Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1776-1784
Rh50 Glycoprotein Gene and Rhnull Disease: A Silent
Splice Donor Is trans to a Gly279
Glu
Missense Mutation in the Conserved Transmembrane Segment
By
Cheng-Han Huang,
Zhi Liu,
Guangjie Cheng, and
Ying Chen
From the Laboratory of Biochemistry and Molecular Genetics, Lindsley
F. Kimball Research Institute, New York Blood Center, New York, NY.
 |
ABSTRACT |
Rhnull disease includes the amorph and regulator types
that are thought to result from homozygous mutations at the
RH30 and RH50 loci, respectively. Here we report an
unusual regulator Rhnull where two G
A nucleotide
(nt) transitions occurred in trans, targeting different
regions of the two copies of Rh50 gene. The nt 836 GA mutation was a missense change located in exon 6; it converted Gly into
Glu at position 279, a central amino acid of the transmembrane segment
9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the
presence of both 836A(Glu279) and 836G(Gly279)
alleles. A detailed analysis of gene organization led to the
identification in the Rh50(836G) allele of a defective donor splice
site, caused by a GA mutation in the invariant GT element of
intron 1. This is the first known example of such mutations that has
apparently abolished the functional splicing of a pre-mRNA encoding a
multipass integral membrane protein. With a silent phenotypic copy in
trans, the negatively charged Glu279 residue may
disrupt TM9 and impair the interaction of the missense protein with
Rh30 polypeptides. To evaluate the significance of the mutation, we
took a comparative genomic approach and identified Rh50 homologues in
different species. We found that Gly279 is a conserved
residue and its adjacent amino acid sequence is identical from
Caenorhabditis elegans to human. These findings
provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in
protein-protein interactions involving Rh complex formation.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
RH30 POLYPEPTIDES and Rh50 glycoprotein
are members of the same family expressed mainly in erythroid cell
lineages.1-5 They share a significant sequence homology, as
well as a similar 12-transmembrane (TM) topology, but their genetic
loci are localized to chromosomes 1p34-36 and 6p11-21.1,
respectively.6-8 The RH30 locus comprises two
tightly linked, highly homologous genes referred to as RHD and
RHCE: the former expresses D antigen and the latter CcEe
antigens in the ce, cE, Ce, or CE allelic combinations.9-13
The RH50 locus appears to harbor a single copy
gene8,14 and its product, the Rh50 glycoprotein, does not
carry Rh antigens, but may serve as a coexpressor by forming a membrane
complex with Rh30s through protein-protein interactions.
The rare occurrence of Rhnull disease, which manifests a
varying degree of chronic hemolytic anemia and
stomatocytosis,15,16 highlights a critical role of Rh
proteins in maintaining the integrity of the erythrocyte membrane.
Classic genetic studies established Rhnull as a recessive
disorder that occurs by two distinct mechanisms.15 The
amorph type arises by mutations at the RH30 locus itself that silence the gene function, whereas the regulator type results from
mutations at a separate suppressor locus. This hypothesis has been
confirmd by the identification of various molecular defects in the Rh30
and Rh50 genes in unrelated Rhnull
patients.14,17-19
Recently we characterized the organization of RH50, showing
that its coding sequence is distributed in 10 exons.14 This information has facilitated our search for the underlying suppressor mutations in regulator Rhnull probands. Here we describe an
unusual example where the phenotypic expression of Rh50 gene is
inactivated by two different mutations in trans configuration.
One mutation is a GA transition at +1 position of intron 1, which inactivates the cis-acting GT element of the donor splice
site. We show that this mutation has apparently abolished RNA splicing
of the mutated Rh50 allele. The other mutation is also a GA
transition, but it causes a Gly279Glu missense
change in the TM9 segment, as recently described by Hyland et
al18 in family studies. By searching Rh50 homologues, we
show that the Gly279Glu missense mutation has
targeted a protein domain conserved from Caenorhabditis elegans to humans.
| |
MATERIALS AND METHODS |
Blood samples, Rh phenotyping, and Western blotting.
Control blood samples were drawn from normal human blood donors. The
Rhnull blood sample was from a female proband (YT) of Australian origin. Serologic test confirmed the null status of Rh
antigens (D-C-E-c-e- and Rh17-). Typing of Rh-related antigens showed
that the proband's red blood cells lacked the 2D10,
LW, and U antigens, but weakly expressed the CD47, S, and s antigens. Western blot was performed using the monoclonal antibodies 2D10 against
Rh50 and LOR-15C9 against RhD, as described.20,21
Southern blotting.
