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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-01-0229.
TRANSFUSION MEDICINE
From the Centre National de Référence des
Groupes Sanguins (CNRGS) and Institut National de la Transfusion
Sanguine (INTS); the Etablissement Français du Sang, La
Réunion; and Institut National de la Santé et de la
Recherche Médicale (INSERM) U76, Paris, France; and
the Centre de la Drépanocytose, Hôpital Henri Mondor,
Créteil; France.
The molecular backgrounds of variants encountered in Afro-Caribbean
black individuals and associated with the production of clinically
significant antibodies against high-incidence antigens (anti-RH18,
anti-RH34) and against Rhe epitopes were determined. We showed that
RH: The RH blood group is the most polymorphic and
immunogenic blood group. The RH locus is composed of 2 highly
homologous genes: the RHD gene, encoding the D polypeptide;
and the RHCE gene, encoding C or c together with either E or
e polypeptides.1-3 Besides the 5 major antigens (D,
C, E, c, e), more than 50 Rh antigens, identified by the corresponding
antibodies, are described. They are schematically divided into low- and
high-incidence antigens.4
Some rare Rh phenotypes are found exclusively in the black population:
the RH: Besides RH: In this work, we studied the serological reactivity and the molecular
background of RBCs from Rhe-positive individuals of Afro-Caribbean
origin producing anti-RH18, anti-RH34, anti-e, anti-ce, and anti-Ce. We
demonstrate the clinical significance of the anti-RH18 for 2 patients
who encountered a lethal outcome following incompatible transfusions
and evaluated the incidence of these rare alleles in a population of
SCD patients. We also describe variants with a decreased expression of
Rhe antigen and for which the risk of immunization against the lacking
e epitopes is unknown, making transfusion counseling difficult.
Samples
Samples 1 to 12 were obtained from unrelated individuals producing
either an antibody reacting with all RBCs expressing the RhCE
polypeptides, or allo-anti-e, anti-ce, and anti-Ce antibodies. Cord
blood was available from newborns of patients 2, 3, and 7. Individuals
7, 8, and 10 were SCD patients. Patients 7 and 8 died after
incompatible transfusions. DNA from the parents of individual 8 was
used for molecular analysis. Samples from siblings and parents of
individual 1 also were available for study.
Samples 13 to 17 exhibited a depressed Rhe antigen that was not
correlated to any known alteration. There was no antibody in the serum.
Blood samples of 146 SCD patients were randomly collected by the Center
of Sickle Cell Disease in Créteil, France. Patients were from the
West Indies and west and central Africa.
Control RBCs were used: normal Rh RBCs and known variant RBCs
[Rhnull, D- -, ceMO, RN,
ce(C48)]5,18-21 were from the Centre National de
Référence des Groupes Sanguins (CNRGS), and RH: Serological analysis
Titers were evaluated for all sera against papain-treated RBCs of different phenotypes (ddccee, DCCee, DccEE) by IgG IAT. Direct antiglobulin test (DAT) and reactivity of each serum against RBCs from the antibody maker (auto-control) were performed to distinguish alloantibodies from autoantibodies. For some samples, autoadsorption studies confirmed that the antibodies were alloantibodies (data not shown). D, C, E, c, and e status of all erythrocytes was established with routine reagents. Rhe antigen reactivity was further analyzed with separate clones (monoclonal antibodies [MoAbs]) from Serologicals (Livingston, United Kingdom): IgM (MS16, MS21, MS63, MS62, MS69) and IgG (MS70). IgG was tested using the IgG IAT on a gel matrix (DiaMed). IgMs were tested on a neutral gel matrix from the same manufacturer. Expression of low-incidence antigens was evaluated with human sera: a serum from the CNRGS containing both anti-RH10 (V) and anti-RH20 (VS) and referred to in this study as anti-RH10/20, and a serum from the CNRGS containing only anti-RH32. Anti-RH50 (FPTT) was obtained by adsorption-elution from Mol serum.22 The high-incidence RH46 antigen was tested with a polyclonal antibody
