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The Human Rh50 Glycoprotein Gene

1998; Elsevier BV; Volume: 273; Issue: 4 Linguagem: Inglês

10.1074/jbc.273.4.2207

1083-351X

Cheng‐Han Huang,

Toxin Mechanisms and Immunotoxins

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 Rhnulldisease. 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 thatRH50 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 RH50facilitated 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 theRH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions. 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 Rhnulldisease. 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 thatRH50 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 RH50facilitated 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 theRH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions. 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 (1Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (92) Google Scholar, 2Cartron J.-P. Agre P. Blood Cell Biochem. 1995; 6: 189-225Crossref Google Scholar, 3Cartron J.-P. Blood Rev. 1994; 8: 199-211Crossref PubMed Scopus (135) Google Scholar, 4Anstee D.J. Tanner M.J.A. Bailliere Clin. Haematol. 1993; 6: 401-422Abstract Full Text PDF PubMed Scopus (88) Google Scholar). 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) 1The 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. topology (50% identity in the putative α-helices) (5Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar, 6Avent N.D. Ridgwell K. Tanner M.J.A. Anstee D.J. Biochem. J. 1990; 271: 821-825Crossref PubMed Scopus (208) Google Scholar, 7Le Van Kim C. Mouro I. Cherif-Zahar B. Raynal V. Cherrier C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10925-10929Crossref PubMed Scopus (246) Google Scholar, 8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar). As nonglycosylated and palmitoylated proteins, RhD and RhCE each contain 417 amino acids, serving as the carriers of D and CcEe blood group antigens (5Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar, 6Avent N.D. Ridgwell K. Tanner M.J.A. Anstee D.J. Biochem. J. 1990; 271: 821-825Crossref PubMed Scopus (208) Google Scholar, 7Le Van Kim C. Mouro I. Cherif-Zahar B. Raynal V. Cherrier C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10925-10929Crossref PubMed Scopus (246) Google Scholar). 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 (8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar, 9Eyers S.A.S. Ridgwell K. Mawby W.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6417-6423Abstract Full Text PDF PubMed Google Scholar, 10Avent N.D. Liu W. Warner K.M. Mawby W.J. Jones J.W. Ridgwell K. Tanner M.J.A. J. Biol. Chem. 1996; 271: 14233-14239Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 (1Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (92) Google Scholar, 2Cartron J.-P. Agre P. Blood Cell Biochem. 1995; 6: 189-225Crossref Google Scholar, 3Cartron J.-P. Blood Rev. 1994; 8: 199-211Crossref PubMed Scopus (135) Google Scholar). 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 (11Race R. Sanger R. Blood Groups in Man. 6th Ed. Blackwell Scientific Publications, Oxford, London1975: 78-260Google Scholar,12Nash R. Shojania A.M. Am. J. Hematol. 1987; 24: 267-275Crossref PubMed Scopus (45) Google Scholar). 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) (1Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (92) Google Scholar, 2Cartron J.-P. Agre P. Blood Cell Biochem. 1995; 6: 189-225Crossref Google Scholar, 3Cartron J.-P. Blood Rev. 1994; 8: 199-211Crossref PubMed Scopus (135) Google Scholar). 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 (11Race R. Sanger R. Blood Groups in Man. 6th Ed. Blackwell Scientific Publications, Oxford, London1975: 78-260Google Scholar, 12Nash R. Shojania A.M. Am. J. Hematol. 1987; 24: 267-275Crossref PubMed Scopus (45) Google Scholar). 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 (13Cherif-Zahar B. Raynal V. Le Van Kim C. D'Amrosio A.-M. Bailly P. Cartron J.-P. Colin Y. Blood. 1993; 82: 656-662Crossref PubMed Google Scholar, 14Carritt B. Blunt T. Avent N. Daniels G. Steers F. Ann. Hum. Genet. 