds in different ethnic groups. It is unlikely,
however, that HDN alone has provided the selective pressure responsible
for the high prevalence of D negativity.
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| Fig 4.
Representation of the genomic organization of the
D-positive haplotype and 3 D-negative haplotypes in Africans.
 indicates RHCE exon;  , RHD exon.
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Acknowledgment |
We are grateful to the Western Province Blood Transfusion Service for
obtaining blood samples from mixed-race donors in South Africa.
| |
Footnotes |
Submitted June 3, 1999; accepted September 2, 1999.
Supported by a grant from DiaMed AG, Cressier-sur-Morat, Switzerland.
Reprints: Geoff Daniels, Bristol Institute for Transfusion
Sciences, Southmead Road, Bristol BS10 5ND, England; e-mail: geoff.daniels{at}nbs.nhs.uk.
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.
| |
References |
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Daniels GL.
Human Blood Groups. Oxford, England: Blackwell Science; 1995.
2.
Issitt PD, Anstee DJ.
Applied Blood Group Serology. 4th ed. Durham, NC: Montgomery Scientific Publications; 1998.
3.
Huang CH.
Molecular insights into the Rh protein family and associated antigens.
Curr Opinions Hemat.
1997;4:94-103[Medline]
[Order article via Infotrieve].
4.
Cartron JP, Bailly P, Le Van Kim C.
Insights into the structure and function of membrane polypeptides carrying blood group antigens.
Vox Sang.
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Abstract
Introduction
