From the Laboratory of Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021
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ABSTRACT |
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The Rh (Rhesus) protein family comprises Rh50
glycoprotein and Rh30 polypeptides, which form a complex essential for
Rh antigen expression and erythrocyte membrane integrity. This article
describes the structural organization of Rh50 gene and identification
of its associated splicing defect causing Rhnull
disease. The Rh50 gene, which maps at chromosome 6p11-21.1, has an
exon/intron structure nearly identical to Rh30 genes, which map at
1p34-36. Of the 10 exons assigned, conservation of size and sequence
is confined mainly to the region from exons 2 to 9, suggesting that
RH50 and RH30 were formed as two separate
genetic loci from a common ancestor via a transchromosomal insertion
event. The available information on the structure of RH50
facilitated search for candidate mutations underlying the Rh deficiency
syndrome, an autosomal recessive disorder characterized by mild to
moderate chronic hemolytic anemia and spherostomatocytosis. In one
patient with the Rhnull disease of regulator type, a
shortened Rh50 transcript lacking the sequence of exon 7 was detected,
while no abnormality was found in transcripts encoding Rh30
polypeptides and Rh-related CD47 glycoprotein. Amplification and
sequencing of the genomic region spanning exon 7 revealed a G A
transition in the invariant GT motif of the donor splice site in both
Rh50 alleles. This splicing mutation caused not only a total skipping
of exon 7 but also a frameshift and premature chain termination. Thus,
the deduced translation product contained 351 instead of 409 amino
acids, with an entirely different C-terminal sequence following
Thr315. These results identify the donor splicing defect,
for the first time, as a loss-of-function mutation at the
RH50 locus and pinpoint the importance of the C-terminal
region of Rh50 in Rh complex formation via protein-protein
interactions.
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INTRODUCTION |
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The Rh (Rhesus) protein family is currently known to consist of
three erythroid-specific integral membrane proteins, the Rh50 glycoprotein and two Rh30 (RhD and RhCE) polypeptides (1-4). Although
their genetic loci are mapped on chromosomes 6p11-21.1 and 1p34-36,
respectively, Rh50 and Rh30 share a clear sequence homology (36%
overall identity) and a similar 12-transmembrane (TM)1 topology (50% identity
in the putative -helices) (5-8). As nonglycosylated and
palmitoylated proteins, RhD and RhCE each contain 417 amino acids,
serving as the carriers of D and CcEe blood group antigens (5-7). By
contrast, the 409-amino acid Rh50 glycoprotein in itself does not carry
Rh antigens but rather interacts with Rh30 polypeptides to form a
protein complex, thereby functioning as a coexpressor to facilitate Rh
antigen disposition in the erythrocyte membrane (8-10).
Apart from being a structural unit of Rh antigen expression, the Rh50 and Rh30 proteins appear to possess some hitherto undefined roles essential for the function and integrity of plasma membranes. This proposal is highlighted primarily by the occurrence of Rh deficiency syndrome, a rare autosomal recessive disorder characterized by a chronic hemolytic anemia of varying severity, a hereditary spherostomatocytosis, and multiple membrane abnormalities (1-3). The Rh deficiency syndrome exists in two conditions in which a complete absence of all Rh antigens defines the Rhnull status and a barely detectable presence defines the Rhmod phenotype (11, 12). Both conditions exhibit an absence or weakened expression of several other membrane glycoproteins or associated antigens, including Rh50, CD47, LW, Duffy (Fy5), and glycophorin B (GPB for SsU) (1-3). Therefore, the Rh deficiency syndrome can be regarded as a disorder of impaired protein-protein interactions.
As shown by family studies, Rh deficiency is almost invariably associated with consanguinity and can occur on different genetic backgrounds (11, 12). The amorph type of Rhnull is thought to arise by silencing mutations at the RH30 locus encoding RhD and RhCE polypeptides, but its underlying molecular defect has remained to be determined (13-15). In contrast, the regulator Rhnull and Rhmod phenotypes are considered to result from suppressor or "modifier" mutations independent of the RH30 locus (16). The genuine interaction of Rh50 with Rh30 proteins in Rh complex formation points to RH50 locus as a primary candidate responsible for the suppressor forms of Rh deficiency. To facilitate the identification of such suppressor mutations, the organization of Rh50 gene has now been delineated. Here, I describe the exon/intron structure of the Rh50 gene and identification of its associated splicing defect as a loss-of-function mutation in one Rhnull patient. The findings reported herein correlate the disease phenotype with an impaired Rh complex formation and provide evidence for the importance of the C-terminal region of Rh50 participating in protein-protein interactions.