1993; 57: 273-279Crossref PubMed Scopus (17) Google Scholar, 15Huang C.-H. Reid M.E. Chen Y. Coghlan G. Okubo Y. Am. J. Hum. Genet. 1996; 58: 133-142PubMed Google Scholar). In contrast, the regulator Rhnull and Rhmod phenotypes are considered to result from suppressor or "modifier" mutations independent of theRH30 locus (16Cherif-Zahar B. Raynal V. Gane P. Mattei M.-G. Bailly P. Gibbs B. Colin Y. Cartron J.-P. Nat. Genet. 1996; 12: 168-173Crossref PubMed Scopus (126) Google Scholar). 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. Blood samples from normal human blood donors with RhD-positive (RhD+) and RhD-negative (RhD−) phenotypes (defined by DCe/DCe anddce/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 (15Huang C.-H. Reid M.E. Chen Y. Coghlan G. Okubo Y. Am. J. Hum. Genet. 1996; 58: 133-142PubMed Google Scholar). Total RNA was isolated from reticulocyte polysomes using the differential cell lysis method (17Goossens M. Kan Y.W. Methods Enzymol. 1981; 76: 805-817Crossref PubMed Scopus (234) Google Scholar), followed by extraction with the Trizol reagent (Life Technologies, Inc.). Genomic DNA was prepared from leukocyte pellets, as described previously (18Huang C.-H. Guizzo M.L. McCreary J. Leigh E.M. Blumenfeld O.O. Blood. 1991; 77: 381-386Crossref PubMed Google Scholar). Southern blot analysis was performed using Rh50, Rh30, and CD47 cDNA probes generated with gene-specific primers (see below) and labeled with [α-32P]dCTP (NEN Life Science Products). 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, andStuI. 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) (19Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Erlich H. Science. 1988; 239: 487-491Crossref PubMed Scopus (13517) Google Scholar). 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).Table Isynthetic oligonucleotides for analysis of Rh50 glycoprotein geneDesignation1-aSuffixes "a" and "s" denote antisense (reverse) and sense (forward) primers, whereas numbers 1 and 2 indicate GSP1 and GSP2, respectively. E10 primers are all located in the 3′-untranslated region downstream of TAA stop codon.Sequence (5′-3′)Nucleotide position1-bAll nucleotide positions are accounted from the first base of ATG initiation codon (Fig. 2).LocationE1–1aCTCAAAGAATATGCCCATGTCTG147-125Exon 1E1–2aCTTGGTGATGTTGAGCTGCTCGAG120-97Exon 1E1–1sAGTGTGCCTCTGTCCTTTGCCACA−25 – −45′-UTRE1–2sATGAGGTTCACATTCCCTCTCATG1-24Exon 1E2–1aCCAAAGCAGCAACGAGTAGGTTG268-246Exon 2E2–2aTGATACCCACACTGCTGAAGCCAT247-224Exon 2E2–1sTGTTCCAAGATGTACATGTTATG158-180Exon 2E2–2sTGTTGGGTTTGGCTTCCTCATGAC186-209Exon 2E3–1aCACTAACCAGGTATTCATTGTGGG481-458Exon 3E3–2aATTGTCATGATCAGCATTTGGGTT437-414Exon 3E3–1sGGAATCAAAAACATGATAAATGCAGAC331-357Exon 2/3E3–2sATAAATGCAGACTTCAGTGCAGCC346-360Exon 3E4–1aCAAGTCTGAGTAGTATGCGGACTC627-604Exon 4E4–2aCTCTTCATTTTCATGCCCCTTTC606-584Exon 4E4–1sGCCTCTGACATTGGAGCATCAATG493-516Exon 4E4–2sAAGAGTCCGCATACTACTCAGAC602-624Exon 4E5–1aGTACGTGTTTACAATGGCCCTGC732-710Exon 5E5–2aCTGTTTGTCTCCAGGTTCAGCAAT708-685Exon 5E5–1sAGCTTTAACTCGGCCATTGCTGAA670-693Exon 5E5–2sAAACAGTGCAGGGCCATTGTAAAC703-725Exon 5E6–1aAGTCAGGAACTTGTATCCAAGCAC945-922Exon 6E6–2aAGACCATTCCTGCAATGCTCCCA919-897Exon 6E6–1sGTTCACATTCAGAATGCCACCCTT808-831Exon 6E6–2sTGTGCGGATATGGCAATTCACCCA856-879Exon 6E7–1aCATTGCATCTGCCACAATGCCTGC1053-1030Exon 7E7–2aGCCTCCCACTACACCAGGTAAG1026-1005Exon 7E7–1sCCACTTTTTACTACTAAACTGAGG946-968Exon 7E7–2sGATACATGTGGGGTCCATAACCTC976-1002Exon 7E8–1aTCATCAGACCTCCAACAACTGCTG1135-1112Exon 8E8–2aCGATAGAGGAACCCAGTGCAGCT1108-1086Exon 8E8–1sTCTATGGCCATGCAGGCAGCTGCA1069-1092Exon 8E8–2sGTTCCTCTATCGGAACAGCAGTTG1097-1120Exon 8E9–1aACCTTCCAATAAACAGAATCATCA1214-1191Exon 9E9–2aAGCAGTTCTGGTCAGATGGCTGT1190-1167Exon 9E9–1sTAATTCTAAAGTTGCCTCTCTGGG1142-1165Exon 9E9–2sCAGCCATCTGACCAGAACTGCTAT1168-1191Exon 9E10–1aAATGGGAAAGGAAGCTGGAGAGCA1321-12983′-UTRE10–2aCCATGTCCATGGAACTGATTGTCA1256-12333′-UTRE10–1sTGGAACCTGAAGTCTAAACACCAT1271-12943′-UTRE10–2sCTTCCTTTCCCATTATCCAGAATC1297-12303′-UTR1-a Suffixes "a" and "s" denote antisense (reverse) and sense (forward) primers, whereas numbers 1 and 2 indicate GSP1 and GSP2, respectively. E10 primers are all located in the 3′-untranslated region downstream of TAA stop codon.1-b All nucleotide positions are accounted from the first base of ATG initiation codon (Fig. 2). Open table in a new tab 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 (20Huang C.