Download Rearrangements of the Blood Group RhD Gene

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Rearrangements of the Blood Group RhD Gene Associated With the
DV'
Category Phenotype
By 1. Mouro, C. Le Van Kim, C. Rouillac, D.J. van Rhenen, P.Y. Le Pennec, p. Bailly, J.P. Canron, andy . Colin
The Rh (Rhesus) blood group antigens, D, Cc, and Ee, are
carried by three unglycosylated membrane proteins of the
human erythrocytes encoded by t w o highly relatedgenes,
D and CcEe. The major antigen, D, is a mosaic composedof
at least ninedeterminants (epD1 through epD9). The lack
of expression of some of these D epitopes at thesurface
of variant red blood cells defines the so-called D category
phenotypes. In thisreport, w e have determined the molecular basis of the D"' category phenotype characterized by
the lack of epitopes D l , D2, D5, D6/7. and D8. Southern
blot analysis and mRNA sequencing showed that the D"'
phenotype is associated with two types ofrearrangement
of the D gene. Of10 D"' genomes investigated, 8 exhibited
a segmental DNA replacement (gene conversion) between
the D fragment encompassing exons 4. 5, and 6 and the
equivalent region of the Cc€e gene. In the two other variants, these threeexons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the
variant RhD transcripts that are translated in 417 and 266
amino acid polypeptides, respectively. A heterogeneity of
category D"' samples basedon variable reactivity of the red
blood cells with anti-D antibodies was previously found t o
be associated with the CDV'eor cDWE
haplotypes. Interestingly, our present results indicated that thisserologic subdivision of the D"' category is correlated to two types of genomic rearrangements of the D gene.
0 1994 by The American Society
of Hematology.
T
In this report, we have shownthat the structure of the D
gene camed by 10 unrelated variants is stronglyaltered after
two differentgenomic rearrangements. The sequence of the
abnormal D protein encoded by the two types of DV' gene
has been deduced from mRNA cloning and sequencing.
HE RhD ANTIGEN is the major antigen of the RH
blood group system. Its presence or its absence at the
human red blood cell(RBC) surface determine the Rh-positive (85% of Caucasians) and Rh-negative (1 5% of Caucasians) phenotypes, respectively. However,
both types of cells
carry antigens of the C/c and E/e series, all definedby specific antibodies.' The D, C/c, and E/e antigens are encoded
by a single RH locus located on chromosome 1p34-~36:~
which is composed oftwo homologous structural genes, D
and CcEe, that have been recently cloned."6The genome of
Rh-positive and Rh-negative individuals can be distinguished, becausethe former carry two genes(Dand CcEe)
and the latter only one ( C C E ~The
) . ~D gene encodes a multispanning membrane protein of 417 amino acids and the
CcEe gene encodes the Cc and Ee proteins, most likelyby a
mechanism ofalternative splicing of a single tran~ript.',~
It is known that some Rh-positive individuals can produce anti-D antibodies in response to immunization by
transfusion with D-positive bloodor by pregnancy witha Dpositive
This is explained by assuming that the D
antigen is a "mosaic" structure and that the RBCs from
some D variant individuals may lackpart of this mosaic and
become immunized to the D epitopes that they do not possess. Rh-positive individuals that make anti-D have been
classified into six main different categories (D"
through DV",
D' being obsolete), each having a different abnormality in
the D antigen.'"I3 Examination of these variants with a
panel of human monoclonal antibodies led to the identification of nine different epitopes on the D antigen, termed
epDl through epD9.I4-I6The number of epitopes varies
from one D category to another, from nine in D"' to three
only in DV'. This explains why category VI is the one that
most frequently fails to react with polyclonal anti-D. Indeed, only 15% of unselected polyclonalanti-D and35% of
selected anti-D made by D-negative subjects reacted with
DV'cells. This observation can leadto a confusion between
the DV' and the D weak (or D') phenotypes, which correspond to qualitative and quantitative alteration of the D antigenicity, respectively. However,D weak individuals never
produce anti-D antibodies, whereas DV'female can be immunized strongly enough to cause hemolytic disease of the
n e ~ b o r n ,as
' ~often observed in case of incompatibilitiesbetween an RhD-positive fetus and an RhD-negative mother.