Genomic DNA was isolated from leukocytes22 and digested
with BamHI and Sph I enzymes. The blots were hybridized
with 32P-cDNA probes labeled by the random primer extension
method.23 The Rh50 probe was a full-length cDNA isolated
from a placenta cDNA library constructed with the Marathon
Amplification Kit (Clontech, Palo Alto, CA). This cDNA contains all of
the coding sequence of 409 amino acids and spans nucleotides (nt) -6 to
1256.14 Rh30 cDNA probes are region-specific and span,
respectively, the 5
(exon 1-3, nt 1-480), middle (exon 4-7, nt
515-1073) and 3 portions (exon 8-10 plus 3-untranslated
region [UTR], nt 1074-1456).24
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA was isolated from hemolysates25 and extracted
with Trizol reagent (GIBCO, Gaithersburg, MD). Rh50 and Rh30
transcripts were analyzed by gene-specific RT-PCR and direct
sequencing.14,24 Primers for cDNA synthesis and PCR are
shown in Table 1. The Rh50 or Rh30 mRNA was
converted into cDNA with a 3-UTR primer and avian myeloblastosis
virus (AMV) RT (Promega, Madison, WI). The cDNA was amplified by two
pairs of specific primers spanning the entire coding region of Rh50 or
Rh30. Conditions for cDNA synthesis and PCR were as detailed
previously.14,24 The cDNA was directly sequenced,
subcloned, and subjected to single strand conformation polymorphism
(SSCP) analysis.
Genomic amplification and mutation screening.
The Rh50 and Rh30 gene sequences were amplified using total genomic DNA
as template. Based on our published report,14 primers were
designed (Table 1) to amplify the Rh50 gene in 12 segments, which
together cover the 5 promoter, 3-UTR, 10 exons, and
flanking splice junctions. Except for 5P2, 5P3, 1Ps,
Ex-6a, and 3-UTa, the others primers all reside in noncoding
introns. The genomic products were analyzed by restriction digestion,
SSCP, subcloning, and sequencing. Exonic sequences of RhD and RhCE were
amplified and analyzed by diagnostic restriction enzymes, as detailed
previously.24
Subcloning and DNA sequencing.
After an initial identification of the two alterations in Rh50 gene,
both cDNA and genomic fragments were subcloned to verify the genotype
of the Rhnull proband. The amplified products were ligated
into the pCR2.1 TA vector according to the supplier's specifications
(Invitrogen, San Diego, CA). After transformation, positive clones with
the insert were screened by color selection with X-Gal and further
identified by PCR with the original amplimers. Multiple clones
were picked and miniprep plasmids were sequenced on both strands on a
Model 373A sequencer (Applied Biosystems, Foster City, CA).
Identification of Rh50 gene homologues.
The discrete domains of human Rh50 were used as queries to search
various databases including dbEST (expressed sequence tags) (National
Biotechnology Information Center, National Institutes of
Health).26 Based on sequence homology, degenerate or exact oligonucleotide primers were designed and used to amplify cDNA or
genomic libraries. For analysis of mouse Rh50, the cDNA library was
generated by using total RNA isolated from the mouse erythroleukemic (MEL) cell line. The PCR products were sequenced, giving further information on primer design and cDNA PCR. For identification of the
Rh50 homologue in C elegans, total RNA was
isolated from mixed stage worms using the standard
method.27 The cDNA was synthesized and amplified with two
exact primers whose sequences were retrieved from GenBank (accession,
Z74026).28 For C elegans Rh-1 homologue,
the following primers were used:
5-CCAGAATGTGGCGGTTCTACATCG-3 (nt -5 to 19, sense, initiation codon underlined), and
5-TTCTCAATCTCTCCTGTATCCCCC-3 (nt 1410-1433, 3-UTR,
antisense). For C elegans Rh-2, the two primers were:
5-CTCTCTTTCAGTAATTCAACC GAAAC-3 (nt -31 to -6, 5-UTR, sense) and 5-CATGAACGACTAACGAGCAATAAAAC-3
(3-UTR, antisense). Full-length cDNA sequences were assembled
and regions relevant to the Rhnull mutation were aligned
for comparison using the DNASIS program (Hitachi, South San Francisco,
CA).
| |
RESULTS |
Gross structure of Rh50 and Rh30 genes.
