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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2535-2540
Rh-Deficiency of the Regulator Type Caused by Splicing Mutations
in the Human RH50 Gene
By
Baya Chérif-Zahar,
Giorgio Matassi,
Virginie Raynal,
Pierre Gane,
Jean Delaunay,
Beatrix Arrizabalaga, and
Jean-Pierre Cartron
From INSERM U76, Institut National de la Transfusion Sanguine, Paris,
France; the Service d'Hématologie et INSERM U299, Hôpital
Kremlin Bicêtre, Le Kremlin Bicêtre, France; and the
Servicio Hematologia, Hospital de Cruces, Bilbao, Spain.
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ABSTRACT |
The Rh polypeptides and the glycoproteins Rh50, CD47, LW, and
glycophorin B, which interact in the red blood cell membrane to form a
multisubunit complex, are lacking or are severely reduced in the
Rh-deficiency syndrome. We previously reported that in several
Rhnull patients the RH50 gene was altered at the
coding sequence level, resulting in either a single amino acid
substitution or the synthesis of a truncated polypeptide. In the
present report, we have detected two mutations in the intronic region
of the RH50 gene that identify a new molecular mechanism
involved in Rh-deficiency. The first mutation affected the invariant G
residue of the 3 acceptor splice-site of intron 6, causing the
skipping of the downstream exon and the premature termination of
translation. The second mutation occurred at the first base of the
5 donor splice-site of intron 1. Both these mutations were found
in homozygote state. RNase protection assays demonstrated that the Rh50
mRNA level was strongly reduced or undetectable in the 3 and
5 splice mutants, respectively. The different mutations
affecting the RH50 gene are indicative of an heterogeneous
mutational pattern, which further supports the hypothesis that the lack
of the Rh50 protein may prevent the assembly or transport of the Rh
membrane complex to the red blood cell surface.
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INTRODUCTION |
THE Rh (Rhesus) ANTIGENS are defined by a
multisubunit complex composed of the unglycosylated Rh polypeptides and
of several glycoproteins (Rh50, CD47, LW, and glycophorin B) held
together by noncovalent linkages. These proteins are collectively
absent or severely reduced on erythrocytes from Rhnull
patients who suffer from a chronic hemolytic anemia known as the Rh
deficiency syndrome.1-3 Among the components of the Rh
membrane complex, Rh, Rh50, and CD47 are of special interest, because
there is no selective deficiency of these proteins presently identified
and, therefore, each of them may play a crucial role in the transport
and assembly of the Rh membrane complex to the red blood cell (RBC)
membrane. In contrast, deficiency of LW and GPB without alteration of
Rh antigen expression is well known,4 suggesting that these
proteins represent dispensable accessory chains of the Rh membrane
complex.
The Rh50 glycoprotein (50 kD) and the RhD/RhCE proteins (30 kD) are
erythroid specific. They share about 36% amino acid identity and a
similar topology of their 12 transmembrane domains.5 These
proteins are encoded by the RH50 and RH genes,
respectively, that belong to the same protein family. Both genes are
composed of 10 exons whose size and exon/intron junctions are highly
conserved.6,7 CD47 has a wide tissue distribution and is
identical to the integrin-associated calcium channel of endothelial
cells.8-11
Rh deficiency may arise from two distinct genetic backgrounds called
amorph (or silent) and regulator.4,12 The amorph type is
very rare and arises by homozygosity of a silent allele at the RH
locus. Recent studies in our laboratory have shown that, in 2 of these
patients, the RHCE gene was altered by different point
mutations, but there was no sequence alteration of either the Rh50 or
CD47 transcripts.13 In the regulator type, the most common
form of Rhnull, which is caused by the homozygosity of a
rare autosomal suppressor gene (X°r) unlinked to the RH
locus, all of the genes and transcripts noted above were normal, except for RH50. The Rh50 protein was absent or severely
reduced because of frameshift, missense mutations, or absence of
detectable transcript.14,15 Because Rh and Rh50 are thought
to interact with each other in the cell membrane,16,17
these findings suggested that, when the Rh50 glycoprotein is lacking,
the Rh membrane complex is not assembled or transported to the cell
surface. Therefore, mutant alleles of the RH50 gene are likely
candidates as suppressors of the RH locus in the Rhnull of
regulator type.