from the CNRGS. An anti-RH19 was obtained as described by Shapiro, by
adsorbing the serum of an immunized RH: Compatibility testing between the sera and RBCs of immunized patients was performed for individuals 3 and 5. The reactivity of serum 3 also was tested after adsorption-elution on RBCs from sample 5. Sera 1, 11, and 12 were tested against RBCs 9. ABO incompatibility, other antibodies, or the small number of samples did not allow compatibility testing for the other samples. For weak D samples, phenotyping for partial D was performed with selected MoAbs known to discriminate categories of partial D. MoAbs were obtained through the Third and Fourth Workshops on MoAbs (Nantes, 1996; Paris, 2001; France). cDNA sequence analysis Reticulocyte RNAs were prepared from 50-mL whole blood as previously described.23 RNA was reverse transcribed using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Uppsala, Sweden). RHCE and RHD cDNA products were amplified by polymerase chain reaction (PCR) with the Klentaq polymerase (Clontech, Palo Alto, CA), using, respectively, primer sets P1-P3 and P4-P5, then reamplified using, respectively, sets P2-P3 and P2-P5 (primers and conditions in Table 1). PCR was performed in a Thermocycler (Robocycler Genomic Gradient 96, Stratagene, La Jolla, CA). PCR products were subcloned into a PCRII vector (TA cloning kit, Invitrogen, Leek, The Netherlands). Recombinant clones and/or PCR products were sequenced with nested sequencing primers (Eurogentec, Herstal, Belgium) and on both strands by the DNA Sequencing Kit (Applied Biosystems, Foster City, CA). Sequences were analyzed on an automated fluorescence-based ABI Prism 310 (Applied Biosystems). Sequences were submitted to GenBank under the accession numbers bankit 464322, 464376, 464386, 464388, 4760047, and 467051.
Genomic DNA analysis Genomic DNA was isolated from peripheral blood leukocytes with a DNA isolation kit (Wizard, Genomic DNA Purification Kit, Promega, Madison, WI).For sequence analysis, exon-specific PCR was performed on genomic DNA. Primer sequences and conditions are described in Table 1. The following primer sets were used: set P6-P7 for nonspecific exon 3; set P8-P9 for RHD exons 4 to 5; set P10-P9 for RHCE exons 4 to 5; set P11-P12 for RHCE exon 6; set P13-P12 for RHD exon 6 (set P13-P14 also was used to amplify RHCE exon 6 from individuals 1 to 8); and set P15-P16 for RHD exon 7. PCR products were subcloned or directly sequenced as described for cDNA analysis. PCR assays Allele-specific primer PCR (ASP-PCR) was designed for the detection of specific mutations in the population of SCD patients. The wild-type (indicated by *) ASP-PCR was performed in parallel to determine the homozygous or heterozygous status of the mutation. Sets P18-P17 and P18-P17* were used to detect G712 and A712*, respectively, in RHCE exon 5. Published oligonucleotides were used in the following ASP-PCR: sets P18-P19 and P18-P19* ASP-PCR detected G733 and C733*, respectively, in RHCE exon 5; sets P20-P21 and P20*-P21 ASP-PCR detected T1006 and G1006*, respectively, in RHCE exon 7; sets P22-P23 and P22-P23* ASP-PCR amplified a hybrid RHD-CE exon 3 and a RHD specific exon 3, respectively.17 Sets P24-P25 and P24*-25 ASP-PCR were designed to detect T667 and G667*, respectively, in RHCE exon 5. Sets P26-P27 and P26*-P27 ASP-PCR were designed to detect T340 and C340*, respectively, in RHCE exon 3. For all PCR assays, an internal control was added, amplifying a common sequence of the RHD and the RHCE genes (set P28-P29).