-H. Chen Y. Reid M.E. Ghosh S. Am. J. Hum. Genet. 1996; 59: 825-833PubMed Google Scholar). 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 (5Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar, 6Avent N.D. Ridgwell K. Tanner M.J.A. Anstee D.J. Biochem. J. 1990; 271: 821-825Crossref PubMed Scopus (208) Google Scholar, 7Le Van Kim C. Mouro I. Cherif-Zahar B. Raynal V. Cherrier C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10925-10929Crossref PubMed Scopus (246) Google Scholar, 8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar, 21Campbell I.G. Freemont P.S. Foulkes W. Trowsdale J. Cancer Res. 1992; 52: 5416-5420PubMed Google Scholar). 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). 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). 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 (22Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17296) Google Scholar). To delineate the structural organization of the Rh50 gene, a bidirectional walking approach was taken to retrieve unknown sequences (Fig.1 A). 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. 1 A 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. The translated sequence of Rh50 was found to be distributed in 10 exons whose size ranges from 15 (exon 10) to 184 bp (exon 2) (Fig.1 B). This global organization is strikingly similar to that of the Rh30 genes (23Huang C.-H. Blood. 1996; 88: 2326-2333Crossref PubMed Google Scholar, 24Cherif-Zahar B. Le Van Kim C. Rouillac C. Raynal V. Cartron J.-P. Colin Y. Genomics. 1994; 19: 68-74Crossref PubMed Scopus (97) Google Scholar) and is essentially conserved in the Rh50 homologues from the mouse and Caenorhabditis elegans. 2Z. Liu and C.-H. Huang, unpublished observations.Comparison of Rh50 with Rh30 showed that their sequence homology is confined mainly to exons 2–9, whereas their 5′ or 3′ regions share little or no sequence similarity. The size of all internal exons except exons 7 and 8 was conserved, and exon 2 of Rh50 was missing codon AGT for Ser99, which is present in Rh30 genes (5Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar, 6Avent N.D. Ridgwell K. Tanner M.J.A. Anstee D.J. Biochem. J. 1990; 271: 821-825Crossref PubMed Scopus (208) Google Scholar, 7Le Van Kim C. Mouro I. Cherif-Zahar B. Raynal V. Cherrier C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10925-10929Crossref PubMed Scopus (246) Google Scholar). Thus, Rh50 and Rh30 show the same assignment of exon/intron junctions except for a difference in their exon 7/exon 8 boundaries (Fig.1 B). The 5′ region of Rh50 has several putative cis-acting elements (Fig. 2), including the TATA boxes that are absent from the proximal promoter of both RhD and RhCE (23Huang C.-H. Blood. 1996; 88: 2326-2333Crossref PubMed Google Scholar, 24Cherif-Zahar B. Le Van Kim C. Rouillac C. Raynal V. Cartron J.-P. Colin Y. Genomics. 1994; 19: 68-74Crossref PubMed Scopus (97) Google Scholar). Multiple transcription initiation sites were identified between the two Ets elements. This mapping result was consistent with the assignment of ATG initiation codon noted in bone marrow Rh50 mRNAs (8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar), although the genomic sequence indicated a potential occurrence of another in-frame ATG codon upstream (nt −96 to −94) (Fig. 2). A detailed study of the Rh50 gene, including the mapping of its introns and dissection of its promoter activity and transcription initiation sites, will be described elsewhere. 3Z. Liu and C.-H. Huang, manuscript in preparation. 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 (25Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7156-7174Crossref Scopus (1977) Google Scholar). 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. 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 aDCe/DCe homozygote. Definition of this Rh genotype by transcript analysis was in full agreement with the result of DNA typing by SphI polymorphisms (15Huang C.