Blood. Vol83, No 4 (February 15). 1994:pp 1 129-1135
MATERIALS AND METHODS
Materials. Restrictionenzymes, bacterialalkaline phosphatase,
and pUC vectors were from Appligene (Strasbourg, France). T4
polynucleotide kinase, DNA polymerase I Klenow fragment, and
radiolabeled nucleotideswere from Amersham (Bucks,
UK). Avian
myeloblastosis virus (AMV) reverse transcriptase were obtained
from Promega Biotec(Madison, WI) and Thermus aquaticus polymerase (Taq polymerase) was from Perkin-Elmer-Cetus(Norwalk,
CT). Random priming labeling kits were from Boehringer Mannheim (Mannheim, Germany) and pUC sequencing kitswere from
Pharmacia (Uppsala, Sweden).
Blood samples. Blood samples from RhD-positive, RhD-negative, and rare DV'donors were collected on heparin or EDTA. Eight
DV' samples (identification numbers 307, 509, 570, 643, 836, 848,
86 1, and 9 13) were provided by the Rode h i s Bloedbank ZuidLimburg(Maastritch,The Netherlands).The two other DV' samples
(DEL and BOUF) were provided by Dr M. Beolet (CRTS Lille,
France) and by the Centre National de Referencepour les Groupes
Sanguins (CNRGS; Paris, France), respectively.
Antibodies and agglutination techniques. Polyclonal antibodies
for Rh blood group typings were from the CNRGS and human
monoclonal anti-D antibodies have been described at the First"
From the Unit6 INSERM U76, Institut National de Transfusion
Sanguine, Paris, France;and the Rode KruisBloedbank Rotterdam,
Rotterdam, TheNetherlands.
Submitted June 7, 1993; accepted October 12,1993.
Supported in part by the Institut National de la SantC et de la
Recherche Mdicale, bythe Caisse Nationaled'Assurance Maladie
des Travailleurs Salarib, and by NATOGrant No. 88/0556.
Address reprint requests to YvesColin,PhD,INSERM
U76,
INTS, 6 rue Alexandre Cabanel, 75015 Paris,
France.
The publication costs ofthis article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0I994 by The American Society
of Hematology.
0006-4971/94/8304-0012$3.00/0
l129
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1130
MOURO ET AL
and Second" International Workshop on Monoclonal Antibodies
Against Human Red Cells and Related Antigens. Agglutination
studies were performed by the antiglobulin test and the papain gel
test according to the protocol of manufacturer (Diamed SA, Morat,
Switzerland) with native or papain-treated cells according to published procedures."
DNA probes. The full-length human RhIXb cDNAencoding
the Cc/Ee proteins4 and the PCR-amplified exon 3-, exon 4-, exon
5-, exon 6-, and exon 7-specific fragments deduced from the CcEe
gene structure analysis2' wereprepared as previously described' and
labeled by the random priming method.
Southern blot analysis. Human genomic DNA extracted from
peripheral leukocytes" were digested with BamHI ( I O U/pg DNA),
resolved by electrophoresis in 0.7% agarose gel, and transferred as
described by Southern to a Zeta probe GT nylon membrane
(BioRad, Richmond, CA). Hybridization with the DNA probes (3
X IO6 cpm/mL) was performed for 24 hours at 65°C in 7% sodium
dodecyl sulfate (SDS), 500 mmol/L NaHPO,, 1 mmol/L EDTA.
Final washes were performed at 65°C for 45 minutes in 5% SDS, 40
mmol/L NaHPO,, I mmol/L EDTA, and for 30 minutes in 1%)
SDS, 40 mmol/L NaHP04, l mmol/L EDTA.