Very few mutations of the RH50 gene have been described so
far.14,15 We describe here two splice-site mutations in the RH50 gene from 2 unrelated patients that identify a new
molecular mechanism leading to the Rhnull phenotype.
Moreover, we trace the inheritance of the mutations by family studies
and provide additional information on the mapping of Rh50 mutations
causing Rh deficiency.
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MATERIALS AND METHODS |
Blood samples.
Rhnull samples of the regulator type and family members
were collected on heparin (10 U/mL) and immediately shipped to Paris, France. Sample AL was provided by L. Lebeck (Loma Linda, CA) and sample
AC, from Bilbao (Spain), was provided by one of us. Both patients
exhibited the typical Rhnull syndrome. Blood samples from
common RhD-positive and RhD-negative phenotypes were obtained from the
Institut National de la Transfusion Sanguine (Paris, France).
Flow cytometry and Western blot analysis.
Immunostaining of intact RBCs was performed as described,18
using murine monoclonal antibodies (MoAbs) directed against CD47 (MoAb
6H9; gift of Dr M. Telen, Durham, NC) and the Rh50 glycoprotein (2D10;
gift of Dr A. von dem Borne, Amsterdam, The Netherlands).
Fluorescein-conjugated F(ab)2 fragments of goat antimouse IgG (Immunotech, Marseille, France) were used and the mean
fluorescence intensity was determined with a FACScan flow cytometer
(Becton Dickinson, San Jose, CA). Determination of antigen site
densities was performed with QIFIKIT microbeads coated with variable
numbers of mouse Ig molecules (Biocytex, Marseille, France).
RBC membrane proteins (60 µg) were separated on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide), transferred onto nitrocellulose sheets, and incubated with the A23
rabbit polyclonal antibody (1:200 dilution) directed against the
N-terminus of the Rh50 glycoprotein (residues 2 through 16) and with a
rabbit anti-p55 antibody13 (1:10,000) used as control. Bound antibodies were detected with alkaline phosphatase-labeled goat
antirabbit IgG (1:800), followed by revelation with the
alkaline-phosphatase conjugate substrate kit (Biorad Laboratories,
Rockville Centre, NY).
Reverse transcription coupled with polymerase chain reaction
(PCR) amplification.
Reticulocyte RNAs from 30 mL of peripheral blood were extracted by
selectively lysing RBCs with the Orskov reaction.19 One microgram of RNA was reverse transcribed in a total volume of 33 µL
using the First Strand cDNA synthesis kit (Pharmacia, Uppsala, Sweden)
following the manufacturer's instructions. Five microliters of cDNA
products was then subjected to PCR in 50 mmol/L KCl, 10 mmol/L Tris (pH
8.3), 0.01% gelatin, 0.2 mmol/L of the four dNTPs, 50 pmoles of each
primer, and 2.5 U of Taq polymerase (Perkin-Elmer Cetus,
Norwalk, CT). PCR conditions were as follows: denaturation for 1 minute
at 94°C, annealing for 1 minute at 55°C, and extension for 1.30 minutes at 72°C for 30 cycles. Relevant PCR fragments were purified
on a 1% low melting agarose gel followed by a Wizard PCR preps
minicolumns (Promega, Madison, WI) and subcloned into a pCR II vector
using the TA cloning kit (Invitrogen, Leek, The Netherlands). Primers
used to amplify Rh (sense, nt  19 to 2; antisense, nt
1,350 to 1,330), Rh50 (sense, nt 22 to 5; antisense, nt
1,262 to 1,243), and CD47 (sense, nt 2 to +14; antisense, nt
1,007 to 989) cDNAs were deduced from published
sequences.5,8,20,21
Genomic DNA analysis and DNA sequencing.