Figure 1 shows blot analysis of
RH50 and RH30 loci. No gross alteration of RH50
was noted in the proband (Fig 1A, left). There was also no apparent
reduction in band intensity, suggesting the presence of two intact
copies of the Rh50 gene. With regard to RH30, the proband
showed a banding pattern conforming to the DCe/DCe genotype29 (Fig 1A, right). This genotype was
verified by transcript sequencing of RhD and RhCe, whose sequence was
identical to that of D+C+c-E-e+ normal subjects. These results excluded
the involvement of a mutated RH30 locus in Rhnull
(YT), suggesting that some subtle alteration(s) had silenced the
phenotypic expression of two copies of the Rh50 gene (Fig 1B).
View larger version (41K):
[in this window]
[in a new window]
| Fig 1.
Gross structure of RH50 and RH30 in
Rhnull as determined by Southern blots. (A) Southern blot
analysis of the genes encoding the Rh50 and Rh30 proteins. Lane 1, RhD-positive; lane 2, RhD-negative; and lane 3, Rhnull(YT).
The BamHI and Sph I blots were probed with the
full-length Rh50 cDNA and Rh30 cDNA (exon 4-7), respectively. Size
markers (in kb) of Hind III-cleaved lambda phage DNA are shown
at the left margin. (B) Schematic representation of the proband's
genotype for RH50 and RH30, whose chromosomal location
is indicated. Note that the proband is homozygous for the two tightly
linked and actively expressed Rh30 genes, RHD and RHCe.
However, the two Rh50 alleles carry different, subtle changes as
suppressor mutations (indicated by crosses).
|
|
Identification of a missense mutation in Rh50 cDNA and Western blot
analysis.
To define the underlying defect, we determined the structure of Rh50
transcripts. Although the Rh50 cDNAs from Rhnull (YT) showed no apparent difference in size (Fig
2A), it contained a point mutation, a GA transition at nt 836 of exon 6 (Fig 2B). This mutation resulted in a
Gly279Glu missense change. Significantly, a
single 836A peak is seen in the sequencing profile (Fig 2B), indicating
the 836A allele as the only Rh50 transcript expressed in
Rhnull cells. To confirm this expression, we subcloned the cDNA, picked 40 independent clones, and performed PCR-SSCP analysis. All of the clones were mutant forms containing the 836A residue (not
shown), suggesting that the 836A(Glu279) change is a
suppressor mutation. To examine the expression of Rh30 and Rh50,
immunoblot analysis of red blood cell membrane proteins
was performed. Compared with controls, no bands with either LOR-15C9 or
2D10 were seen in the proband (Fig 2C), suggesting that no significant
amount of Rh30 or Rh50 was present in plasma membranes prepared from Rhnull cells.
View larger version (45K):
[in this window]
[in a new window]
| Fig 2.
Identification of nt 836 GA
(Gly279Glu) missense mutation in the Rh50
transcript. The strategy for synthesis and amplification of Rh50 cDNA
is shown. The Rh50 mRNA was reverse-transcribed with the 3-UTa
primer and then amplified with two pairs of upstream primers. (A) The
resultant cDNA products, designated by the primers used, were
electrophoresed on 1.8% agarose gel. Size markers of
HaeIII-cleaved X174 DNA are shown at left. Lanes are
designated as in Fig 1. No difference in size is seen between normal
subjects and Rhnull(YT). (B) DNA sequencing profiles for
the nt 836 GA missense change. The G residue is seen in
normal subjects, whereas the A residue is seen in
Rhnull(YT) only. (C) Immunoblot analysis of RhD and Rh50 in
red blood cell membranes. Lanes are the same as in
(A). The antibodies used are indicated. Note that no band is seen in
Rhnull (YT), even though a normal RhD and a missense Rh50
transcript were expressed. Note also that RhD-negative control did not
react with LOR-15C9.21
|
|
Heterozygosity of nt 836A mutation.
As shown by Southern analysis (Fig 1A), the proband carried two copies
of Rh50 gene. The finding that only the 836A transcript was expressed
(Fig 2B) suggested that the proband was either a homozygote of the
mutation or a double heterozygote with an additional mutation in the
other copy of Rh50 gene. To distinguish these possibilities, we
determined the genomic sequence encompassing exon 6 (Fig 3A). The exon 6-containing fragment
from Rhnull(YT) harbored both G and A at nt 836 (not
shown). This allelic coexistence was further indicated by SSCP analysis
showing two single-stranded forms (Fig 3B). Sequencing of the subcloned
inserts verified the heterozygosity for 836G and 836A alleles in
Rhnull (YT) (Fig 3C).
