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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1458-1463
A Novel Single Missense Mutation Identified Along the RH50 Gene in a
Composite Heterozygous Rhnull Blood Donor of the Regulator
Type
By
C.A. Hyland,
B. Chérif-Zahar,
N. Cowley,
V. Raynal,
J. Parkes,
A. Saul, and
J.P. Cartron
From the Australian Red Cross Blood Service, Brisbane, Queensland;
Queensland Institute of Medical Research, Herston, Queensland,
Australia; and Unite Institut National de la Santé et de la
Recherche Médicale U76, Institut National de la Transfusion
Sanguine, Paris, France.
 |
ABSTRACT |
Rare individuals who lack all of the Rh blood group antigens are
called Rhnull and may be classified as "regulator" or
"amorph" types. The suppression of Rh antigen expression for
regulator types may be attributed to mutations of the RH50
gene, which is independent of the RH locus. The RH50 gene
encodes a glycoprotein that interacts with the Rh proteins to form a
functional complex within the red blood cell membrane. This report
describes an RH50 gene mutation for a previously unclassified
Rhnull donor. Sequencing cDNA clones from Rh50 mRNA
revealed a single base change (G836A) yielding a missense and
nonconservative mutation (Gly279Glu) within a predicted hydrophobic
domain for this membrane protein. Genomic DNA studies using polymerase
chain reaction (PCR) restriction analysis and sequencing showed that
the Rhnull propositus was a composite heterozygote for this
mutation, carrying two alleles with the A and G at nucleotide 836, respectively. In contrast, cDNA studies showed that only the A836
sequence was present, suggesting that the second allele with G836 was
apparently silent (no transcript detected). Family studies showed that
the mutant RH50 allele (836A) was inherited maternally, whereas
the silent RH50 allele (836G) was from paternal transmission.
These findings provide further evidence that rare but diverse genetic
alterations may occur along the RH50 gene where the
Rhnull syndrome of the regulator type occurs. The single
amino acid change (Gly to Glu) provides insight into the critical value
of these residues for assembly of the Rh antigen complex within the
membrane.
| |
INTRODUCTION |
RARE INDIVIDUALS WHOSE red blood cells
lack all of the known Rh antigens found within the Rh blood group
system are defined as Rhnull.1 These donors
exhibit a mild clinical syndrome, called Rh-deficiency syndrome,
characterized by a chronic hemolytic anemia in which the red blood
cells have a stomatocytosis morphology, an increased osmotic fragility,
an altered ion transport system, and abnormal membrane phospholipid
organization.2,3 Family studies show that two classes of
Rhnull types exist and arise from independent genetic
events. The "amorph" type is caused by homozygosity for a silent
allele at the RH locus, whereas the more common "regulator" type
is apparently caused by homozygosity for an autosomal suppressor gene
(Xor) that is genetically independent of the RH
locus.
The RH locus is located on chromosome 1p34.3-1p36.1 and contains two
highly homologous genes, D and CE, which have been
cloned and sequenced.4,5 The RhD antigen polymorphism
generally arises from complete absence of the D gene, and
individuals therefore may carry zero, one, or two copies of the
D gene, although the CE gene is almost invariably
present in two copies.6 Sequencing studies have shown that
the coding regions for RH genes are completely normal not only
for Rhnull individuals of the regulator type, as expected,
but also for one sample of the amorph type, suggesting that the
abnormalities in the latter arise from a transcriptional defect.7
The D and CE genes encode two major Rh proteins,
designated Rh30, that carry D and C/c, E/e specificities,8
although minor species arising by alternative splicing may also
occur.9 Within the red blood cell membrane, Rh30 proteins
interact with a number of other proteins that include a glycoprotein of
50 to 100 kD (designated Rh50), CD47, glycophorin B, and the
glycoproteins carrying the LW and Fy antigens.4,5 Protein
defects of the Rhnull cell membranes include the lack of
Rh30 and LW polypeptides and the absence or severe reduction of Rh50
and CD47.3 In addition, a reduced expression of the S/s
antigens on glycophorin B and of other blood group antigens
(Fy5, U, and Duclos) has been documented.10 The
gene encoding the Rh50 protein has been cloned and sequenced and maps
to chromosome 6p11-p21.1.11,12 Both the RH50 and
CD47 genes are candidates for the postulated gene suppressing Rh
antigen expression in Rhnull of the regulator type. In
support of this, three distinct abnormalities have now been described
along the RH50 gene for four regulator Rhnull.12
This report describes a novel single point mutation along the
RH50 gene for an Rhnull individual, Y.T., for which
the regulator or amorph type has never been formally documented,
although the donor's cells were used in several biochemical
studies.7,13-16 Preliminary family studies showed that
functional D and C antigens were transmitted from Y.T. to three
children, suggesting that Y.T. belonged to the regulator type. The
following molecular studies show the pattern of inheritance of the
novel mutation and also show that Y.T. was a composite heterozygous
donor, carrying one mutant RH50 allele and one
transcriptionally silent RH50 allele.