Reverse transcription coupledwith polwnerase chain reaction
(PCR)amplification. Total RNAs were extracted from I O mL of
whole blood by the acid-phenol-guanidium method.22 RNA samples prepared from heparinized blood were treated with heparinase
I to avoid further inhibition of the Taq polymerase.23RNA ( 1 pg)
was firstincubated for 5 minutes at 70°C with random primers (0.5
pg) and then cooled to room temperature. Reverse transcription
was performed at 42°C for 60 minutes in a reaction mixture (25
pL) containing 50 mmol/L Tris HCI (pH 8.3), 50 mmol/L KCI,
I O mmol/L MgC12, 20 mmol/L DTT, 0.5 mmol/L spermidine, 1
mmol/L of each deoxynucleotide triphosphate (dNTPs), 25 U of
ribonuclease inhibitor (RNasine), and 15 U of AMV reverse transcriptase. One-fifth of the cDNA products were subjected to PCR
amplifi~ation~~
in 50 mmol/L KCI, IO mmol/L Tris (pH 8.3). 3
mmol/L MgC12, 0.001% (wt/vol) gelatin, 0.2 mmol/L of the four
dNTPs, 50 pmol of each primer, 2.5 U of Taq polymerase. Oligonucleotide sequences, deduced from the human RhDcDNA clone:
were 5'TGT CGG TGC TGA TCT
CA3' (sense primer, exon 3 specific) and 5'GGC TCC GACGGT ATCY (antisense primer,exon 7
specific).Thirty cycles of amplification were performed in a PerkinElmer Cetus thermal cycler under the following conditions: denaturation for I minute at 94"C, primer annealing at 48°C for I minute, and extension at 72°C for I minute. Amplified cDNA products
were purified on agarose gels, phosphorylated with the polynucleotide kinase, and then subcloned in pUC18 vectors.
DNA sequencing. Inserts fromrecombinant
pUCl8 vectors
were sequenced on both strands by the dideoxy chain termination
method25with a Pharmacia T7 sequencing kit.
Membrane proteins analysis. For immunostaining analysis,
SDS-lysates from RBC membrane preparations prepared as described26were separated by polyacrylamide gradient gel (10% to
20%) electrophoresis (BioRad), transferred to nitrocellulose sheets,
and incubatedas described" with a rabbit polyclonal antibody
raised against asynthetic Rh peptide (MPC8 reacting with the
COOH-terminal region of the Rh proteins).** Bound antibodies
were detected with alkaline phosphatase-labeled goat antirabbit IgG
diluted (1:800) followed by revelation with the alkaline-phosphatase conjugate substrate kit (BioRad Laboratories, Rockville Center, NY).
RESULTS
Immunochemical characterization of the D"' category
cells. Ten unrelated RBC samples classified as DV' in pre-
Table 1. D Epitopes on D"' Category Cells Analyzed
With Human MoAbs
Epitopes
epD 1
epD2
epD3
epD4
epD5
epD6/7
epD8
epD9
NS
NS
D"' Cells
~
-
+
+
~
-
+
-
.I-*
MoAb-D
NT
23\1\13
NT
1 ow3
6W5
26\1\16
105
NA
89
113
D'" RBCs are reactive (+) or unreactive (-) with anti-D MoAbs (MoAbD) according to the presence or the absence of D epitopes'6,'eon their
membrane.
Abbreviations: NS, not suitablefor epitope determination; NT, not
tested; NA, not available.
Antibody 1 13 reacts with all DV'samples, except DEL and 3 0 7 .
liminary investigations were collected and tested simultaneously withpolyclonal and monoclonal anti-D antibodies.