View larger version (45K):
[in this window]
[in a new window]
| Fig 3.
Heterozygosity for the 836G and 836A Rh50 alleles in
Rhnull(YT). The genomic region encompassing exon 6 of the
Rh50 gene was amplified by two pairs of primers In-5s/Ex-6a and
In-5s/In-6a (Table 1). (A) 1.8% agarose gel electrophoresis of the
amplified In-5s/Ex-6a (246 bp) and In-5s/In-6a (380 bp) fragments.
Lanes are designated as in Fig 1. (B) SSCP analysis of the In-5s/Ex-6a
fragments followed by silver staining. The shifted band seen in
Rhnull(YT) (lane 3, arrow-indicated) is the single-stranded
form containing 836A. (C) Sequencing profiles of the subcloned
In-5s/In-6a inserts derived from Rhnull(YT). The presence
of both GGA and GAA codons (denoted by two arrows) confirmed
the proband to be heterozygous for the missense mutation.
|
|
Exon/intron structure and splice donor mutation.
To search for the unknown mutation in the Rh50(836G) allele, genomic
fragments for the 5 and 3 regions, all exons, and
flanking exon/intron junctions were amplified and sequenced
(Fig 4A). This screening identified a
single GA transition at +1 position of the donor splice site
in intron 1 (Fig 4B). This point mutation was also present in a
heterozygous state, as the strength of A and G signals was equal (not
shown). Because the GA transition introduced a novel
BspHI site (TC/GTGA to TC/ATGA), a
diagnostic assay was done. As shown, the amplified fragments from
Rhnull gave rise to two smaller bands for the mutant allele
and one undigested band as seen in normal subjects
(Fig 5A, see page 1780). Subcloning and
sequencing of the proband's genomic products verified the presence of
both wild-type and mutant donor splice sites (Fig 5B, see page 1780).
Thus, although the Rh50(836G) allele had a wild-type coding sequence,
the splice donor mutation had blocked the functional splicing of its
pre-mRNA.
View larger version (47K):
[in this window]
[in a new window]
| Fig 4.
Exon/intron structure and donor splice site mutation of
the silent Rh50 (836G) allele. Mutation screening of the silent
Rh50(836G) allele in Rhnull (YT) was performed by genomic
PCR. The amplified products were each designated by the two primers
used (Table 1). (A) 1.8% agarose gel electophoresis of the 11 genomic
products. Note that segment 5P2/5P3 is upstream of and
overlapping with segment 1Ps/In-1a. (B) The exon/intron structure of
the silent Rh50 (836G) allele. Nucleotide sequences of the acceptor and
donor splice sites are shown and compared with the consensus ones at
bottom. Intronic nucleotides are denoted by lower-case letters.
The ga mutation at +1 position of intron 1 that destroys
the invariant gt-element of the donor splice site is bolded and marked
by an arrow.
|
|
View larger version (46K):
[in this window]
[in a new window]
| Fig 5.
Heterozygosity for the splicing donor mutation as shown
by Bsp HI analysis and subcloning. (A) Restriction digestion of
genomic segments 1P/In-1a by Bsp HI. Lanes 1 and 2, normal;
lanes 3 and 4, Rhnull (YT). ( ), uncut; and (+),
Bsp HI cut. Note that no digestion is seen in controls. For
Rhnull (YT), the size of the uncleaved fragment from the
Rh50(836A) allele and the two smaller fragments expected of the
Rh50(836G) allele is indicated. Shown schematically at the bottom are
the wild-type or mutant exon 1/intron 1 boundary (triangles) and
recognition sequence of Bsp HI. (B) Sequencing profiles of the
subcloned fragments 1P/In-1a from Rhnull (YT). Two types of
the inserts that carry either the G or A residue at +1 position of
intron 1 (IVS) were identified, confirming the proband to be
heterozygous for the splice donor mutation.
|
|
Localization of Gly279Glu mutation to a
conserved protein domain.