| |
MATERIALS AND METHODS |
Blood samples and Rh typing.
Blood samples were collected from the Rhnull propositus
Y.T., her husband, their three children, and Y.T.'s parents. The
parents are not related, and this is supported by the family history. Y.T.'s paternal grandfather was English and the paternal grandmother was French. They met in France during World War I and migrated to
Australia. Y.T.'s maternal great-grandparents both migrated from
Ireland and Scotland to a separate region of Australia. Y.T.'s parents
therefore grew up in these separate regions. The propositus Y.T., aged
49, is in good health. She was diagnosed with non-insulin-dependent diabetes in 1995. Her hemoglobin level (114 to 134 g/L between 1984 and
1991 and 105 to 116 g/L between 1995 and 1997) is at or below the lower
range of normal (normal range for females, 115 to 165 g/L). For Rh
phenotyping, standard serology techniques were used. D gene
dosage was performed by D genotyping using DNA amplification of exon 7 of the D gene as previously described.17
Flow cytometric analysis of Rh D antigen site density.
A suspension of 5% red blood cells in phosphate-buffered saline (PBS)
was prepared from frozen cells. Monoclonal anti-D IgG (Diagast, Lille,
France) was diluted 1:7.5 in PBS. The 5% (vol/vol) red blood cell
suspensions (50 µL) were mixed with 100 µL anti-D or with 5%
bovine serum albumin (BSA). The latter served as control. These
mixtures were incubated for 1 hour at 37°C and then washed three
times in saline. The anti-human Ig F(ab)2 fraction
conjugated to fluorescein (Silenus, Hawthorn, Victoria, Australia) was
diluted 1:20 in saline; 100 µL of this mixture was mixed with the
washed and sensitized cells and with the respective negative control for the cell blocked with BSA. Reaction mixtures were incubated at room
temperature for 1 half-hour, washed three times, and resuspended in 2 mL PBS before determining the fluorescence. A relative measure of D
antigen expression was established by determining the mean channel
fluorescence signals on a Coulter Epics XL-MCL Flow Cytometer (Coulter
Electronics, Sydney, Australia). The controls were samples for which
the absolute D gene dosage measurement was described previously.18
Reverse transcription coupled with PCR amplification.
Reticulocyte RNAs from 30 mL peripheral blood from the propositus Y.T.
were extracted by selectively lysing red blood cells by the Orskov
reaction.19 Total RNAs from different members of Y.T.'s
family were extracted from 10 mL fresh EDTA whole blood using the RNA
Isolation Kit (Stratagene, La Jolla, CA). One microgram of RNAs 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
were then subjected to PCR in a 50-µL reaction mixture containing
1× Taq buffer (50 mmol/L KCl, 20 mmol/L Tris (pH 8.3), 1.5 mmol/L MgCl2, and 0.01% gelatin), 0.2 mmol/L of the four
dNTPs, 50 pmol of each primer, and 2.5 U Taq polymerase
(Perkin-Elmer-Cetus, Norwalk, CT). RH50 cDNA from the
propositus Y.T. was amplified using the primer pair X and Y (Table
1) spanning the full coding sequence. PCR
conditions were as follows: denaturation for 1 minute at 94°C,
annealing for 1 minute at 55°C, and extension for 90 seconds at
72°C for 30 cycles. Relevant PCR fragments were purified on a 1%
low-melting agarose gel followed by a Wizard PCR preps minicolumn
(Promega, Madison, WI), and subcloned into a PCR II vector using the TA
cloning kit (Invitrogen, San Diego, CA). cDNA products
prepared from the family members of Y.T. were further amplified between
D and C primers (Table 1). PCR conditions were as follows: denaturation
for 5 minutes at 94°C, and 30 cycles of denaturation for 30 seconds
at 94°C, annealing for 45 seconds at 54°C, and extension for 30 seconds at 72°C. Primers D and C amplify sequences within an exon;
therefore, with this primer combination, the mRNA extract was treated
with DNAse before cDNA preparation to avoid genomic contamination.