DNA was prepared from peripheral leukocytes by the SDS/proteinase K
extraction procedure.22 A 316-bp fragment, encompassing the
exon 1 to intron 1 junction of the RH50 gene, was obtained using an appropriate primer pair (sense, nt 2 to +15; antisense intronic, 5ccaggtaagccgtggaggttatgg3) after 30 cycles
each, including 1 minute at 94°C, 30 seconds at 50°C, and 30 seconds at 72°C. A 1.3-kb fragment encompassing the exon6/exon7
junctions was also amplified between specific primers (sense,
A3, nt 857 to 880; antisense, A4, nt 1,013 to
990) with 2.5 U of enzyme mix (Taq & Pwo DNA polymerases) using the
Expand long template PCR system (Boehringer Mannheim, Mannheim,
Germany) under the following conditions: 2 minutes at
94°C (1 cycle); 10 seconds at 94°C, 30 seconds at 65 °C,
and 4 minutes at 68°C (10 cycles); 10 seconds at 94°C, 30 seconds at 65 °C, and 4 minutes at 68°C plus cycle elongation
of 20 seconds for each cycle (15 cycles); and an elongation time up to
7 minutes at 68°C. PCR products were purified through a microcon ultrafiltration membrane (Amicon, Beverly, MA). Sequencing was performed on an automated ALF express sequencer (Pharmacia) using
either the Cy5 Autoread sequencing kit (Pharmacia) with plasmid DNA or
the Thermo Sequenase sequencing kit (Amersham, Bucks, UK) for direct
sequencing of PCR products, following the manufacturer's instructions.
Ribonuclease protection assay.
Two DNA fragments of 270 bp between nucleotides (nt) 162 and 431 of the
Rh50 cDNA5 and of 207 bp between nt 616 and 822 of the XK
cDNA encoding the Kx antigen23 were amplified and cloned
into a pCR II vector using the TA cloning kit (Invitrogen). From the
linearized recombinant plasmids, antisense RNA probes were synthesized
using [ -32P]-UTP (800 Ci/mmol; NEN, Boston, MA) in an
in vitro transcription system (Riboprobe Core System; Promega). The
full-length transcripts were purified from a 5% acrylamide, 8 mol/L
urea gel with 0.5 mol/L ammonium acetate, 1 mmol/L EDTA, and 1% SDS.
Rh50 and XK RNA probes (8 × 104 and 1 × 104 cpm, respectively) were hybridized with
12.5 µg of total reticulocyte RNAs overnight at 52°C and digested
with RNase A and T1, following the manufacturer's instructions (RPAII;
Ambion, Austin, TX). The protected RNA fragments were separated through
a 5% denaturing polyacrylamide gel. Hybridizing signals on the
autoradiograph were quantified using the NIH-Image software.
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RESULTS |
Cell surface expression of the antigens and proteins from the Rh
membrane complex.
RBC samples from the Rhnull propositus and members of two
families (C and L) were analyzed by flow cytometry with MoAbs specific for Rh (D, C, c, E, and e) and LW antigens as well as MoAbs directed against Rh50 and CD47. RBC samples from patients AC and AL did not
react with anti-Rh, anti-LW, and anti-Rh50 reagents, but reacted only weakly with CD47 (2 to 3 × 103
v 35 to 39 × 103 molecules/cell) as compared
with controls. In contrast, RBC samples from the family members reacted
normally (data not shown).
Analysis of the Rh and Rh50 transcripts in Rhnull
patients.