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EXPERIMENTAL PROCEDURES |
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Blood Samples--
Blood samples from normal human blood donors
with RhD-positive (RhD+) and RhD-negative
(RhD) phenotypes (defined by DCe/DCe and
dce/dce genotypes) were used as controls. The
Rhnull blood sample was obtained from a Japanese patient
(T. T.). Preliminary studies showed that the propositus was a
homozygote for the regulator type of Rhnull disease and no
Rh antigen was detectable by serologic testing. Furthermore, Southern
blot analysis demonstrated that the RH30 locus was grossly intact without apparent gene deletion or rearrangement (15).
Nucleic Acid Isolation and Southern Analysis--
Total RNA was
isolated from reticulocyte polysomes using the differential cell lysis
method (17), followed by extraction with the Trizol reagent (Life
Technologies, Inc.). Genomic DNA was prepared from leukocyte pellets,
as described previously (18). Southern blot analysis was performed
using Rh50, Rh30, and CD47 cDNA probes generated with gene-specific
primers (see below) and labeled with [-32P]dCTP (NEN
Life Science Products).
Characterization of Exon/Intron Structure of the Rh50 Gene-- To determine the structural organization of the Rh50 gene, genomic DNA from a normal person was digested separately with restriction endonucleases EcoRV, HincII, PvuII, SmaI, SspI, and StuI. The total digests of each restriction enzyme were ligated to the same adaptor to generate a genomic library using the Marathon amplification kit (CLONTECH). The exon and its adjacent intron sequences were then amplified in two steps using the Taq DNA polymerase chain reaction (PCR) (19). The first step employed the adaptor primer (AP1) and a Rh50 gene-specific primer (GSP1), whereas in the second step, nested AP2 and GSP2 were used. The resultant products were analyzed by agarose gel and sequenced after purification by 5% polyacrylamide gel electrophoresis. When new sequence information became available, new primers were designed for further bidirectional walking (Table I).
RT-PCR Analysis of Rh50, Rh30, and CD47 Transcripts--
To
determine the structure and expression of Rh50, Rh30 and CD47
transcripts in normal and Rhnull erythroid cells, cDNAs
were synthesized from total RNA and amplified by RT-PCR, as described (20). The cDNA was reverse-transcribed with an oligo(dT) primer or
a gene-specific primer located in the 3-untranslated region (3
-UTR);
the entire coding sequence was then amplified in two overlapping
segments with four 5
amplimers. All nucleotide (nt) positions of sense
(s) and antisense (a) primers are counted from the first base of ATG
codon in the respective cDNAs (5-8, 21). The Rh50 primers were: 1)
3
-UTR, 5
-AATGGGAAAGGAAGCTGGAGAGCA-3
(nt 1321-1298); 2) amplimers:
1s, 5
-AGTGTGCCTCTGTCCTTTGCCACA-3
(nt
27 to
4, 5
-UTR of exon 1);
5a, 5
-CTGTTTGTCTCCAGGTTCAGCAAT-3
(nt 708-685, exon 5); 4s,
5
-GAAGAGTCCGCATACTACTCAGAC-3
(nt 601-624, exon 4); 7s,
5
-CCACTTTTTACTACTAAACTGAGG (nt 946-969, exon 7); and 10a,
5
-CCATGTCCATGGAACTGATTGTCA-3
(nt 1256-1233, exon 10). The Rh30
primers were: 1) 3
-UTR of RhD, 5
-GTATTCTACAGTGCATAATAAATGGTG-3
(nt
1458-1432, exon 10); and 3
-UTR of RhCE,
5
-CTGTCTCTGACCTTGTTTCATTATAC-3
(nt 1388-1363, exon 10); 2)
amplimers: 1s, 5
-ATGAGCTCTAAGTACCCGCGGTCTG-3
(nt 1-25, exon 1); 5a,
5
-TGGCCAGAACATCCACAAGAAGAG-3
(nt 663-640, exon 5); 4s,
5
-CCAAAATAGGCTGCGAACACGTAGA-3
(nt 515-539, exon 4), and 10a,
5
-TTAAAATCCAACAGCCAAATGAGGAAA-3
(nt 1254-1228, exon 10). The CD47
primers were: 1) 3
-UTR, 5
-TCACGTAAGGGTCTCATAGGTGAC-3
(nt
1120-1197); 2) amplimers: Is, 5
-ATGTGGCCCCTGGTAGCGGCGCT-3
(nt
1-23); Ia, 5
-CACTAGTCCAGCAACAAGTAAAGC-3
(nt 555-534); IIs, 5
-CTCCTGTTCTGGGGACAGTTTGGT-3
(nt 460-483); and IIa,
5
-CAAATCGGAGTCCATCACTTCACT-3
(nt 1001-977).