Genomic DNA analysis of the RH50 locus.
Genomic DNA was extracted from peripheral whole blood collected onto
EDTA.20 Allele-specific PCRs with wild-type and mutant specific primers were performed using primer combinations A and C or B
and C (Table 1) to amplify the 836G wild-type allele or the 836A mutant
allele, respectively (expected size, 89 bp). PCR mixtures were as
already described using 200 ng genomic DNA. PCR cycles were as follows:
denaturation for 5 minutes at 94°C, and 30 cycles of denaturation for
30 seconds at 94°C, annealing for 45 seconds at 63°C for primers A
and C or 57°C for primers B and C, and extension for 30 seconds at
72°C. PCR products were subjected to electrophoresis on 4% Nusieve
(FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide
staining.
PCR-RFLP assay.
PCR products amplified from genomic DNA or cDNA with D and C primers
(101 bp) were digested for 1 hour at 37°C with 10 U MnlI in
the buffer provided (New England Biolabs, Beverly, MA) made up to 20 µL and including 100 mg/mL BSA. After digestion, the enzyme was
inactivated for 10 minutes at 65°C and the products were visualized
on 4% Nusieve gel electrophoresis.
Sequencing of PCR products from genomic DNA and from cDNA.
PCR products were separated by electrophoresis on 2% Nusieve and
excised and purified using the QIAEX II Agarose Gel Extraction Kit
(Qiagen, Chatsworth, CA). Sequencing of PCR products was
performed as suggested by the manufacturer. PCR extension products were purified by ethanol precipitation and sequenced on the Applied Biosystems (Foster City, CA) 373A DNA sequencer.
| |
RESULTS |
Rh antigen expression among family members of the Rhnull
propositus Y.T.
The Rh phenotype and genotype for the family members, deduced from
serology and D gene dosage measurements,17,18 are
shown in Table 2. The parents of the
Rhnull propositus Y.T. were both DCe/DCe, and Y.T.
therefore could only inherit DCe haplotypes. All three of
Y.T.'s children were DCe/ce genotypes. The ce
haplotype must have been transmitted from the father, E.T., as Y.T.
could only transmit the DCe haplotype. The D and
C antigens were expressed on red blood cells from all three
children, indicating that the D and Ce genes carried by
Y.T. were structurally normal. Interestingly, the flow cytometric
studies on two children, M.P. and P.T., indicate that the level of
expression of D antigen was below the normal range established
from 39 donors with DCe/ce types (Table 2). Values for M.P. and
P.T. were 71 and 91, compared with 174 ± 39 for the 39 controls,
indicating a reduction in Rh antigen expression for the obligate
heterozygote carrier of the regulator null trait. Additional typing
analysis with specific monoclonal antibodies indicated that red blood
cells from Y.T. lacked Rh50 glycoprotein and expressed only a low level
of CD47 (3,000 v 30,000 to 50,000 mol/cell), as expected for
Rhnull erythrocytes (data not shown). That the red blood
cells from Y.T. lack the Rh50 protein was reported previously,16 and is confirmed.
Analysis of the RH50 transcript in donor Y.T.
Reticulocyte RNAs isolated from Y.T. were reverse-transcribed, and the
RH50 cDNA was amplified using the X and Y primer pair. Sequence
analysis of the cloned PCR product (1.28 kb), corresponding to the
complete coding region of the cDNA, revealed a G to A transition at
nucleotide 836, creating a glycine to glutamic acid missense mutation
at residue 279 of the Rh50 protein.
Genomic detection of the RH50 mutation.