Owing to the difficulty in expressing the Rh complex, we took a
comparative genomic approach to assess the significance of the
Gly279Glu mutation and to infer its contribution
to the Rhnull phenotype. We reasoned that if
Gly279 is critical to the function of Rh50, it would be
conserved in Rh50 homologues from different species. Homologues were
identified in C. elegans and mice and their full-length cDNA
sequences were deposited in GenBank. Figure 6 shows the alignment of amino acid sequences from the three species in
regions relevant to the 836GA
(Gly279Glu) mutation. Gly279 is
indeed a conserved residue from C elegans to human and its adjacent amino acid sequence is identical in a contigous 7-residue stretch among the three species. Notably, the region also is highly conserved in the Rh30 proteins9-13 and in the
marine sponge Rh homologue.30
View larger version (39K):
[in this window]
[in a new window]
| Fig 6.
Conservation of Gly279 and TM9 in Rh50
homologues from three species. Comparison of amino acid sequences
(equivalent to residues 270-315 in humans) encoded by exon 6 of Rh50
homologues. The Gly279Glu (G279E) mutation found
in Rhnull (YT) is indicated by an arrow. h,
Homo sapiens; m, Mus musculus; c,
C elegans; and g, Geodia cydonium. Identical
amino acids are indicated by colons. Note that exon 6 is identical in
size between the human and mouse Rh50, but splits into two exons in
C elegans Rh-1. Predicted TM9 and TM10 are boxed. The sequences
for human Rh30s, RhCE (upper), and RhD (lower) are also aligned. Note
the two consecutive glycine residues (G279G280)
and their flanking amino acids are conserved in the Rh family of
proteins. In the TM9 of Rh50 homologues, each pair of the three
substitutions (A275S, V283I, and T285S) is similar in nature, rendering
a 100% similarity between C elegans and Homo
sapiens.
|
|
| |
DISCUSSION |
Family studies show that the Rhnull phenotype is often
displayed by homozygotes of a given mutation transmitted on
consanguineous background.15 However, the regulator form of
Rhnull may also result from heterozygosity of compounded
mutations in the Rh50 gene. Rhnull (TB) appeared as a case
like this, although the exact defect in one of two copies of
RH50 had not been defined.17 Here we characterized
the first double heterozygote of regulator Rhnull, showing
that RH50 harbored one splice donor mutation and one missense
mutation. These two mutations must occur in trans or otherwise
the expression of a wild-type allele should be observed. This
conclusion is supported by the pattern of family inheritance that is
not known to be consanguineous.18 It is also evident that
both mutations have contributed to the null phenotype, as neither Rh
antigens nor Rh30 and Rh50 proteins were detectable in the red
blood cell membrane. Our findings suggest a molecular model in which the mutations each affect RH50 at a different
level of gene expression (Fig 7).
View larger version (31K):
[in this window]
[in a new window]
| Fig 7.
Model for regulator Rhnull caused
by two mutations in trans configuration. Of the two mutant
copies of RH50 (vertical bars), one contains the 836A mutation
and the other carries a defective splice donor in intron 1 (circled a).
The RH30 locus is also illustrated whose genomic structure and
transcript expression are apparently normal. Diagrammed are the two
different levels at which the expression of RH50 is affected
(crossed arrows denote blocked steps). The inactive donor is
assumed not to alter transcription, but prevents pre-mRNA splicing.
This, in turn, leads to degradation of the accumulated hnRNA precursors
and no production of mature mRNAs. As a defect likely affecting
posttranslational events, E279 (Glu279) may disrupt TM9 of
Rh50, alter its conformation (indicated by a notch), and impair the
interaction with Rh30. As such, the Rh complex could not be assembled
in the cell membrane. Changes of the Rh50 mutant in stability or
intracellular routing might also contribute to the failure of Rh
complex formation.
|
|
The intronic GA transition resides in the invariant GT
element of splice donor that directs the processing of exon 1 and, therefore, defines a posttranscriptional splicing defect. Several lines
of evidence indicate that the mutation may totally prevent the
Rh50(836G) allele from functional splicing in Rhnull cells. (1) No aberrant (whether truncated or elongated) cDNA forms containing 836G were detected. (2) Direct sequencing showed that only the 836A(Glu279) allele was expressed. (3) Forty independent
cDNA clones were shown by SSCP analysis to be derived from the 836A allele, but not the 836G allele. As the half-life of hnRNAs is fairly
short, the failure to outsplice intron 1 caused by the inactive donor
site may lead to a rapid degradation of the mutant pre-mRNA (Fig 7).