Serological analysis Rh terminology for RH18 (Hr), RH19 (hrS), RH34 (HrB), and RH31 (hrB) is as follows: RH18 and RH34 define high-incidence antigens produced by the RHCE gene. Anti-RH18 and anti-RH34 are the antibodies produced by RH: 18
and RH:34 individuals, respectively. As defined by
Shapiro,7 these antibodies can be distinguished after
adsorption of anti-RH18 and anti-RH34 on DccEE RBCs. The remaining
specificity of anti-RH18 resembles anti-e or anti-ce and is named
anti-RH19. The remaining specificity of anti-RH34 resembles anti-Ce and
is named anti-RH31.7 In this study, anti-RH19 is used as a
reagent that is negative with all RH:18 RBCs but also with some rare variants of Rhe antigen.18
We categorized blood samples of individuals 1 to 12 into 3 groups
according to the antibody specificities (Table
2) and the RBC Rh phenotypes (Table
3): group A: samples 1 to 8; group B: samples 9 and 10; group C: samples 11 and 12. Origin of immunization is
indicated in Table 2. A fourth group (group D: samples 13 to 17) had
heterogeneous Rh reactivity (Table 3). No antibody was detected in
patients in group D, but no immunization challenge could be documented.
Group A sera (samples 1 to 8) reacted with all common RBCs. Sera 1, 5, 6, 7, and 8 were negative with Rhnull and D- - RBCs, indicating antibodies recognizing common RHCE gene
products. Sera 2, 3, and 4 were negative with Rhnull RBCs
but positive with D- - RBCs, indicating that sera contained
antibodies recognizing the common RHCE gene products as well
as anti-D. After adsorption of sera on Rhe-negative RBCs (DccEE), in
all cases, the remaining antibody was an anti-e-like antibody reacting
with all normal Rhe-positive RBCs. These data met criteria defined by
Shapiro to identify anti-RH18.7 Anti-RH18 specificity was
confirmed for sera 3, 4, 5, 7, and 8 that could be tested negative with known RH: In this group, RBCs were typed Dccee. Samples 1 to 6 had a normal reactivity with anti-Rhe routine reagents and were negative with the anti-RH19 reagent, but exhibited different patterns with anti-e MoAbs and anti-RH10/20 serum. Compatibility testing between RBCs and sera from samples 3 and 5 showed that serum from sample 5 agglutinated RBCs from sample 3, whereas serum from sample 3 failed to react with RBCs from sample 5. When serum from sample 5 was adsorbed on RBCs from sample 3 and then eluted, the reactivity was anti-e. Variants 1 to 5 exhibited the specific profile for the DAR partial phenotype when tested with a panel of anti-D MoAbs (data not shown).25 This result was consistent with the anti-D found in sera 2 to 4. Group B sera (samples 9 and 10) also reacted with all RBCs tested,
except those from Rhnull. Serum 9 reacted with D- - RBCs, indicating that an anti-D was associated with the antibody against the
common products of the RHCE gene, whereas serum 10 did not react with D- -, eliminating the presence of an anti-D. When sera were
absorbed on Rhe-negative RBCs (DccEE), the remaining reactivity was
anti-Ce. As defined by Shapiro,7 these results suggest that sera 9 and 10 both contained anti-RH34 antibody. No
RH: In group C, individual 11 serum contained anti-e and anti-ce.