Reticulocyte RNAs isolated from patients AC and AL were reverse
transcribed and the Rh and Rh50 cDNAs were amplified using appropriate
primer pairs (see Materials and Methods). As expected for Rh
transcripts, three PCR products were detected in the RhD-positive control (Fig 1). The 1.3-kb fragment
corresponds to both RHCE and RHD full-length cDNAs. The
1.2- and 1.0-kb products are derived from RHD spliceoforms, as
previously described.24-26 In the RhD-negative sample, the
1.3-kb and the faint 1.2-kb amplified products correspond to the
full-length RHCE cDNA and to the Rh4 spliceoform of the RHCE gene,27 respectively. The amplification
pattern distinctive of the RhD-positive phenotype was obtained in AC
and AL samples, indicating that both RH genes are present and
transcribed in these patients (Fig 1). Moreover, sequence analysis of
the full length Rh transcripts (1.3 kb) from AC and AL showed no
alteration (data not shown).
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| Fig 1.
Reverse transcription-PCR (RT-PCR)
amplification of reticulocyte RNAs from AC and AL Rhnull
patients. The Rh and Rh50 cDNAs were amplified by RT-PCR from
reticulocyte mRNAs as described in Materials and Methods. The Rh50
amplification products showed a smaller size in patient AC as compared
with RhD-negative or RhD-positive controls, and no product is present
in AL.
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The amplification product from the Rh50 cDNA was smaller in size in the
AC sample (1.1 kb) with respect to the controls (1.2 kb), whereas no
product was detected in AL (Fig 1), as previously suggested.14 Sequencing of the Rh50 cDNA amplified from AC
mRNAs showed a loss of 122 bp (positions 946 to 1067) corresponding to
the entire transcribed region of exon 7, as compared with the normal
RH50 sequence5 (Fig 2).
This deletion introduced a frameshift after the threonine codon at
position 315 and resulted in a premature stop codon, 108 bp downstream,
located in the exon 9 region of the gene. The predicted truncated
protein is 351 amino acids long (instead of 409) and includes 36 novel
residues at the C-terminus. This result raised the hypothesis that the
lack of Rh50 expression in AC erythrocytes, determined by flow
cytometry analysis (see above), may result from the failure of the
anti-Rh50 antibody (2D10) to bind to the mutated region of the protein.
This possibility was ruled out by Western blot analysis, using a
polyclonal antibody directed against the Rh50 N-terminus (see Materials
and Methods), which confirmed the absence of the Rh50 glycoprotein in
the RBC membrane (Fig 3). In addition, the
CD47 cDNA from AC and AL patients were amplified and their sequences
were found to be identical to that already published.8
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| Fig 2.
Rh50 transcript analysis in patient AC. In the control
sample, nt 945/946 correspond to the exon 6-exon 7 junction. In patient
AC, a 122-bp deletion (nt 946 to 1067, ie, the entire transcribed exon
7 region) results in a new junction, nt 945/1068. This deletion
introduced a frameshift resulting in 36 novel amino acids (in bold
italics) at the C-terminal region, after the threonine at position 315. Ctrl, normal Rh50 cDNA.5 The star identifies the stop codon
located in exon 9. Junctions of exons 6-7 and exons 7-8 are shown.
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| Fig 3.
Immunostaining of RBC membrane proteins from AC and AL
erythrocytes. The Rh50 glycoprotein carries a N-linked
polylactosylaminyl carbohydrate chain and is shown as a diffuse band of
45 to 75 kD in the RhD-positive control (Ctrl) using an antibody
directed against the Rh50 N-terminus. No signal was detected in both AC
and AL. All samples exhibited equivalent amounts of p55.
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RH50 gene analysis.
No obvious abnormality of the RH50 locus was observed by
Southern blot analysis on genomic DNA extracted from AC and AL blood samples (not shown). Therefore, the 122 nt deletion of the Rh50 cDNA in
patient AC may be attributed to an exon skipping during the processing
of the pre-mRNA that may well be the result of a 3 acceptor
splice-site mutation localized upstream from nucleotide position 946. To test this hypothesis, the intron 6 regions in AC and RhD-positive
genomic DNAs were amplified using primers located in exons 6 and 7 (A3 and A4, see Materials and Methods) and the
1.3-kb PCR genomic product was directly sequenced
(Fig 4). Patient AC was found to be
homozygous for a G A point mutation at the invariant G residue
of the 3 acceptor splice-site of intron 6. All members of the
family C, sequenced at the genomic level, were found to be heterozygous
(G or A) for the same mutation (not shown).