This is in contrast to the GA mutation in the splice donor of
intron 7 that results in exon skipping and production of a shortened
mRNA in another regulator Rhnull patient.14
In a survey of human disease genes related to RNA
splicing,31 most mutations occur in internal donor GT and
acceptor AG invariant elements.32 Such mutations generally
cause a total skipping of the affected exon and may in some cases lead
to aberrant splicing by activating cryptic splice sites. Mutations
rarely target the splice donor for the extreme 5 exon. To our
knowledge, the splice donor mutation described here is the first
example involving a multipass membrane protein gene. The two other
examples of such splicing defects are found in human
thalassemic 
-globin genes that totally abolish the production of
mature mRNAs.33,34 This comparison suggests that the splice
donor mutations of intron 1 have a more profound effect on pre-mRNA
processing than those of internal introns. In fact, the mechanism
governing the splicing of 5 exons in a multiexon gene is not
fully understood; the present mutation may offer an interesting model
to address how the extreme 5 exon is scanned and whether its
splicing couples with transcription.35,36
As shown, the 836A missense allele was expressed as a normally spliced
transcript. Given its location, the point mutation may not impede
protein initiation or translation. Thus, its loss of function is more
likely related to the Gly279 Glu change causing some defects in posttranslational events (Fig 7).
Gly279 is predicted to lie at center of TM9 of the Rh50
protein,8 and its replacement with a negatively charged,
relatively bulky Glu would break up the continuity of hydrophobicity of
TM9. This perturbation alone may be sufficient to cause a
conformation change, disrupting the assembly of Rh30 and Rh50 as a
complex in the cell membrane. Second, Glu279 may
render the missense protein defective in cellular routing, as those
seen in cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporin-2 mutants.37,38
Third, the Glu279-containing mutant itself and Rh30
proteins may become vulnerable to proteolysis if they fail to form a
stable, correctly folded complex. Although these hypotheses remain to
be examined, the apparent disruption of the Rh complex by the missense
change provides evidence for the presence of additional interacting
sites in the C-terminal half of Rh50.39
The identification of Rh homologues in the mouse and C
elegans shows a high degree of sequence conservation in the TM
domains (Fig 6). This finding substantiates our viewpoint that the
Gly279Glu missense change occurs as a
loss-of-function mutation. Relevant to this study is the recent
characterization of an Rh homologue in the marine sponge G. cydonium.30 These observations together envisage an
ancient occurrence of the Rh protein family, reinforcing the hypothesis
that the Rh complex and its homologues possess an essential function
yet to be identified.1-5 This function is unrelated to
antigen presentation, but is critical to the architecture and
physiology of plasma membranes, including the red
blood cell membrane. Coincidentally, recent database
search has related human Rh50 and Rh30 to a large family of genes
encoding ammonia transporters.40 It is hoped that a full
appreciation of the structure/function relations of the Rh family will
emerge from a detailed molecular catalogue of Rhnull
mutations along with studies of Rh homologues in other organisms.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted April 24, 1998.
Supported in part by Grant No. HL54459 from the National Institutes of
Health.
Address reprint requests to Cheng-Han Huang, MD, PhD, Laboratory of
Biochemistry and Molecular Genetics, Lindsley F. Kimball Research
Institute, New York Blood Center, New York, NY 10021; e-mail:
chuang{at}nybc.org.
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 are grateful to Marion Reid for the Rhnull blood sample
and blood typing, Zhibing Wang for MEL cell total RNA, Anthony Radice
for nematode total RNA, and Tian Ye for assistance in library construction. We thank Antonie Blancher for monoclonal antibody LOR-15C9 and Albert von dem Borne for monoclonal antibody 2D10. Thanks
are also due to Olga Blumenfeld for comments on the manuscript and
Tellervo Huima-Byron and Yelena Oskov for production of photoprints.
| |
REFERENCES |
1.
Agre P,
Cartron J-P:
Molecular biology of the Rh blood group antigens.
Blood
78:551,
1991[Abstract/Free Full Text]
2.
Anstee DJ,
Tanner MJA:
Biochemical aspects of the blood group Rh (rhesus) antigens.
Bailliere's Clin Haematol
6:401,
1993[Medline]
[Order article via Infotrieve]
3.
Cartron J-P:
Defining the Rh blood group antigens: Biochemistry and molecular genetics.
Blood Rev
8:199,
1994[Medline]
[Order article via Infotrieve]
4.