Amplification and Analysis of the Genomic Region Encompassing
Exon 7 of Rh50 Gene--
To assay the donor splice site mutation, the
genomic region spanning exon 7 of RH50 in normal and
Rhnull was amplified. For PmlI digestion, the
fragment was amplified with intron primers 6s and 7a: intron 6s,
5-GCCCAGCTATAGCTGTGTTTCAGT-3
(nt
80 to
56 upstream of exon 7);
and intron 7a, 5
-CTAATGATCTTCTCTCAGGCGCGT-3
(nt 128-152 downstream
of exon 7). For restriction analysis with NlaIII, the
fragment was amplified with exon 7 primer 7s (nt 946-969, see above)
and intron 7 primer 7a
, 5
-ATGGGACCACAGGGGCTGA-3
(nt 22-40
downstream of exon 7).
Direct Nucleotide Sequencing and Sequence Analysis-- All amplified cDNA and genomic DNA products were purified by native 5% polyacrylamide gel electrophoresis and sequenced with either amplimers or nested primers. Nucleotide sequence determination was carried out using fluorescent dye-tagged chain terminators on an automated DNA sequencer (model 373A, Applied Biosystems). The resultant nucleotide sequences were analyzed by the DNASIS program (Hitachi), and the deduced amino acid sequences were assessed for hydropathy character using the Kyte-Doolittle plotting method (22).
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RESULTS |
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Organization of Rh50 Gene and Comparison with Rh30 Gene-- To delineate the structural organization of the Rh50 gene, a bidirectional walking approach was taken to retrieve unknown sequences (Fig. 1A). 40 synthetic primers that cover various coding sequences (Table I) were used in combination to amplify the adaptor-ligated, restriction enzyme-specific genomic libraries. Fig. 1A shows a representative panel of the resultant Rh50gene products, each spanning a unique exon/intron junction. They range in size from several hundred base pairs (bp) to several kilobase pairs, depending on the distribution of restriction sites. Sequencing of these amplified products revealed the features of the Rh50 gene and confirmed no coamplification from the related Rh30 genes.
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Sequence of Splice Sites and Exon/Intron Junctions in the Rh50
Gene--
Fig. 3 schematically shows the
nucleotide sequence of splice sites as well as the structure of
exon/intron junctions in the Rh50 gene. All the 5 donor and 3
acceptor splice sites conform to the "GT-AG" rule and possess the
consensus splicing signals (25). Of the 10 exons identified, only exon
6 is symmetrical, having intraexon codons GTT (Val270) and
ACT (Thr315) at its 5
and 3
ends, respectively, whereas
the other exons have either one or two split interexon codons (Fig. 3).
One potential consequence of this type of exon/intron arrangement is
that skipping of any single internal exon, except exon 6, during the
splicing of Rh50 pre-mRNA would result in a shift in open reading
frame and, therefore, alter the encoded amino acid sequence downstream of the skipped exon.
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Expression of Rh50, Rh30, and CD47 mRNAs in Normal and Rhnull Cells-- To identify the molecular defect underlying the Rhnull disease, the expression of candidate genes encoding the Rh50, Rh30, and CD47 proteins was characterized by RT-PCR and nucleotide sequencing. The full-length cDNA of Rh30 or CD47 was readily detectable in normal and Rhnull erythroid cells (gels not shown), indicating a comparable expression of the corresponding mRNA. Sequencing showed that the Rh30 or CD47 cDNA from Rhnull was normal and that the Rh30 cDNA contained both RhD and RhCe, indicating that the patient was a DCe/DCe homozygote. Definition of this Rh genotype by transcript analysis was in full agreement with the result of DNA typing by SphI polymorphisms (15). These data showed that the RH30 or CD47 locus itself is not responsible for the disease phenotype.