The G836A mutation abolishes an MnlI restriction site, and this
polymorphism was used to determine the presence of the mutation in the
genomic DNA of Y.T., the zygosity, and the inheritance of the mutation
in Y.T.'s family. A 101-bp fragment encompassing nucleotide 836 was
amplified from the genomic DNA of all family members using primer pair
D and C. MnlI cleavage of the 101-bp resulted in two fragments
of 79 and 22 bp. This pattern was observed for Y.T.'s father J.H.,
husband E.T., and children D.T. and P.T., consistent with their
carrying two RH50 alleles with the G836 (Fig
1). In contrast, Y.T.'s mother A.H., Y.T.
herself, and her daughter M.P. showed the 101-bp fragment present after
digestion in addition to the 79- and 22-bp cleaved products, consistent with the presence of two alleles with and without the G836 (Fig 1).
This indicated that the A836 mutation was present in the genomic DNA of
Y.T. at the heterozygous state. Sequencing the 101-bp products for
A.H., Y.T., and M.P. showed mixed sequences at nucleotide 836, with
both A and G present (data not shown).
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| Fig 1.
Family tree for the Rhnull donor Y.T.,
showing inheritance of the RH50 allele containing the G836A
mutation. PCR products were prepared from genomic DNA using primers D
and C that span nt 820 to 908. Primer D has a 12-base tail added. The
101-bp PCR product obtained from each family member was digested with
MnlI, and the fragments were analyzed on agarose gel.
Products containing the G836 sequence are cut into 22- and 79-bp
fragments, whereas those with A836 are uncut. The polymorphism at nt
836 (A or G) is indicated below each family member.
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Expression of the RH50 gene among Y.T.'s family members.
To determine whether the two RH50 alleles carried by Y.T. were
both transcribed into mRNA, the 101-bp PCR product was amplified from
the cDNA of Y.T. using D and C primers. A.H. and M.P. cDNAs were used
as the controls, as both carry the A836 mutant and their second
RH50 allele (G836) must therefore be normal. The PCR products from A.H., M.P., and Y.T. cDNAs were subjected to restriction analysis
with MnlI. After digestion, A.H. and M.P. cDNAs yielded two
products at 101 and 79 bp, respectively, consistent with both carrying
two alleles (G836 and A836) and both alleles being transcribed into
mRNA (Fig 2). In contrast, the 101-bp
fragment from Y.T. cDNA was not cut with MnlI. All PCR
fragments were also directly sequenced to further check whether the
mRNA could be detected from both RH50 genes. A.H. and M.P. were
again used as controls. A.H. and M.P. showed mixed sequences at
nucleotide 836, with both A and G present. In contrast, Y.T. showed
only one sequence corresponding to A836, which was consistent again
with the second RH50 gene (G836) not being transcribed. This
silent allele must have been inherited from the father J.H.
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| Fig 2.
Expression studies of the G836 or A836 alleles. cDNA was
prepared from RNA extracts of blood samples and amplified by PCR using
primers D and C. The 101-bp PCR products were digested with MnlI, and the fragments were analyzed on agarose gel. Products containing the G836 sequence are cut into 22- and 79-bp fragments, whereas those with A836 are uncut. After digestion, only the 101-bp fragment was detected with Y.T., indicating that only the allele carrying the A836 mutation is transcriptionally expressed. In contrast,
both the 101- and 79-bp fragments were present in A.H. and M.P.,
indicating that both A836 and G836 are expressed.
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Frequency of the G836A transition in the population.
Allele-specific PCR was used to test for the frequency of the G836A
transition in blood donors from the Brisbane Center. Using the primer
combination pair A and C specific for the wild-type 836G nucleotide, an
89-bp product was amplified for all 44 random individuals tested. In
contrast, no product was obtained among these 44 donors using the
primer pair B and C specific for the mutant 836A nucleotide (data not
shown). E.T. and Y.T. were used as controls in these experiments. E.T.
showed a product with the A and C primer pairs only, and as expected,
Y.T. showed products with both primer pairs A and C and B and C. This
result indicated that the G836A mutation did not correspond to a
frequent polymorphism.
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DISCUSSION |
In this study, molecular alteration of the RH50 gene that
encodes a major membrane component of the Rh membrane complex was characterized in a previously undescribed Rhnull family.