Antibodies reacted with RBCs from sample 9 (considered as RH: Individual 12's serum contained anti-e and anti-Ce that reacted with
RH: In group D (samples 13 to 17; Table 3), distinct serological patterns
were found. Sample 13, typed Dccee with weak e, was similar to ceMO in
terms of Rhe reactivity and RH19 reactivity (RH: cDNA sequence analysis Complete sequence of RHD and RHCE transcripts were analyzed for samples 1, 3 to 6, 9 to 12, 14, and 15. Known transcripts as well as new transcripts were identified (Table 4, Figure 1). For individual 1, 3 different transcripts were found: (1) the DAR allele,25 carrying 602C>G (Thr201Arg) in exon 4, 667T>G (Phe223Val) in exon 5, and 1025T>C (Ile342Thr) in exon 7; (2) the ceAR allele25 carrying 48G>C (Trp16Cys) in exon 1; 712A>G (Met238Val), 733C>G (Leu245Val), 787A>G (Arg263Gly), and 800T>A (Met267Lys) in exon 5 and 916A>G (Ile306Val) in exon 6; (3) the new ceEK allele, carrying 48G>C (Trp16Cys) in exon 1; 712A>G (Met238Val), 787A>G (Arg263Gly), and 800T>A (Met267Lys) in exon 5. In sample 3, we found the DAR and the ceEK alleles. In sample 4, we found the DAR and the ceAR alleles. Individuals 3 and 4 were homozygous for ceEK and ceAR, respectively, as shown by the absence of double peak when direct sequencing of PCR products was performed. In individual 6, 4 different transcripts were characterized: (1) DAR; (2) the new D(667) allele carrying 667T>G in exon 5 (Phe223Val); (3) ceEK; (4) the new ceBI allele, carrying 48G>C in exon 1 (Trp16Cys); 712A>G in exon 5 (Met238Val); 818C>T in exon 6 (Ala273Val), and 1132C>G in exon 8 (Leu378Val). For individuals 1, 3, and 4, transcript analysis could not determine if DAR was in single or double dose. Analysis of transcripts from individuals 9 and 10 showed the known (C)ces haplotype (ces stands for a ce allele that produced the RH20 antigen and is associated with the 733C>G mutation).16 As described by Faas et al,16 (C)ces is composed of 2 altered genes: (1) a hybrid D-Ce-D transcript in which exons 1 and 2, the first 3 polymorphic positions of exon 3 (361, 380, and 383), and exons 9 and 10 derived from the RHD gene. The hybrid transcript carries 4 extra nucleotide substitutions: 186G>T in exon 2 (Leu62Phe), 410C>T in exon 3 (Ala137Val), 733C>G in exon 5 (Leu245Val), and 1006G>T in exon 7 (Gly336Cys); (2) and a ces transcript carrying 733C>G in exon 5 but also 1006G>T (Gly336Cys) in exon 7. Individuals 9 and 10 were homozygous for (C)ces as shown by the absence of double peak when direct sequencing of PCR products was performed.
Transcript analysis of sample 11 identified the RN and ceMO alleles (ceMO carries C48 in exon 1 and T667 in exon 5)6,18 with a normal RHD transcript. In sample 12, a normal cE allele was associated with a new ces allele referred to as ces(340) and carrying 733C>G in exon 5 but also 340C>T in exon 3 (Arg114Trp). A new D(674) transcript carrying 674C>T in exon 5 (Ser225Phe) was also found for individual 12. Individual 14 carried a normal RHD gene, a hybrid Ce-D(4)-Ce allele characteristic of an RN haplotype, and a new ces allele referred to as ces(748) with 733C>G and 748G>A (Val250Met) in exon 5. Individual 15 carried a normal RHD gene, one RN allele, and one hybrid ce-D(4-9)-ce allele. Genomic DNA analysis The transcript analysis was confirmed on genomic DNA (except for C48 in exon 1, very frequent in black individuals).RHD and RHCE exons 4 to 5, RHCE exon 6, and RHD exon 7 were sequenced from individuals 2, 5, and 7. Individual 2 carried DAR, ceAR, and ceEK alleles, whereas individuals 5 and 7 carried DAR and ceAR alleles. Individual 8 genotype (DAR-ceAR/DAR-ceAR) was deduced from genomic analysis of the parents, both who carried a DAR allele plus a normal RHD gene and one ceAR allele plus a normal ce allele. Family study (Figure 2) of individual 1 (Rh phenotype and genomic sequencing) showed that DAR was in
single dose and also that individual 1 inherited a DAR-ceAR
haplotype from the mother and a noD-ceEK haplotype from the
father.