-H. Reid M.E. Chen Y. Coghlan G. Okubo Y. Am. J. Hum. Genet. 1996; 58: 133-142PubMed Google Scholar). These data showed that theRH30 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′ portion of Rh50 cDNA encompassing exons 1–5, the 3′ portion of Rh50 cDNA encompassing exons 4–10 always showed a truncation in the Rhnull patient (Fig.4 A). This finding indicated that the Rh50 mRNA from Rhnull could be an aberrantly spliced form lacking a portion of the 3′ sequence. Indeed, sequencing showed that the 122-bp sequence of exon 7 was excluded from the truncated cDNA, resulting in the connection of exon 6 to exon 8 (Fig. 4 B). To determine whether the skipping was complete or partial, a 3′ RACE reaction was carried out using 7 s and 3′-UTR primers. A cDNA product of expected size (376 bp) was found in normal controls but not in the Rhnull patient (Fig.4 C), indicating that no splicing of exon 7 occurred for the Rh50 primary transcript. Further studies showed that this exon skipping was not seen in 15 normal subjects nor in other Rhnullpatients examined; thus, it could not be a constitutive splicing or regulated alternative splicing event. 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. 5 A). 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. Because the mutation abolished a PmlI restriction site (CAC↓GTG) (Fig. 3) and introduced a novel NlaIII site (↓CATG), a direct diagnostic assay was performed on amplified exon 7-containing fragments. The two enzymes showed an opposite cleavage pattern in normal and Rhnull fragments (Fig.5 B), confirming the mutation at the splicing junction. To demonstrate that loss of the PmlI site was not caused by PCR spurious mutations, Southern blot of native genomic DNAs was hybridized with a probe spanning the exon7/intron 7 junction. As shown, thePmlI specific band was seen in normal but not in Rhnull (Fig. 5 C). Given the observation of no dosage reduction in RH50, these results confirmed that the patient is homozygous for the G → A splicing mutation. Such a genotype assignment is consistent with the inheritance of Rhnull syndrome in an autosomal recessive fashion. 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.6 A). 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. 6 A). 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. 6 A). Compared with the wild-type Rh50 protein (8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar), hydropathy plot analysis of the mutant form suggested two possible alterations in membrane organization of the C-terminal region (Fig. 6 B). (i) Deletion of the exon 7-coding sequence abolishes the 5th intracellular loop as well as the 11th TM segment. (ii) The inherent frameshift and premature termination further eliminates the last TM domain, and the resulting new sequence would face the cytoplasmic side due to lack of a continuous stretch of hydrophobic residues. Apparently, loss of a normal C-terminal portion of the Rh50 protein is the major cause for the perturbation of Rh complex formation in the Rhnullerythrocyte. Rh50 glycoprotein is a critical coexpressor of Rh30 polypeptides, the carriers of erythrocyte Rh antigens (1Huang C.-H. Curr. Opin. Hematol. 1997; 4: 94-103Crossref PubMed Scopus (92) Google Scholar, 2Cartron J.-P. Agre P. Blood Cell Biochem. 1995; 6: 189-225Crossref Google Scholar, 3Cartron J.-P. Blood Rev. 1994; 8: 199-211Crossref PubMed Scopus (135) Google Scholar, 4Anstee D.J. Tanner M.J.A. Bailliere Clin. Haematol. 1993; 6: 401-422Abstract Full Text PDF PubMed Scopus (88) Google Scholar). 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 (23Huang C.-H. Blood. 1996; 88: 2326-2333Crossref PubMed Google Scholar, 24Cherif-Zahar B. Le Van Kim C. Rouillac C. Raynal V. Cartron J.-P. Colin Y. Genomics. 1994; 19: 68-74Crossref PubMed Scopus (97) Google Scholar). 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 (5Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar, 6Avent N.D. Ridgwell K. Tanner M.J.A. Anstee D.J. Biochem. J. 1990; 271: 821-825Crossref PubMed Scopus (208) Google Scholar, 7Le Van Kim C. Mouro I. Cherif-Zahar B. Raynal V. Cherrier C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10925-10929Crossref PubMed Scopus (246) Google Scholar, 8Ridgwell K. Spurr N.K. Laguda B. MacGeoch C. Avent N.D. Tanner J.M.A. Biochem. J. 1992; 287: 223-226Crossref PubMed Scopus (114) Google Scholar), 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. 