However, RT-PCR analysis of Rh50 gene expression in erythroid cells revealed an important difference between the normal and Rhnull patient. Although there was no apparent change in size of the 5
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Identification of Rhnull-associated Donor Splice Site
Mutation in Rh50 Gene--
The complete absence of exon 7 associated
with Rh50 cDNA suggested strongly that either a splicing defect or
a genomic deletion was present in the cognate gene. To define the
nature of the underlying mutation, amplification from
Rhnull genomic DNA of a segment encompassing exon 7 of the
Rh50 gene was attempted. A fragment of 354 bp in size was detected,
excluding the possibility of gene deletion. Sequencing of this fragment
on both strands led to the identification of a single G A mutation
in the invariant GT element (+1 position) of the 5
donor splice site
attached to exon 7 (Fig. 5A).
Sequencing of other exon/intron junctions amplified with
intron-specific primers (data not shown) confirmed this mutation to be
the only structural alteration in the Rh50 gene.
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Deduced Primary Sequence and Predicted Membrane Topology of Rh50
Mutant Protein--
To gain information on the primary structure of
Rh50 glycoprotein, the Rhnull-associated Rh50 cDNAs
were sequenced to completion. Compared with normal Rh50, no point
mutation other than an absence of the sequence encoded by exon 7 was
observed in the Rhnull patient (Fig.
6A). Because exon 7 is
asymmetric in codon distribution at the 5 side (Fig. 3), its complete
skipping and the subsequent joining of exon 6 with exon 8 inevitably
resulted in an open reading frame shifting (Fig. 6A). In
turn, the deduced translation product would be truncated and
prematurely terminated, containing only 351 amino acid residues. This
includes the loss of 41 amino acid residues encoded in exon 7 and gain
of an entirely new sequence of 36 residues following Thr315
(Fig. 6A).
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DISCUSSION |
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Rh50 glycoprotein is a critical coexpressor of Rh30 polypeptides,
the carriers of erythrocyte Rh antigens (1-4). Here, the exon/intron
structure of Rh50 gene has been delineated, which should facilitate
identification of mutations underlying the suppressor forms of Rh
deficiency syndrome. A homology-based approach coupling with
bidirectional walking revealed that Rh50 is a single copy gene with 10 exons and has a global organization strikingly similar to its related
Rh30 members (23, 24). Both the structural conservation and sequence
homology of the two genes are confined mainly to exons 2-9, while
their 5 and 3
regions, including the promoter and untranslated
sequences, share little or no similarity. Since Rh50 and Rh30 genes are
located on different chromosomes (5-8), these findings suggest that
the two genetic loci might be formed by a rare transchromosomal
insertion event. Our recent studies suggest that Rh50 and Rh30 genes
originated from a common ancestor and were linked to each other
following their initial duplication; later, one was translocated and
diverged as the independent locus on a separate
chromosome.4 Comparative
analysis of the Rh50 and Rh30 gene orthologues in lower organisms
should help decipher the evolutionary pathway ultimately leading to the
establishment of two genetic loci encoding the Rh family proteins in
Homo sapiens.
The extreme rareness, recessive nature, and consanguineous background of Rh deficiency syndrome (11, 12) point to a heterogeneous spectrum of the underlying mechanisms. At present, the molecular defect at RH30 locus responsible for the amorph type of Rhnull remains unknown (13-15). Nevertheless, several lines of evidence suggest that the RH50 locus is the prime target of suppressor mutations resulting in the regulator Rhnull disease. (i) Rh50 is thought to directly interact with Rh30, and the deficiency of the two proteins in the plasma membrane occurs in parallel (9, 26). (ii) Despite a close link of Rhnull with absence or deficiency in GPB, Duffy, or LW, the erythrocytes lacking these glycoproteins per se exhibit no change in the Rh antigen expression and no apparent perturbations in membrane physiology and cell morphology (27-30). Presumably these proteins are casually associated components not essential for the interaction and membrane assembly of Rh family proteins. (iii) Although CD47 is also reduced in Rhnull state, its low level of expression is restricted to erythroid cells but not to other hematopoietic cells (31, 32), suggesting that CD47 deficiency occurs as the consequence of, rather than the cause for, the defect in Rh complex formation. (iv) More recently, two small DNA deletions causing frameshift in the Rh50 gene have been found to be associated with the regulator Rhnull phenotype in unrelated patients (16).