RHCE exon 3 and exons 4 to 5 were sequenced from samples 13, 16, and 17. Individual 13 carried a ceMO allele and one hybrid ce-D(5)-ce allele. Individual 16 (with a serological profile similar to that of individual 11) carried one RN allele associated to a ceMO allele. Individual 17 (with a serological profile similar to that of individual 12) carried a normal cE allele and a ces(340) allele. Sequencing of RHD exons 3 to 7 (in which the more frequent mutations in partial D are localized) was normal for samples 16 and 17. For sample 13, we found a normal RHD gene next to an RHD gene carrying mutations previously described in DIVa: 455A>C and 1048G>C mutations. RHD exon 2 was not sequenced, but expression of RH30 confirmed the hypothesis of a DIVa gene for individual 13 (data not shown). Screening of SCD patients for rare RH alleles Since the carriers of some rare RH alleles may develop antibodies following transfusion or pregnancy, the frequency of such alleles was estimated in a population of 146 SCD patients. For the detection of ceEK, ceAR, and ceBI alleles, the presence of the common G712 mutation was determined by a specific PCR assay. Among 6 patients carrying G712, sequencing of RHCE exons 4, 5, and 6 PCR products showed that 4 and 2 patients were found heterozygous for ceAR and ceEK, respectively. Three patients carried ceMO heterozygously, as they carried both the T667 mutation and the wild-type G667 mutation. To identify the (C)ces haplotype and the ces(340) allele, a first step was performed by screening the G733 mutation in RHCE exon 5. Among the 75 patients positive for the G733 mutation, one also carried T340 heterozygously in exon 3 [ces(340) allele], and 10 carried both T1006 and a hybrid exon 3 [(C)ces]. Among patients carrying the (C)ces haplotype, one was homozygous, as shown by absence of normal RHD exon 3, wild-type G1006 mutation, and wild-type C733 mutation. From these findings, we calculated the incidence of the rare alleles and haplotypes in our population of 146 SCD patients as follows: ceEK: 1.4%; ceAR: 2.7%; ceMO: 2%; (C)ces: 7.5%; and ces(340): 0.7%.
This report shows that a variety of RHCE alleles are present in Afro-Caribbean black individuals, among which 4 are new alleles: ceEK, ceBI, ces(340), and ces(748). Some of the alleles found in this ethnic group are associated with the loss of immunogenic epitopes, and there is a risk of immunization if exposition to normal Rh antigens occurs. Some of the antibodies produced are clinically significant. Therefore, for efficient transfusion purposes, a complete characterization of the variants is required. Serological diagnosis of these variants is difficult, especially when different combinations of altered alleles occur. The serological and molecular data described here led us to propose a procedure to detect these variants within a population of SCD patients and black blood donors, to ensure patient transfusion safety. Clinical issues must be addressed for patients homozygous for
ceEK, ceAR, ceBI,
(C)ces, ceMO, and
ces(340) or for those who are composite
heterozygous, as we showed that they can produce anti-Rh
antibodies. Individuals homozygous for ceAR within
a DAR-ceAR haplotype can produce a clinically significant
anti-RH18, as illustrated by the fatalities of individuals 7 and 8 following incompatible transfusion. In both cases, these SCD
patients were first transfused with DccEE RBCs because of the presence
of an antibody that reacted mainly to Rhe-positive RBCs, in addition to
anti-C for patient 7. As the transfusion was inefficient, a further
transfusion with DccEE blood units was decided upon, despite complete
incompatibility. Hemolysis and death occurred rapidly for both
patients. Patient 7 was a pregnant woman who received transfusions
after a cesarean delivery. The serum obtained after the last
transfusion contained anti-RH18, anti-C, anti-Fya, and
anti-S. The DAT was strongly positive (IgG and complement). The
anti-RH18 (serum titer: 256 on ddccee RBCs) was eluted from the RBCs.