The affected propositus Y.T. is a composite heterozygote, carrying two
distinct RH50 alleles, one with the 836G nucleotide and one
with the 836A nucleotide (Fig 3). The
abnormality for the RH50 allele carrying the 836G nucleotide
most likely resides at either the transcription or the
posttranscription level, as no mRNA could be detected in the
reticulocytes from Y.T. It follows that the nucleotide sequence for the
coding region of this allele could not be deduced from the cDNA. The
presence of transcriptionally silent RH50 genes has been
reported for two other Rhnull of the regulator type, one
homozygous and one heterozygous.12 The molecular basis for the abnormality has not been clarified and may require additional studies that analyze promoter and splice junction sites. The second RH50 allele of Y.T. carries the A836 nucleotide and is
successfully transcribed into mRNA, although no Rh50 protein could be
detected at the red blood cell surface. The cDNA sequence of this
mutant is identical to the previously published sequence, with the
exception of the single nucleotide substitution G836A. Two other
examples of structural abnormalities along the RH50 gene have
been described previously for Rhnull types.12
These involved, first, a two-nucleotide change and two-nucleotide
deletion for two Rhnull individuals from South Africa, and
second, a single nucleotide deletion observed for one
Rhnull individual of Swiss origin. In both cases, the changes produced a frame shift of the coding sequence with a premature stop codon, and the Rh50 protein was not detected on red blood cells.12
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| Fig 3.
RH and RH50 genotypes of family YT. RH50 alleles are as
follows: +, wild-type carrying a G at nt 836; s, transcriptionally silent allele carrying a G at nt 836; *, mutant allele carrying an A at
nt 836. The RH genotype of the Rhnull propositus Y.T. is
shown in brackets since it is not phenotypically expressed.
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The single nucleotide substitution described in the family of Y.T.
results in a single amino acid change from Gly279Glu in the Rh50
protein. This change may be attributed to the Rhnull defect
for three reasons. First, this change was not detected among a random
selection of the community, indicating that it is not a frequently
occurring polymorphism. Second, it is the only alteration along an
otherwise normal RH50 gene that is transcribed successfully.
Third, hypothetical models to explain the membrane topology of the Rh50
protein indicate that the Rh50 glycoprotein has 12 hydrophobic
domains.11,21 The nonconservative change described in this
report from a Gly to a Glu occurs within the ninth hydrophobic domain,
and by introducing a charged residue, it has the potential to disrupt
the conformation of the protein. This conformational change could
potentially affect either the transport into the membrane
and/or the insertion and interaction with other proteins within
the membrane. This would provide an explanation for the previous
studies, confirmed here, showing that neither Rh50 nor Rh30 proteins
can be detected in the membrane of Y.T.14,15 In addition,
this hypothesis is consistent with more recent studies showing that the
Rh50 glycoprotein interacts with the Rh30 protein during biosynthesis,
and this appears essential for transport of the Rh30 protein across the
membrane compartments.15,21
While this report suggests that a single missense mutation in the
RH50 gene for Y.T. prevents expression of the Rh50 glycoprotein in the membrane, it does not necessarily conflict with the possibility that a truncated Rh30 protein isoform, resulting from alternative splicing along the RH30 gene or from a posttranscriptional
modification, may be inserted into the membrane.9
Conceivably, the structural requirement permitting correct membrane
insertion may be preserved in such a protein isoform, although it is
lost in the Rh50 mutant protein of Y.T.
It is of interest that a mutant membrane protein, aquaporin-2, showed a
single nonconservative change along the gene giving rise to a serine to
proline transition in a hydrophobic membrane-spanning domain. This
mutation was identified in a patient with nephrogenic diabetes
insipidus, and the inability to facilitate water transport was
attributed to an impaired routing to the plasma membrane.22
It is also of interest that one other example of a single nucleotide
change in the RH50 gene has been described for an individual with reduced Rh antigen expression classified as
Rhmod.12 The nucleotide change resulted in a
serine to asparagine change at position 79 along the second hydrophilic
segment of the Rh50 glycoprotein that protrudes within the cytosol of
the red blood cell. Low amounts of Rh50 protein were detected in the
membrane, consistent with a reduced transport and assembly process. It
is likely therefore that a range of substitutions along the
RH50 gene may be responsible for the varying degree of
modulated Rh antigen activity observed.12 However, the
change observed in Y.T. occurred in a critical position preventing any
expression. Definitive proof that the missense mutation G836A is
sufficient to account for the Rhnull phenotype of Y.T. will
require mutagenesis and expression of the RH50 mutant in an
expression system in which the RhD expression may also be examined. One
such system is now available for D expression in the erythroleukemic
K562 cells transduced with retroviral vectors; however, these have an
endogenous Rh50 protein.8 There is currently no evidence
that both RhD and RH50 (or mutants) may be expressed in a
nonerythroid cell line. Studies of such systems are under investigation
in Paris.
