Download Lack of RHCE-Encoded Proteins in the D

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1518
CORRESPONDENCE
Lack of RHCE-Encoded Proteins in the D-- Phenotype May Result
From Homologous Recombination Between the Two RH Genes
To the Editor:
by
TheRh blood groupantigens D, C/c,andE/earecarried
atleastthree red blood cellmembraneproteinsencodedbytwo
homologousgenes, RHD and RHCE. Thesetwogenesarepresent
thein Rh-positive
only
individuals,
whereas
RHCE
encoding
gene
the Cc and Ee proteins
is present in Rh-negative cells.' Rare homozygousindividualscarrying the D-- genecomplexarecharacterized
by atotalabsence o f C/candE/eantigensand an excessrepresentation o f the D antigen.* The RH locus o f three D-- individuals
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CORRESPONDENCE
(French. Icelandic. and Italian donors) had been previously analyzed
by Southern blot and Rh cDNAs sequencing.'" The results indicated
that the lack of C/c and E/e antigens resulted from either a reduced
transcriptional activity' or from an internal gene deletion','of the
RHCE gene. We describe here a third mechanism leading to this
phenotype by analyzing the RH locus of a D-- individual (LM)
of Italian origin. We discovered that this donor was independently
studied by Huang et al.' but our results are different. because we
ascertained that this D-- gene complex most likely resulted from
a homologous recombination event between RHD and RHCE genes.
as indicated by sequence analysis and by a reverse transcriptasepolymerase chain reaction (RT-PCR) assay that specifically detected
hybrid gene products (see below).
Southern blot analysis with Rh cDNA and exon-specific probes
showed an alteration of the RHCE gene of LM. because the RomHI
and Hind111 restriction fragments carrying exons 4 through 6 and
exons 5 through 7, respectively. were lacking (data not shown).
Reticulocyte RNAs from LM were reverse transcribed to cDNA and
amplified by PCR using a primer located in the S' untranslated region
(S'UT; nt -19 to -2; + I representing A of the initiation ATG
codon) common to the RHD and RHCE genes and a primer specific
of the 3' untranslated sequence of the RHD (3'UTD: nt1,421 to
1.437) or RHCE (3'UTCE: nt 1.363 to 1.380) genes. respectively
(Fig IA). The cDNA amplified between the S'UT and 3'UTD primers (1.45 kb) carried three base substitutions (G,&. T,,,,,C. and
AII',IT) that were previously assigned to exons I and 9 of the RHCE
gene. This cDNA was also identified by Huang et al.'
Sequencing of the PCR product amplified between the S'UT and
3'UTCE primers ( I .40 kb) showed a hybrid cDNA composed of an
RHD sequence transcribed from exons 1 to 9 and an RHCE sequence
corresponding to exon IO. This cDNA was not identified by Huang
et al.'Both
cDNAs carried the G520A transition, resulting in a
V174M substitution. These data indicated that the Rh RNAs from
LM cells were transcribed from two rearranged RH genes (Fig IA).
To identify the sequence upstream nt48 in the two cDNAs (1.45 and
1.40 kb), we have amplified by PCR a 815-bp fragment, including
676 bp upstream the intiation ATG codon, using a S' primer located
in the promoter region (nt -676 to -659) and a 3' primer in exon
I (nt 122 to 139). because several substitutions (nt -597. -554.
-390, - 1 4 4 . and -132) allow us to differentiate the RHD from
the RHCE promoter (our unpublished data). Sequencing analysis
indicated that the 1.45-kb cDNA was transcribed from the RHCE
promoter. whereas the I .40-kb cDNA was transcribed from the RHD
promoter. These results suggest an unusual complexity of the RH
locus from LM, which we assume as being composed of two hybrid
genes. C€-D-C€-D (a similar complex hybrid between the genes
encoding the erythrocyte glycophorins A and B has been described")
and D-C€. The proteins encoded by these rearranged genes would
carry D but not CcEe epitopes. thus explaning the serologic properties of the D-- complex (greater than the normal amount of D and
a total absence of C c E e antigens).
To confirm the presence of the D-C€hybrid transcript in LM
reticulocytes. a hybrid RT-PCR using 5' primers located in exon 5
of RHD or RHCE sequence (E'D, nt 695 to 712: or E'CE, nt 650
to 667) and the 3'UTCE primer was setup (Fig IB). The primer
pair E5D/3'UTCE amplified a 690-bp product from LM cDNA (as
well as 556- and 403-bp isoforms). but it did not amplify any product
from the cDNA of a normal Rh-positive (DCe) control (Fig IB).
Conversely. the primer pair E5CE/3'UTCE amplified a 733-bp product (as well as several minor isofonns) from the RHCE gene of the
Rh-positive (DCe) control, but it did not amplify any product from
LM cDNA (Fig IB).