All cells gavepositive reactions with the selected polyclonal
IgG anti-D, but the agglutination titers in the antiglobulin
tests performed at low ionic strength were significatively
lower than those given by common D-positive samples (titers 8 to 32 v 2,048). Subsequently, human monoclonal
anti-Ds antibodies (anti-D MoAbs), definedat the First and
the Second International Workshop on Monoclonal Antibodies Against Human Red Cellsand Related Antigens,I8.l9
were used in the papain-gel test (Diamed). MoAbs 23W3,
6W5, and 26W6, which recognize epitopes D2, D5, D6/7,
and D8, respectively, and MoAb 89, which does not react
with DV' cells, were unreactive with the 10 D variant cells
under study. On the otherhand, MoAb 10W3, which recognizes epitope D4, gave positivereactions of similar intensity
as compared with the polyclonal antibodies. These results
clearly established that all the 10 D variants cells belongto
the DV' category (Table 1). Moreover, these cells were not
reactive with two anti-Ds made by other DV'individuals and
some individuals included in this study havedeveloped
anti-D after immunization. Antibody 1 13 was peculiar because it reacted (although weakly) in the antiglobulin test
and the papain gel test with allthe DV'samples, except DEL
and 307, a behavior indicating heterogeneity inside DV'
samples, as previously reported.29
Full RBC phenotyping with anti-Rh reagents (anti-C, -c,
-E, and -e) indicated that DEL and 307wereDV'ccEe,
whereas the other samples were Dv'Ccee. Thus, all samples
are heterozygous for D with the mostlikely genotypes
cDV'E/cde (DEL and 307) and CDV'e/cde (others), respectively. DNAstudies have confirmed these expectations (see
below).
Southern blot analysis ojthe RH locus in D" phenotypes.
Genomic DNA from the I O DV'individuals were digested
with BamHI restriction enzyme and subjected to Southern
blot analysiswith the RhIXb cDNA probe. DNA from
RhD-positive (DCCee) and RhD-negative (ddccee) donors
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REARRANGEMENTS OF THE RH
D"
1131
GENE
m
U
m
0
: : a
n
A
A
B
Fig 1. Southem blot analysis of D"' genomic DNAs and comparison with RhD-positive and RhD-negative genomes. DNAs from
two D"' individuals and from RhD-positive (DCCee) and RhD-negative (ddccee) donors were digested with the restriction enzyme
BamHl and hybridized on aSouthern blot with the RhlXb cDNA (A)
and with the exon 4-specific probe (B). Rehybridizationof the same
blot withexons 5- or 6-specific probes gave the same pattern. Of
the 10 D"' DNA samples studied, 8exhibited the restriction pattern
of sample 848 and 2 had the patternof DEL (not shown).
were included as control. As shown in Fig 1A, two types of
genomic polymorphisms were shown when the hybridization patterns of the DNAs fromD"' and RhD-positive individuals were compared. In eight D"' variants, represented
by sample 848, a 4.5-kb BamHl fragment wasmissing,
whereas an additional fragment of 5.1 kb appeared (Fig1A).
In the two other D"' genomes, representedby sample DEL,
the 4.5-kb BamHI fragment was also missing,
but no abnormal band was detected. Previous Southern blot analyses
have indicated that the 4.5-kb BamHI fragment is specific
for the RhD gene, becauseit is present inthe D-positive genomes and absentin the D-negative DNAs' (Fig1A).
To better characterizethe 4.5-kb polymorphic fragment,
the same blot was successively rehybridized with several
exon-specificprobesdeducedfrom
CcEe gene structure
analysis (see Materialsand Methods). Because of the colinearity and of the high degree of identity(96%)between the
D and non-D cDNAs,~it has been assumedthat the intron/
exon organization of the D and CcEe genes is most likely
identicalZoand, therefore, the exonic probesused are able to
detect equivalent coding regions of both the D and CcEe
genes. Results from partial sequence analysis the
of D gene
support this hypothesis (our unpublished results).The 4.5kb BamHl fragment was detected inthe RhD-positive con-
trol DNA by probes specific of exons 4, 5, and 6 (Fig 1 B).