Cartron J-P,
Agre P:
Rh blood groups and Rh deficiency syndrome.
Blood Cell Biochem
6:189,
1995
5.
Huang C-H:
Molecular insights into the Rh protein family and associated antigens.
Curr Opin Hematol
4:94,
1997[Medline]
[Order article via Infotrieve]
6.
Cherif-Zahar B,
Mattei G,
Le Van Kim C,
Bailly P,
Cartron J-P,
Colin Y:
Localization of the human Rh blood group gene structure to chromosomal region 1p34.3-1p36.1 by in situ hybridization.
Hum Genet
86:398,
1991[Medline]
[Order article via Infotrieve]
7.
MacGeoch C,
Mitchell CJ,
Carritt B,
Avent ND,
Ridgwell K,
Tanner MJA,
Spurr NK:
Assignment of the chromosomal locus of the human 30 kDa Rh (Rhesus) blood group-antigen-related protein (Rh30) to chromosome region 1p36.13-p34.
Cytogenet Cell Genet
59:261,
1992[Medline]
[Order article via Infotrieve]
8.
Ridgwell K,
Spurr NK,
Laguda B,
MacGeoch C,
Avent ND,
Tanner MJA:
Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (Rhesus) blood group antigen expression.
Biochem J
287:223,
1992
9.
Cherif-Zahar B,
Bloy B,
Le Van Kim C,
Blanchard D,
Bailly P,
Hermand P,
Salmon C,
Cartron J-P,
Colin Y:
Molecular cloning and protein structure of a human blood group Rh polypeptide.
Proc Natl Acad Sci USA
87:6243,
1990[Abstract/Free Full Text]
10.
Avent ND,
Ridgwell K,
Tanner MJA,
Anstee DJ:
cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood group-antigen expression.
Biochem J
271:821,
1990[Medline]
[Order article via Infotrieve]
11.
Le Van Kim C,
Mouro I,
Cherif-Zahar B,
Raynal V,
Cherrier C,
Cartron J-P,
Colin Y:
Molecular clnoning and primary structure of the human blood group RhD polypeptide.
Proc Natl Acad Sci USA
89:19925,
1992
12.
Arce MA,
Thompson ES,
Wagner S,
Coyne KE,
Ferdman BA,
Lublin DM:
Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals.
Blood
82:651,
1993[Abstract/Free Full Text]
13.
Kajii E,
Umenishi F,
Iwamoto S:
Isolation of a new cDNA clone encoding an Rh blood group antigen.
Hum Genet
91:157,
1993[Medline]
[Order article via Infotrieve]
14.
Huang C-H:
The human Rh50 glycoprotein gene: Structural organization and associated splicing defect resulting in Rhnull disease.
J Biol Chem
273:2207,
1998[Abstract/Free Full Text]
15. Race RR, Sanger R: The Rh blood groups, in
Blood Groups in Man (ed 6). Oxford, UK, Blackwell, 1975, p 220
16.
Nash R,
Shojania AM:
Hematological aspect of Rh deficiency syndrome: A case report and a review of the literature.
Am J Hematol
24:267,
1987[Medline]
[Order article via Infotrieve]
17.
Cherif-Zahar B,
Raynal V,
Gane P,
Mattei M-G,
Bailly P,
Gibbs B,
Colin Y,
Cartron J-P:
Candidate gene acting as a suppressor of the RH locus in most cases of Rh deficiency.
Nat Genet
12:168,
1996[Medline]
[Order article via Infotrieve]
18.
Hyland CA,
Cherif-Zahar B,
Cowley N,
Raynal V,
Parkes J,
Saul A,
Cartron JP:
A novel single missense mutation identified along the RH50 gene in a composite heterozygous Rhnull blood donor of the regulator type.
Blood
91:1458,
1998[Abstract/Free Full Text]
19.
Huang C-H,
Chen Y,
Reid ME,
Seidl C:
Rhnull disease: The amorph type results from a novel double mutation in RhCe gene on D-negative background.
Blood
92:664,
1998[Abstract/Free Full Text]
20.
Mallinson G,
Anstee DJ,
Avent ND,
Ridgwell K,
Tanner MJA,
Daniels GL,
Tippett P,
von dem Borne AEGKr:
Murine monoclonal antibody MB 2D10 recognizes Rh-related glycoproteins in human red cell membrane.
Transfusion
30:222,
1990[Medline]
[Order article via Infotrieve]
21.