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| Fig 4.
RH50 gene analysis in patient AC. The G residue at the
3 acceptor splice-site of intron 6 is mutated to A in patient
AC, thus resulting in the skipping of exon 7. Normal splicings of
introns 6 and 7 are indicated by the broken lines. The approximate
position of primers A3 and A4, used to amplify
intron 6, is indicated. Intron and exon sequences are in lowercase and
uppercase, respectively.
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To characterize the mutation affecting the RH50 gene in AL, a
316-bp genomic fragment, encompassing exon 1 as well as 157 bp of
intron 1, was amplified (see Materials and Methods) and cloned into the
PCR II vector. Sequence analysis showed a GA transition at
the invariant first base of the 5 donor splice-site of intron 1 (Fig 5A). This point mutation created a
novel Rca I restriction site polymorphism in genomic DNA, which
was used to test the inheritance of the RH50 mutation in family
L by a PCR-restriction fragment length polymorphism (RFLP)
assay (Fig 5B). As expected, the 316-bp PCR product remained uncut in
BL (wife of the propositus) as well as in control DNAs, whereas it was
cleaved into two fragments of 158 bp each in AL (homozygous for the
mutation). The daughter LL exhibited a heterozygous pattern in which
both uncleaved and cleaved amplification products are present.
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| Fig 5.
Mutation of the RH50 gene in patient AL and its
inheritance in the family. (A) The G nucleotide of the 5 donor
splice-site of intron 1 in the control sequence is replaced by an A in
AL, thus creating a new Rca I site. (B) Tree of family L and
Rca I restriction pattern of the genomic PCR products are
shown. The 316-bp PCR product (see Materials and Methods) encompassing
the splice-site mutation shown in (A) was cleaved into two 158-bp
fragments in AL, demonstrating the homozygosity of the mutation in this
patient. As expected, the product from a control DNA was uncut. The
arrow shows the Rhnull proposita AL. The initials of each
family member are indicated. Intron and exon sequences are in lowercase
and uppercase, respectively.
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Quantification of Rh50 mRNA in Rhnull patients.
To elucidate the functional significance of the mutations affecting the
RH50 gene in AL and AC, the Rh50 mRNAs from these patients were
quantified by ribonucleotide protection assay (RPA) using the XK mRNA
as an internal standard. The results, shown in
Fig 6, definitely demonstrate that no Rh50
mRNA was detectable in the AL patient. Moreover, the amount of Rh50
mRNA in the AC sample was found to be reduced to about 54% of the
level detected in the RhD-positive control.
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| Fig 6.
Ribonuclease protection analysis of Rh50 mRNAs. Total
reticulocyte RNAs (12.5 µg) protected a 270 nt Rh50 fragment and a
207 nt XK fragment. Rh50 mRNA levels in AC, compared after normalizing
them to the XK internal standard, were about 54% of those detected in
the RhD-positive control. No Rh50 mRNA was detected in AL. RNA probes
of 396 nt and 333 nt for Rh50 and XK, respectively, migrate very close
to each other in the gel. A radiolabeled HaeIII digest of phage
 X174 was used as a size marker.
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DISCUSSION |
In the Rhnull patients of the regulator type studied so
far, the RH50 gene is altered by mutations that most likely
account for the lack or the severe reduction of RBC surface expression of the Rh50 glycoprotein.14,15 Therefore, this protein may be essential for the correct expression of the Rh antigens on RBCs.5,14 In the present study, we have identified new
mutations affecting different splice-sites of the RH50 gene in
2 unrelated Rhnull individuals.
