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 Rh_null status and a barely detectable presence defines the Rh_mod 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 Rh_null 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 Rh_null and Rh_mod 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 Rh_null 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.

	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 Rh_null blood sample was obtained from a Japanese patient (T. T.). Preliminary studies showed that the propositus was a homozygote for the regulator type of Rh_null 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 Rh_null 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 Rh_null 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).

	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.

View larger version (49K): [in this window] [in a new window]   Fig. 1.   Amplification of exon/intron junctions and organization of the Rh50 gene. A, strategy for amplification of exon/intron junction segments. The transcript and genomic structures of Rh50 are schematically shown (not to scale). Initiation ATG and stop TAA codons in the cDNA and upstream ATG leading a potential open reading frame in the genomic DNA are denoted. Bent arrows illustrate the 40 primers anchored to 10 exons in either sense (s) or antisense (a) direction (Table I). Shown below is a representative 1.8% agarose gel electrophoresis of sequenced genomic products. The exon (E)/intron (IVS) content and restriction enzyme usage of amplified fragments are indicated. Note that lanes 8 and 13 are products amplified directly from total genomic DNA, which encompass the whole intron 4 and intron 6, respectively. Bands seen in other lanes each were obtained by two rounds of PCR using AP1+GSP1 and AP2+GSP2 (Table I). M,  (HindIII) and X174 (HaeIII) DNA markers. B, organization of the Rh50 gene and comparison with the Rh30 gene. Exons are denoted by solid or open bars and introns by broken lines (not to scale). The size of coding sequence for each exon (in base pairs) is shown; exon 1 is counted from ATG and exon 10 ends before the TAA codon (marked by asterisks).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of the 5 portion of the Rh50 gene. The 5 region and exon sequences are shown by uppercase letters and the intron 1 sequence by lowercase letters. For brevity, the intron 1 sequence, whose size is grater than 15 kilobase pairs in size, is omitted (shown by dots). Putative cis-acting motifs in the promoter are marked and underlined. Note that TATA boxes are not found in the promoter of both RhD and RhCE genes (23, 24). Note also that there is a strong strand asymmetry in the region. Multiple transcription initiation sites occur between the two putative Ets binding sites (see Footnote 3). The first position of ATG codon assigned for translation initiation of the erythroid-specific Rh50 protein (8) is denoted. The encoded amino acids of exon 1 and exon 2 (partial) are shown below the nucleotide sequence.

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.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Sequence of exon/intron junctions and assignment of splice sites in the Rh50 gene. The 409-amino acid coding sequence of Rh50 gene is distributed in 10 exons (boxed). The nucleotide positions marking the beginning and end of each exon are numbered: nt 1 denotes the first nucleotide of erythroid ATG initiation codon and nt 1230 the third base of TAA stop codon. "aataaa" indicates one of the polyadenylation signals present in the 3-UTR. Exon sequences are denoted by uppercase letters, whereas intron sequences, including the 3-acceptor and 5-donor splice sites, are indicated by lowercase letters. Interval exon sequences are omitted (shown by dots). Amino acids encoded by the respective exon/exon boundaries are indicated below the middle position of the triplet code.

Expression of Rh50, Rh30, and CD47 mRNAs in Normal and Rh_null Cells-- To identify the molecular defect underlying the Rh_null 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 Rh_null erythroid cells (gels not shown), indicating a comparable expression of the corresponding mRNA. Sequencing showed that the Rh30 or CD47 cDNA from Rh_null 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.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Analysis of Rh50 transcript expression in normal and Rhnull erythroid cells. RT-PCR analysis of Rh50 transcript was carried out using 3-UTR primer for cDNA synthesis and two pairs of amplimers for cDNA amplification. The location, direction, and designation of primers with respect to the structure of Rh50 are specified. A, agarose gel electrophoresis of amplified Rh50 cDNA products from RhD+, RhD, and Rhnull. The size of segment 4s-10a from Rhnull is smaller than that of controls, indicating a deletion in the region spanning exons 5-10. Note that the Rhnull lanes were overloaded. B, nucleotide profiles of the exon/exon boundary associated with exon skipping. Exon boundary is indicated by a vertical arrow. In normal, exon 6 is joined to exon 7, whereas in Rhnull exon 7 is absent, resulting in exon 6 to exon 8 connection. C, 3 RACE assay for the functional splicing of exon 7 in Rh50 pre-mRNAs. A primer anchored in exon 7, 7s, was coupled with 3-UTR primer for 3 RACE reaction. The expected cDNA product of 376 bp is clearly seen in control lanes but not the Rhnull lane, confirming a complete exclusion of exon 7 from the latter.

Identification of Rh_null-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 Rh_null 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.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Identification of Rhnull-associated donor splice site mutation in intron 7 of Rh50 gene. A, amplification and sequencing of the genomic segments encompassing exon 7 of the Rh50 gene. The G  A transition at the +1 position of intron 7 in the nucleotide profiles is indicated by two vertical arrows. Shown at bottom are the PmlI site located exactly at exon 7/intron 7 junction (CACGTG) of wild-type Rh50 and the NlaIII site (CATG) in mutant Rh50, resulting from the 5 donor splice site mutation. The direction and position of primers are indicated. B, a diagnostic analysis of the donor splice site mutation with PmlI and NlaIII enzymes. The 6s-7a fragment was cleavable with PmlI in normal but not Rhnull. In contrast, the 7s-7a fragment of Rhnull has an extra NlaIII site. C, Southern blot analysis of native genomic DNAs from normal person and Rhnull patient. Genomic DNAs were digested the enzymes indicated and hybridized with an exon7/intron 7 junction probe. The PmlI cleavable fragments are seen in normal persons but not in Rhnull patient.

Deduced Primary Sequence and Predicted Membrane Topology of Rh50 Mutant Protein-- To gain information on the primary structure of Rh50 glycoprotein, the Rh_null-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 Rh_null 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).

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Amino acid sequence and predicted membrane topology of mutant Rh50 in Rhnull disease. A, comparison of the primary structure between the wild-type (wt) and mutant (mt) Rh50 glycoproteins. The mutant lacks 41 amino acids (dashes) encoded by exon 7, but gains a new 36-amino acid sequence (bold) due to a frameshift and premature termination (preterm). Note that in both the mutant and wild types, the amino acid at position 242 is occupied by Asn (N with an asterisk) but not by Asp (D) as reported (8). This Asn is seen in all unrelated normal and Rhnull individuals examined (n > 15). B, model for membrane topology of the mutant Rh50 protein. Also shown is the hydropathy profile of wild-type Rh50 with 12 TM domains connected by short loops on either side of the lipid bilayer. "Y" indicates the N-glycan on Asn37 (9). The mutant Rh50 protein lacks the last two TM domains and carries an extended C-terminal sequence most likely facing the cytoplasmic space. Its features, including the underlying defect and associated structural alterations, are summarized at right margin.

	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 Rh_null 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 Rh_null 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 Rh_null 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 Rh_null 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 Rh_null phenotype in unrelated patients (16).

Our previous studies showed that this Rh_null 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 Rh_null patient. However, transcript analysis showed that there was no expression in the Rh_null 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 Rh_null 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 Rh_null 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 Rh_null 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 Rh_null 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 Rh_null 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.