These probes also
showed a band of 5.3 kb
in both the RhDpositive and RhD-negative genomes, which corresponded
to the CcEe gene fragment encompassing exons 4,5, and
6. In the DEL sample, only the CcEe 5.3-kb fragment was
detected, suggesting that the restriction fragment carrying
exons 4,5, and 6 of the D gene is deleted.
In the 848 sample,
the CcEe 5.3-kb fragmentwas shown togetherwith the abnormal 5. l-kb fragment, suggesting either that the fragment
carrying exons4,5, and 6 of the D gene is rearranged
or that
one of the BamHI sites generating this restriction fragment
is polymorphic.
Ithasbeenpreviouslyshown
that hybridization with
exon-specific probes allows us to determine the homozygous or heterozygous status for the D gene bycomparing the
intensities of the CcEe-and D-specific signals.30 Using
either
exon 4 or exon 5 or exon 6 as probes, the CcEe- and Dspecific fragments were detected withthe same intensity in
the homozygous D-positivecontrol DNA (two copies of the
D gene and two copies of the CcEe gene), whereasa 1 :2 gene
dosage effect was observed
in the 848 DNA sample (FigI B),
indicating the D heterozygosity of this donor. Similarly, hybridizationperformed withexon 7 probe (not shown)
showed that the nine other D"' variants were heterozygous
for the D gene, as expected (see above).
Analysis gf the Rh transcripts in DV'phenotypes. Total
RNAs extracted from peripheral blood
of two D"'individuals (samples 848and DEL) and of RhD-positive and RhD-
+ 701 bp
+ 248 bp
Fig 2. PCR amplification of the D"' transcripts and comparison
with the amplified products of RhD-positiveand RhD-negativetranscripts. Total RNAs extracted from theperipheral blood of the two
types of D"' individuals (DEL and 848 samples) and of RhD-positive
and RhD-negative donors were reverse transcribed. The cDNAs
were subjected to a PCR amplification between an oligonucleotide
common to D and CcEe cDNAs in the exon 3 and a D-specific oligonucleotide in the exon 7 (see Materials and Methods). The PCR
products were resolvedon an agarose gel.
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1132
MOURO ET AL
E4
D
DvI(848)
Dvl(DEL)
D
Dvl(848)
Dvl(DEL)
210
TATIPSLSAM
R-----"--
E5
220
LGALFLWMFW
-_________
******************
230
PSFNSALLRS
--V----"-
**********
240
PIERKNAVFN
--Q"--"-
**********
E5
E
6
2
250
60 280
270
TYYAVAVSVVTAISGSSLAHPQGKISKTYVHSAVLAGGVA
----L""- _ _ - _ _ _ _ --R---"-__
**********
R*********
**********
__________
**********
L"_
E6
D
Dvl (848)
Dvl(DEL)
290
310
300
VGTSCHLIPSPWLAMVLGLVAFLISVGGAKYLPGCCNRV--""C-"1""
"
"
"
"
"
"
"
"
"
"
********** ********** ********** ***
(417
GCCNRV- - - ( 4 1 7
GCCNRV---(266
aa)
aa)
aa)
Fig 3. Amino acid sequence comparison ofD, DV'848, and D'" DEL encoded proteins in the affected region. The D proteinwas deduced
from the recently cloned RhD cDNA.' Identical positions
noted
are as dashes.The bars abovethe protein sequence delineate
the amino acids
encoded by exons3 through 7 (E3 through E7). Amino acid sequencefrom position 163 to position 3 1 3 of the D"' 848 protein corresponds
to the amino acid sequence of
the Ce or ce encodedp r ~ t e i n .In
~ ,D"'
~ DEL, amino acids1 6 3 to 3 1 3 encoded by exons
4,5, and 6 are deleted
(*) and, therefore, amino acid
1 6 2 is linkedto amino acid3 14 inthe new protein variant.
negative donors were converted to cDNAs and enzymatically amplified. The PCR amplification was performed between a first primer corresponding to an exon 3 sequence
common to the D and non-D cDNAs and a second primer
designed froma D-specific sequencein exon 7 (see Materials
and Methods).6As expected, no amplification product was
obtained with the RhD-negative cDNA, whereas a 701-bp
fragment was amplified fromthe RhD-positive cDNAtemplate (Fig2).