Therefore, the hybrid RT-PCR method appears to be useful to
in Rh variants. Accorddetect hybrid gene structure(s) present
ingly, we found that the E5D/3'UTCE primer pair gave positive
1519
EsCE / 3'UTCE
E5D
I3'UTCE
B
Fig 1. RH locus and detection of hybrid transcripts in Rh variants by RT-PCR analysis. (A) Schematic structure of RH haplotypes
from Rh-positive (DCe) and Rh-negative (dce) individuals compared with RH haplotypes from variants with a total or partial
defect of Clc andlor Ele antigen expression. RHD and RHCE sequences are indicated by solid and open boxes, respectively. Small
boxes upstream each gene correspond to therespective promoter.
Numbers above gene boxes refer to exon regions of the RHD (solid
symbols) or RHCE (open symbols) genes involved in the rearrangements. Arrows indicate primers used in the RT-PCR assays. Fulllength D and non-D cDNAs were amplified with primers 5'UT
(arrow heads, common to RHD and RHCE genes), 3'UTD (solid
arrow, D-specific), and 3'UTCE (open arrow, CE-specific) and sequenced (see text). (B) Detection of hybrid Rh transcripts by RTPCR analysis. PCR (30 cycles of 94°C for 30 seconds, 57°C for 30
seconds, and 72°C for 1 minutel were performed with the primer
pairs E5CE/3'UTCE and E5D/3'UTCE, which were designed to amplify 733- and 690-bp fragments from theCE and hybrid D-CE transcripts, respectively. Primer ESCE(open arrow, sense primer) is
located in exon 5 and is CE specific, whereas primer E5D (solid
arrow, sense primer) is located in exon 5 and is D-specific. The
primer 3'UTCE (open arrow) is CE-specific and was used in both
assays as antisense primer. PCR products amplified from DCe,
D-- (LM), D-- (Gou), DC- (Bol), and D . ' ( D a v )were resolved on
2% agarose gel and visualized under UV light after staining with
ethidium bromide. Sizes of the CE (left) and hybrid D-CE (right)
amplification products are indicated. The 556- and 403-bp fragments are isoforms lacking exon 7 and exons 7 through 9, respectively.
signals(Fig I B ) with the cDNAsprepared from homozygous
DC- (Bol.) and D - - ( D a v . ) individuals,whichcarrya
hybrid
CE-D(e.rort 4 to 9)-CE gene' and a D(e.ror? /-6)-CE(e.rorl7 - I O )
hybrid gene (our unpublished data), respectively. However.
no
signal was detected with the D-- sample (Gou) that carries an
intact RHCE gene.' On the other hand. the E'CE13'UTCE primer
pair could amplify a 733-bp RHCE gene product from the D-sample. but therewasnoamplilication
with the DC- and
D * samples (Fig I B).
In conclusion. our results show the heterogeneity
of the phenotypes with a total absence of RhCclEe antigen expression. They
also show that the hybrid RT-PCR assay is useful to detect hybrid
gene structures. suggesting that this method can be generalized
to identify gene rearrangementsin many examples of R h variants.
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CORRESPONDENCE
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ACKNOWLEDGMENT
We thank Dr Georgio Reali (Immunoematologia e Transfusionale,
Genova, Italy) andDr Marcela Contreras (North London Blood
Transfusion Service, London, UK) for the generous gift of D-(LM) and D (Dav) samples, respectively.
Baya Cherif-Zahar
Virginie Raynal
Jean-Pierre Cartron
INSERM U76
Institur National de Iu Transfusion Sanguine
Paris,France
REFERENCES
1. Cartron JP, Agre P: Rh blood group antigens: Protein and gene
structure. Semin Hematol 30:193, 1993
2. Race RR, Sanger R: Blood Groups in Man. Oxford, UK, Blackwell Scientific, 1975
3. Cherif-Zahar B, Raynal V, D’Ambrosio AM, Cartron JP, Colin
Y: Molecular analysis of the structure and expression oftheRH
locus in individuals with D--, DC-, and DC”- gene complexes.
Blood 84:4354, 1994
4. Blunt T, Steers F, Daniels G, Carritt B: Lack of RHClE expression in the Rhesus D-- phenotype is the result of a gene deletion.
Ann Hum Genet 58:19, 1994
5. Huang CH, Reid ME, Chen Y: Identification of a partial internal deletion in the RH locus causing the human erythrocytes D-phenotype. Blood 86:784, 1995
6. Huang CH, Lomas C, Daniels G, Blumenfeld 00:Glycophorin
He (St;‘) of the humanredblood cell membrane is encoded by a
complex hybrid gene resulting from two recombinational events.
Blood 83:3369, 1994
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
1996 88: 1518-1520
Lack of RHCE-encoded proteins in the D--phenotype may result from
homologous recombination between the two RH genes [letter;
comment]
B Cherif-Zahar, V Raynal and JP Cartron
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