Apoil PA,
Reid ME,
Halverson G,
Mouro I,
Colin Y,
Roubinet F,
Cartron J-P,
Blancher A:
A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblot.
Br J Haematol
98:365,
1997[Medline]
[Order article via Infotrieve]
22.
Huang C-H,
Guizzo M-L,
McCreary J,
Leigh N,
Blumenfeld OO:
Typing of MNSs blood group specific sequences in the human genome and characterization of a restriction fragment tightly linked to S-s-alleles.
Blood
77:381,
1991[Abstract/Free Full Text]
23.
Feinberg AP,
Vogelstein B:
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem
137:266,
1984[Medline]
[Order article via Infotrieve]
24.
Huang C-H:
Alteration of RH gene structure and expression in dCCee and DCW- red blood cells: Phenotypic heterozygosity versus genotypic heterozygosity.
Blood
88:2326,
1996[Abstract/Free Full Text]
25.
Goossens M,
Kan YW:
DNA analysis in the diagnosis of hemoglobin disorders.
Methods Enzymol
76:805,
1982
26.
Altschul SF,
Gish W,
Miller W,
Myers EW,
Lipman DJ:
Basic local alignment search tool.
J Mol Biol
215:403,
1990[Medline]
[Order article via Infotrieve]
27.
MacLeod AR,
Karn J,
Brenner S:
Molecular analysis of the unc-54 myosin heavy-chain gene of Caenorhabditis elegans.
Nature
291:386,
1981[Medline]
[Order article via Infotrieve]
28.
Wilson R,
Ainscough R,
Anderson K,
Baynes C,
Berks M,
Bornfield J,
Burton J,
:
2.2 Mb of contigous nucleotide sequence from chromosome III of C. elegans.
Nature
368:32,
1994[Medline]
[Order article via Infotrieve]
29.
Huang C-H,
Reid ME,
Chen Y,
Couglan G,
Okubo Y:
Molecular definition of red cell Rh haplotypes by tightly linked Sph I RFLPs.
Am J Hum Genet
58:133,
1996[Medline]
[Order article via Infotrieve]
30.
Seack J,
Pancer Z,
Muller IM,
Muller WEG:
Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium.
Immunogenetics
46:493,
1997[Medline]
[Order article via Infotrieve]
31.
Krawczak M,
Reiss J,
Cooper N:
The mutational spectrum of single base-pair substitutions in mRNA splice juctions of human genes: Causes and consequences.
Hum Genet
90:41,
1992[Medline]
[Order article via Infotrieve]
32.
Shapiro MB,
Senapathy P:
RNA splice junctions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression.
Nucleic Acids Res
15:7155,
1987[Abstract/Free Full Text]
33.
Kazazian Jr HH:
The thalassemia syndromes: Molecular basis and prenatal diagnosis in 1990.
Semin Hematol
27:209,
1990[Medline]
[Order article via Infotrieve]
34.
Treisman R,
Orkin SH,
Maniatis T:
Specific transcription and RNA splicing defects in five cloned -thalassemia genes.
Nature
302:591,
1983[Medline]
[Order article via Infotrieve]
35.
Green MR:
Biochemical mechanisms of constitutive and regulated pre-mRNA splicing.
Annu Rev Cell Biol
7:559,
1991
36.
Miwa M,
MacDonald CC,
Berget SM:
Are vertebrate exon scanned during splice-site seletion?
Nature
360:277,
1992[Medline]
[Order article via Infotrieve]
37.
Cheng SH,
Gregory RJ,
Marshall J,
Paul S,
Souza DW,
White GA,
O'Riordan CR,
Smith AE:
Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.
Cell
63:827,
1990[Medline]
[Order article via Infotrieve]
38.
Deen PMT,
Croes H,
van Aubel RAMH,
Ginsel LA,
van Os CH:
Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.
J Clin Invest
95:2291,
1995
39.
Eyers SAC,
Ridgwell K,
Mawby WJ,
Tanner MJA:
Topology and organization of the human Rh (Rhesus) blood group-related polypeptides.
J Biol Chem
269:6417,
1994[Abstract/Free Full Text]
40.
Marini A-M,
Urrestarazu A,
Beauwens R,
Andre B:
The Rh (Rhesus) blood group polypeptides are related to NH4 transporters.
Trends Biochem Sci
22:460,
1997[Medline]
[Order article via Infotrieve]
Abstract
Introduction
Abstract