Amplification performed with the transcripts from the
DEL donor yielded a PCR product of 248 bp (Fig 2). Sequence analysis indicated that this cDNA fragment lacked
nucleotides 487 to 939 of the normal RhD cDNA (+ 1 taken
as the first residue of
the initiator methionine.6Based on the
analysis ofthe R h gene@)organization (Chirif-Zahar et alZo
and our unpublished data) we deduced that the 5' and 3'
boundaries of the deletion in the transcription product corresponded to the exon 3lintron 3 and intron6/exon l transitions, respectively. This result, together with the Southern
analysis described above, showed that the genomic region
encompassing exons 4, 5 , and 6 of the D gene is deleted in
donor DEL. Becauseintrons 3 and 6 interrupt the D coding
sequence after the third and before the first nucleotide of a
codon triplet, respectively,the reading frame of the mRNA
resulting fromthe fusion of exons 3 and 7 in the DEL gene
was not changed. Therefore, the deduced DV' (DEL)-encoded protein represents a shortened form of the normal D
polypeptide composed of 266 amino acids that lacks residues 163 to 3 13 (Fig 3).
The size ofthe PCR products obtained after amplification
of the D transcript from the D"' (848) and the RhD-positive
donors were identical (70 l bp; Fig 2). Sequence comparison
between these products showed 17 nucleotide substitutions
all located within the regions transcribed from exons 4, 5 ,
and 6. No other polymorphisms were identified in the remaining coding region of the 848 cDNA (not shown). Interestingly, allthe substitutions between 848 and the normal D
cDNAs corresponded to that previously identified between
the D and non-D transcript^.^.^ These findings, together
with the Southern blot analysis, indicated that the variant
848 is characterized by a rearranged RH locus in which the
region carrying exons 4, 5 , and 6 of the D gene has been
replaced by an equivalent fragment of the CcEe gene. The
deduced protein encoded by this hybrid D-CcEe-D gene isa
4 17 amino acid polypeptide that differs fromthe normal D
polypeptide by 15 amino acid substitutions (Fig 3).
Western blot analysis of the R h polypeptides in D" phenotypes. The calculated molecular mass of the normal D
and variant D"' (DEL) polypeptides being significatively
different (45.5 and 29.2 kD, respectively), a comparative
Western blot analysis
was performed with a polyclonal antibody raised against a synthetic peptide derived from the
COOH sequence common to the D and non-D polypep
tides.** Surprisingly, evenafter migration in gradient poly-
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REARRANGEMENTS OF THE RH D"' GENE
1133
(calculated Mr,45.5 kD). However,the shortened polypep
tide could not be separated from the normal Rh polypep
tides expressed in common phenotypes, which all migrate
with an apparent Mr of 32 kD on SDS-polyacrylamide gel
electrophoresis (SDS-PAGE).This is most likely explained
because these proteins are highly hydrophobic molecules
that exhibit an abnormal behavior in SDS-PAGE."' Thisobservation may explain why the Cc and Ee proteins that are
thought to be translatedfromdifferentsplicingisoform
transcripts of the CcEe gene, one of which precisely lacks
exons 4, 5, and 6,8*9 could not be distinguished from each
other by SDS gel electrophoresis.