While the Rh50 glycoprotein appears essential for transport
and/or expression of Rh30 polypeptides, the Rh50 glycoprotein does not have such a strict requirement for coexpression with the Rh30
polypeptides. Indeed, a low amount of the Rh50 glycoprotein is
detectable with a murine monoclonal antibody on red blood cells from a
subgroup of Rhnull individuals classified as U-positive (reacting weakly with anti-U antibodies), but not on those that are
U-negative.23 Rhnull U-positive samples are
less frequent than Rhnull U-negative samples, but occur for
all amorph types and occasionally for regulator types of
Rhnull.15,24 Red blood cells from both types
exhibit a variable degree of suppression of Ss (and U) antigens, which
has been correlated with a 60% to 70% decrease of glycophorin
B.25 Although Rh30 proteins are undetectable on these
cells, they still carry a small amount of Rh50 glycoprotein. One such
example (patient D.A.A., Rhnull amorph type) with an intact
RH50 transcript was reported in our previous report12 and showed that minor amounts of Rh50 glycoprotein may still be expressed at the red blood cell surface even in the absence of Rh30 proteins. Why reduced Rh50 expression is restricted to
some Rhnull cells only, which apparently have a similar
content of glycophorin B,24 is presently not understood.
That some Rhnull cells may have a structurally altered
glycophorin B is unlikely, but has not been formerly excluded. It is
possible but not proven that Rh50 expression in certain
Rhnull cells is permitted by some unidentified factor(s).
This will result in the Rhnull U-positive phenotype,
following protein interaction between glycophorin B and the residual
amount of Rh50 glycoprotein, as previously postulated.21 Clarification of the chemical nature of the U antigen may help to solve
this issue.
The family study for Y.T. showed that the transcriptionally silent
RH50 allele was inherited on the paternal side and was transmitted from the Rhnull mother (Y.T.) to one daughter,
D.T., and one son, P.T. The second allele, carrying the hitherto
unreported mutation G836A, was inherited from the mother (A.H.) and
transmitted on to one daughter, M.P. The RH genes for Y.T. were
normal, as the D and C antigens expressed by all three children could
only have been inherited from the mother. However, the D antigen
expression was reduced for both the daughter (M.P.) carrying the A836
gene and the son (P.T.) carrying the silent RH50 gene, as
initially reported for obligate Rhnull
heterozygotes.26 This again would be consistent with their
carrying only a single dose of functional RH50 gene.
These findings definitively classify Y.T. as a regulator
Rhnull with a DCe/DCe genotype. Most cases of
Rhnull arise by consanguinity and result from the
homozygosity for a defective gene.7 However, the donor
reported here represents one of the rare cases in which two distinct
alleles inherited from nonconsanguineous parents have produced the null
trait. The family history supports these findings. The results from
these studies provide further evidence that abnormalities along the
RH50 gene are responsible for the Rhnull trait.
They also provide some insight into the critical nature of the protein
structure before successful assembly and expression of the Rh antigen
complex can occur.
| |
FOOTNOTES |
Submitted July 21, 1997;
accepted October 15, 1997.
Supported by the National Health and Medical Research Council of
Australia, The Alexander Steele Young Memorial Lions Foundation, and
Brisbane North Regional Health Authority Liver Transplant Unit.
Address reprint requests to C.A. Hyland, PhD, Australian
Red Cross Blood Service, Queensland, PO 10325, Brisbane, 4000, Australia.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We warmly and gratefully acknowledge the support and interest that Y.T.
and her family have continued to show throughout these studies.
| |
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Abstract
Introduction
Abstract