The newborn typed (DCcee) was healthy despite a strongly positive DAT
(IgG) and elution of the anti-RH18. Patient 8 received transfusions for
acute anemia. After the last transfusion, the serum contained only
anti-RH18 (titer 2000 on ddccee RBCs) and was eluted from the RBCs. No
anti-D was found in the sera of the 2 patients despite their partial D
phenotype (DAR) and transfusion of D-positive RBCs. No rare RH: The other Afro-Caribbean individuals who produced anti-RH18 in group A
shared a similar Dccee phenotype but exhibited different reactivity
profiles with the anti-e MoAbs and the anti-RH10/20 serum. The
RHCE genes were as follows: ceAR/ceEK
(individuals 1 and 2), ceEK/ceEK (individual 3), and
ceEK/ceBI (individual 6). The RH haplotype was
precisely defined for individual 1, for whom the family study showed
one DAR-ceAR haplotype and one noD-ceEK haplotype
(noD stands for no RHD gene). The ceEK
allele also can cosegregate with DAR, as individual 3, who
is homozygous for ceEK, carried at least one DAR
allele. Since the 3 RH: Anti-RH18 was eluted from RBCs from the newborn of individual 2 (ceAR/ceEK), but no hemolysis was detected. No antibody was eluted from RBCs of the newborn of individual 3 (ceEK/ceEK). For the other newborns, only clinical data were available. They all were healthy. As opposed to the findings of another author,10 we did not find anti-RH18 involvement in any hemolytic disease of the newborn. Clinical issues also are to be taken into account for the other alleles described in this study, as the carriers may develop anti-Rh antibodies. Patients 9 and 10, homozygous for the (C)ces haplotype, produced an anti-RH34. Patient 9 was a pregnant woman. Despite the high titer of the antibodies (RH34 and anti-D), the newborn was healthy, but no biologic data (newborn phenotype, DAT) were available. Patient 10 was a young SCD patient who received only 3 transfusions (phenotype of blood units unknown) before production of the antibody. For this patient, it was strongly advised that no more transfusions occur, as the anti-RH34 titer was 128 on DCCee RBCs. Among the immunized patients, individual 11 carried the
ceMO allele next to a RN allele. We
showed already that ceMO encoded a weak Rhe
antigen18 and bring evidence here that it may encode a
partial Rhe antigen, as individual 11 produced anti-e and anti-ce
antibodies. We showed previously that variants homozygous for
ceMO or with a ceMO/cE genotype were typed
RH: The ces(340) allele was found in individual 12, who produced allo-anti-e and allo-anti-Ce antibodies (through
pregnancies). Her serum was not compatible with RH: The evidence that ceEK, ceAR, ceBI,
(C)ces, ceMO, and
ces(340) but also RN may
induce partial phenotypes with risk of immunization in blacks led us to
evaluate the frequency of these alleles in a population of black SCD
patients. Incidence of the corresponding rare phenotypes has been
deduced from frequency of the alleles: RH:
In group D (samples 13 to 17), patients were nonimmunized. Clinical issues have been discussed above for patients 16 and 17, as they were similar in terms of phenotype and genotype as patients 11 and 12, respectively, but also for patient 13, who carried ceMO next to a hybrid ce-D(5)-ce allele. Two other composite heterozygous individuals have been found. Individual 15 carried one RN haplotype associated to a ce-D(4-9)-ce allele, which probably encodes only a few epitopes of both c and e antigens and no RH46 antigen. It can be assumed that the absence of RH46 antigen makes this phenotype clinically significant for transfusion. Individual 14 carried a new ces(748) allele next to an RN allele. Alterations found in individual 14 have not been associated, so far, to the production of Rh antibodies. Therefore, transfusion counseling is even more complex because the risk is unknown. In conclusion, we characterized rare Rh phenotypes found in black
individuals as we determined the serological reactivity and the
molecular background of RH:
We are grateful to Martine Verdier, MD, Jérôme Babinet, MD, Danièle Vanahaeke, MD, and Lucienne Mannessier, MD (Etablissement Français du Sang, France) for referring to our laboratory the donor samples. We also acknowledge the SCARF contribution to this work.
Submitted January 25, 2002; accepted July 3, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-01-0229.
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.
Reprints: France Noizat-Pirenne, 20 rue Bouvier, 75522 Paris Cedex 11 France; e-mail: pirenne{at}ints.fr.
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STEM, a new low-frequency Rh antigen associated with the e-variant phenotypes hrS- (Rh: | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