In the type I1 variants, representedby the 8 other DV'samples, a different rearrangementof the D gene was identified,
because exons 4, 5, and 6 were present but camed by a restriction fragmentof abnormal size. cDNA sequence analysis indicatedthat the region ofthe DV'transcript encoded by
Fig 4. lmmunostaining of the RBC membrane proteins from
these three exonscorresponded to that of the non-D
DCCee, D"' 848, and D"' DEL donors. Total membrane proteins (60
mRNAs, and more precisely to the transcription productof
pg) separated on a polyacrylamide gradient (1
gel
0%to 20%)were
the Ce or ce alleles accordingto the presence of an Alanine
transferred to nitrocellulose sheets and incubated with a rabbit
anti-Rh protein antibody (MPC8: 1/4,000). After washing, the niresidue at the E/e-associated polymorphic position 226.9
trocellulose membrane was incubated with a goat antirabbit IgG
These results strongly suggested that the DNA segment of
conjugated to alkaline phosphatase. Antibodies bound to the Rh
the D gene encompassing exons 4, 5, and 6 has been reproteinswere visualized by an alkaline-phosphatase conjugate subplaced inthese variants by the equivalent region ofthe CcEe
strate kit. The arrow on the right side indicates
the migration of the
gene. Becausethe D and CcEegenes are sequence related?*'
detectable in
Rh polypeptides.(*) Undefined molecular species not
Rh-null sampleswith the MPC8 polyclonal antibody(P. Bailly. perit is assumedthat such a DNA transfer occurred after chrosonal communication).
mosomal misalignment between the two Rh genes during
meiosis (Fig 5B). Whether
the resulting hybrid"D-CcEe-D"
gene isthe product of an intergenic double crossing-over
or
acrylamide gels (1 0% to 20%), no difference in the electroofa combination ofgene conversion between the CcEegene
phoreticmobility ofDELversusnormal
D membrane
(as donor) and the D gene (as acceptor) and branch migraproteins could be detected (Fig
4).
tion cannot be determined because the second product of
these gene rearrangements is not available.However, gene
DISCUSSION
conversion providesa simpler modeland is thus commonly
favored when,as in the present case,the length of the region
We have analyzedthe organization and expression of the
RH blood group locus in 10 variants of the DV' category
affected is ofthe order of a few kilo base^."^ Homologous rephenotype, which represents the most frequently detected
combination through a mechanism of gene conversion has
variant of the D categories."*'*Our results clearly estabbeen widely reported ina number of multigene familiesin
lished that the DV' phenotype can
be associated withat least
humans, includingglobin,33haptoglobin,"* Ig,34.35major
two typesof genomic rearrangementsof the D gene, called
histocompatibility complex,36 complement ~ystem,~'
and
here typesI and 11.
glycophorin gene c l u ~ t e r s . ' ~Analysis
* ~ ~ of the evolution of
the mammalian &like globin genes indicated
that segmental
In the type I variants, represented by 2 of the 10 samples
investigated, Southern blot analysis indicated that the D
DNA replacement, by transfemng several base substitugene fragment encompassing exons
4,5, and 6 was deleted.
tions in a single event,can lead to wrong phylogenetic relaSequence analysisof the Rh mRNA transcribed from one
tionships of specie^.^.^' Therefore, the present description
ofthe type I variantsconfirmedthis result and indicated that
of such a rearrangement inthe Rh gene cluster shouldgive
the breakpoints of the deletion were located withinintrons
rise to special caution during the phylogenetic analysis of
3 and 6 of the D gene. We assumed that, as frequently dethe RH locus.
scribed in case of partial gene deletion,
this defective D gene
It
is
noteworthy
that the DV'-associated
gene
remost likely results from
an unequal crossing over caused
by
arrangements of types I and I1 both involve exons4,5, and
recombination between homologous intronic regions (Fig
6. This result suggests that the D-specific amino acids en5A). However, these putative repeated sequences have not
coded by these three exons ( 15 residues; see Fig 3) might
be
yet been characterized. Accordingto this model, the recip
critical for the reactivity with the different MoAbs characrocal product of the recombination event canbe predicted
terizing epitopesD I , D2, D5,D6/7, and D8 that are camed
to be an abnormal D gene with a duplication of exons 4,5,
by the normal D but not by the D"' protein.I5.l6 Conversely,
and 6 that remains to be identified.The deduced translation
the remaining part of the D"' protein still cames epitopes
product of the deleted D" gene is a 266 amino acid protein
D3, D4, and D9, which, however, have not yet been prethat lacks 15 1 amino acids (calculated Mr,
29.2 kD) as comcisely mapped.