Our previous studies showed that this Rhnull patient had a
grossly intact RH30 locus occurring in the form of
DCe/DCe haplotype combination (15). The present study
confirmed this assignment and showed further that the RH30
locus gave rise to expression of both RhD and RhCe transcripts with
sequences identical to that from normal subjects. These results,
together with the identification of a normal CD47 gene, exclude the
involvement of mutations of RH30 or CD47 locus in
this Rhnull patient. However, transcript analysis showed
that there was no expression in the Rhnull cells of any
full-length form of Rh50 mRNAs except the shortened one specifically lacking the sequence of exon 7. Genomic sequencing revealed the occurrence of a homozygous G A mutation in the invariant GT element of 5
donor splice site as the only alteration in
the Rh50 gene. These findings establish the pre-mRNA splicing defect, for the first time, as the suppressor mutation of
RH50 leading to a loss-of-function phenotype characteristic
of the regulator form of Rhnull disease.
Mutations in the GT and AG motifs of the donor and acceptor splice sites, the cis-acting elements essential for pre-mRNA splicing (33), portray an important mechanism for the origin of human genetic diseases (34). The donor splice site mutation described here has caused a complete skipping of exon 7 from the mature form of Rh50 mRNA in the Rhnull patient. Significantly, such a splicing event not only excluded a coding sequence for 41 amino acids but resulted in a frameshift after the codon for Thr315 and a premature chain termination after the codon for Ile351. Therefore, the deduced Rh50 mutant protein contains only 351 amino acids, including a stretch of 36 new residues at the C terminus. Correlation of these primary changes with regulator Rhnull disease provides new insight regarding how different mutations might act as suppressors to disrupt or modify the protein-protein interactions that dictate the Rh complex formation.
Prior studies suggested that there may be a direct contact between Rh50 and Rh30 via their N-terminal sequences (9, 10). Nevertheless, additional interacting sites are likely to be present in the Rh protein complex. For the Rh50 mutant reported here, its only difference from the wild-type lies C-terminal to the 10th putative TM domain (Fig. 6). This suggests that the C-terminal half may also participate in the interaction directly and/or confer required conformation to stabilize that interaction. In support of this notion, we have identified in unrelated Rhnull patients several missense mutations that are clustered in the C-terminal region of the Rh50 protein.4 It is of further interest to note that such mutations all target the TM domains in the C-terminal half that are conserved in the Rh50 homologues from the mouse to C. elegans. Currently, little is known about how the disruption of the Rh protein complex causes the multiple facets of structural and functional abnormalities in the Rh-deficient erythrocytes. There is also a lack of general information regarding the involvement and coordination of possible intracellular factor(s) in the functioning of the Rh membrane complex. A full description of Rhnull disease mutations and assessment of their phenotypic effects in model systems, such as C. elegans, should lead to a better understanding of the membrane assembly and structure/function relations of the Rh family of proteins.
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ACKNOWLEDGEMENT |
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I am particularly grateful to Y. Okubo and M. Reid for providing and typing the Rhnull blood sample used in this investigation. I thank Y. Chen for technical assistance, and T. Ye for help in the construction of human Marathon genomic libraries. I also thank O. O. Blumenfeld and C. Redman for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL54459.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031548, AF031549, AF031550, and AF031551.
To whom correspondence should be addressed: Laboratory of
Biochemistry and Molecular Genetics, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E. 67th St., New York, NY 10021. Tel.: 212-570-3388; Fax: 212-737-4935; E-mail: chuang{at}nybc.org.
1 The abbreviations used are: TM, transmembrane; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; UTR, untranslated region; nt, nucleotide(s); bp, base pair(s); RACE, rapid amplification of cDNA ends.
2 Z. Liu and C.-H. Huang, unpublished observations.
3 Z. Liu and C.-H. Huang, manuscript in preparation.
4 C.-H. Huang, J. Cheng, Y. Chen, and Z. Liu, unpublished observations.
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REFERENCES |
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