Patient AC was found to be homozygous for a GA transition at
the invariant base of a 3 acceptor splice-site, which leads to
the skipping of the downstream exon 7 (Fig 4). Interestingly, the RPA
analysis showed that AC Rh50 mRNA was reduced to about half of the
amount found in the RhD-positive phenotype (Fig 6). This could be due
to either the lower transcriptional activity of the mRNA or its lower
stability. The abnormality of the Rh50 mRNA, disrupted by a 122 nt
deletion, seems more likely to favor the second hypothesis. The
presence of potential crytpic splice-sites in the intron 6 or exon 7 regions is ruled out by the fact that no PCR products were detected in
AC in amplification reactions encompassing the cDNA region
corresponding to exon 6 and exon 7 (not shown).
The premature termination of translation is expected to alter the
structure and conformation of the protein. Indeed, the predicted truncated protein would be 351 amino acids long (v 409 residues for the normal Rh50 protein), including a C-terminus region containing 36 new amino acids. Moreover, hydropathy analysis of the deduced protein sequence predicted a reverse orientation of the C-terminal region, which may become exposed extracellularly (not shown). However,
the mutated protein was not found on the RBC surface, because no
reactivity of both the 2D10 and the anti-N-terminus antibodies could
be detected by flow cytometry and Western blot analyses, respectively.
These results suggest that the mutant protein is either unstable
(degradation) or that it is not transported to the cell surface. In the
latter case, one may speculate that the C-terminal region of the Rh50
protein is critical for the assembly or transport of the Rh membrane
complex to the plasma membrane. Experimental confirmation of this
hypothesis will be necessary when an efficient system for the
coexpression of both Rh and Rh50 proteins will became available.
The mutation described in patient AL occurs at a critical position of
the 5 splice-site of intron 1 of the RH50 gene and therefore completely inactivates splicing. Indeed, mutations at the
canonical GT dinucleotide at the donor splice-site alter RNA processing
and can lead to several splicing defects.28-30 As clearly demonstrated by RPA analysis (Fig 6), the mutation affecting AL results
in the lack of Rh50 mRNA. This may be accounted for by the instability
and the consequent degradation of the misspliced RNA in the nucleus due
to either the inclusion of intron 1 (about 17.7 kb)7
and/or the appearance of a premature stop codon. Reduction of
the levels of mRNA associated with splice mutations and premature
termination codon has been reported for several mutant
alleles31-33 (and references therein).
In conclusion, we have described a novel type of mutation affecting two
different splice-sites of the RH50 gene, which identify an
additional molecular mechanism responsible for Rh deficiency. These
findings further substantiate the hypothesis that the RH50 mutants represent distinct alleles of the X°r gene, the
so-called Rh suppressor gene,34 which most likely function
by preventing the assembly and transport of the Rh complex to the RBC
membrane. Clearly, these mutants do not act as a transcriptional
regulators of the RH locus, because in all Rhnull regulator
individuals analyzed so far, the Rh transcripts are normally present
and their sequence is not altered. Mapping the Rh50 mutations in
Rhnull patients, therefore, might help to define critical
positions or domains of the Rh50 protein that are involved in the
assembly of the Rh membrane complex and the expression of Rh antigens.
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FOOTNOTES |
Submitted January 27, 1998;
accepted May 19, 1998.
Supported in part by a EC Grant No. BIO2-CT93-0348. G.M. thanks ORTHO
Clinical Diagnostics for financial support.
Address reprint requests to Jean-Pierre Cartron, PhD,
INSERM U76, Institut National de la Transfusion Sanguine, 6 rue
Alexandre Cabanel, 75015 Paris, France; e-mail: cartron{at}infobiogen.fr.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors thank L. Lebeck for the gift of blood sample AL and P. Bailly for the gift of the A23 antibody and helpful discussion.
| |
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Abstract
Introduction
Abstract