pared withthe 4 I7 amino acids ofthe normal D polypeptide
The D"' polypeptides encodedby the type I and type I1
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1134
MOURO ET AL
D gene
CcEe gene
A
16
+
D"' type I (DEL)
D gene
13
CcEe gene
E
B
E l E2 E3 E4 E5 E6 E7 E8
+
D"' type It (848)
Fig 5. Schematic representation showing possible genetic rearrangements resulting
in D"' genes. The high degreeof similarity between
that the D and the &€e genes have a similar organization?
Exons (El
the CcEe gene andthe D gene' aswell as preliminary studies suggest
through E l 0)from CcEe and D genes are represented by open and
dotted boxes, respectively (not
to scale). 13 and I6 denote introns3 and 6,
respectively. The given position ofthe D and CcEe genes onthe chromosome isarbiirary. (A) Intragenic unequal crossingover occurring
6 of the other chromosome giving rise
to a new chromosome carryinga deletion of exons 4,5,
within intron 3 of one chromosome and intron
and 6. as observedin D"' DEL. (B) lntergenic double crossing over or gene conversion occurring
between a D and a Cc€e gene giving rise
to a
hybrid D-&€e-D gene, as observedin D"' 848. Brackets indicatethat the genetic rearrangementmay have occurredbetween a D-positive
chromosome and a D-positive orD-negative
a
chromosome. Reciprocal gene rearrangements generated by these recombination events are
not shown (see
text).
variants might well be discriminated by immunologic methods because the deletion and conversion process generated
unusual D proteins characterizedby a new amino acid junction or peptide insertion, respectively. As a matter of fact,
some heterogeneity among DV' phenotypes has been suspectedserologically.
Indeed, it has been reported that
cDV'E/cde but not CDV'e/cde samples failed to react with
one of two monoclonal anti-G antibodies and that CDV'e/
cde but not cDV'E/cde cells expressa "new" low frequency
antigen inherited as a mendelian character.42It was found
also that CDV'e/cde cells often gave
stronger reactions than
cDV'E/cde cells with some anti-D MoAbs.'' Interestingly,
these subdivisions of the DV' category appear to be correlated at the molecular levelwith the twotypes ofrearrangement of the D gene, becausewe found here that the
cDV'E and CDV'e haplotypes are associated with the deleted" (type I) or the "converted" (type 11) forms of the D
gene, respectively.Our results strongly suggest
that the serologic heterogeneity of the DV' phenotype described above
is based on different primary, and most likely secondary,
structures of the DV*polypeptides expressed at the surface
of the variant RBCs. Further investigationsof partial D variants should help to delineate the structure of the D epitopes
on D polypeptides and to determine if some play a predominant role inthe high immunogenicity ofthe D antigen.
ACKNOWLEDGMENT
We thank Dr Baya Cherif-Zahar for providing
data on the structure of the Rh gene beforepublication, Dr Marylise Beolet (CRTS
Lille, France) for the gift of DEL sample, Drs Pascal Bailly and Patricia Hermand for providingthe MPC8 antibody, and Marie-The&se Klein(CNRGS, Paris) for technical assistance.
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1994 83: 1129-1135
Rearrangements of the blood group RhD gene associated with the DVI
category phenotype
I Mouro, C Le Van Kim, C Rouillac, DJ van Rhenen, PY Le Pennec, P Bailly, JP Cartron and Y
Colin
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