4C.-H. Huang, J. Cheng, Y. Chen, and Z. Liu, unpublished observations. 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 inHomo sapiens. The extreme rareness, recessive nature, and consanguineous background of Rh deficiency syndrome (11Race R. Sanger R. Blood Groups in Man. 6th Ed. Blackwell Scientific Publications, Oxford, London1975: 78-260Google Scholar, 12Nash R. Shojania A.M. Am. J. Hematol. 1987; 24: 267-275Crossref PubMed Scopus (45) Google Scholar) point to a heterogeneous spectrum of the underlying mechanisms. At present, the molecular defect atRH30 locus responsible for the amorph type of Rhnull remains unknown (13Cherif-Zahar B. Raynal V. Le Van Kim C. D'Amrosio A.-M. Bailly P. Cartron J.-P. Colin Y. Blood. 1993; 82: 656-662Crossref PubMed Google Scholar, 14Carritt B. Blunt T. Avent N. Daniels G. Steers F. Ann. Hum. Genet. 1993; 57: 273-279Crossref PubMed Scopus (17) Google Scholar, 15Huang C.-H. Reid M.E. Chen Y. Coghlan G. Okubo Y. Am. J. Hum. Genet. 1996; 58: 133-142PubMed Google Scholar). 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 (9Eyers S.A.S. Ridgwell K. Mawby W.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6417-6423Abstract Full Text PDF PubMed Google Scholar, 26Mallinson G. Anstee D.J. Avent N.D. Ridgwell K. Tanner M.J.A. Daniels G.L. Tippett P. von dem Borne A.E.G. Transfusion. 1990; 30: 222-225Crossref PubMed Scopus (51) Google Scholar). (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 (27Huang C.-H. Johe K.K. Moulds J.J. Siebert P.D. Fukuda M. Blumenfeld O.O. Blood. 1987; 70: 1830-1835Crossref PubMed Google Scholar, 28Tournamille C. Colin Y. Cartron J.-P. Le Van Kim C. Nat. Genet. 1995; 10: 224-228Crossref PubMed Scopus (579) Google Scholar, 29Mallinson G. Soo K.S. Schall T.J. Pasacka M. Anstee D.J. Br. J. Haematol. 1995; 90: 823-829Crossref PubMed Scopus (135) Google Scholar, 30Hermand P. Le Pennec P.Y. Rouger P. Cartron J.-P. Bailly P. Blood. 1996; 87: 2962-2967Crossref PubMed Google Scholar). 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 (31Avent N.D. Judson P.A. Parsons S.F. Mallison G. Anstee D.J. Tanner M.J.A. Evans P.R. Hodges E. Maciver A.G. Holmes C. Biochem. J. 1988; 251: 499-505Crossref PubMed Scopus (80) Google Scholar, 32Lindberg F.P. Lublin D.M. Telen M.J. Veile R.A. Miller Y.E. Donis-Keller H. Brown E.J. J. Biol. Chem. 1994; 269: 1567-1570Abstract Full Text PDF PubMed Google Scholar), 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 (16Cherif-Zahar B. Raynal V. Gane P. Mattei M.-G. Bailly P. Gibbs B. Colin Y. Cartron J.-P. Nat. Genet. 1996; 12: 168-173Crossref PubMed Scopus (126) Google Scholar). Our previous studies showed that this Rhnull patient had a grossly intact RH30 locus occurring in the form ofDCe/DCe haplotype combination (15Huang C.-H. Reid M.E. Chen Y. Coghlan G. Okubo Y. Am. J. Hum. Genet. 1996; 58: 133-142PubMed Google Scholar). The present study confirmed this assignment and showed further that the RH30locus 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 ofRH50 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 (33Padgett R.A. Grabowski P.J. Konarska M.M. Seiler S. Sharp P.A. Annu. Rev. Biochem. 1986; 55: 1119-1150Crossref PubMed Google Scholar), portray an important mechanism for the origin of human genetic diseases (34Krawczak M. Reiss J. Cooper D.N. Hum. Genet. 1992; 90: 41-54Crossref PubMed Scopus (1131) Google Scholar). 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 Rhnulldisease 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 (9Eyers S.A.S. Ridgwell K. Mawby W.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6417-6423Abstract Full Text PDF PubMed Google Scholar, 10Avent N.D. Liu W. Warner K.M. Mawby W.J. Jones J.W. Ridgwell K. Tanner M.J.A. J. Biol. Chem. 1996; 271: 14233-14239Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 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 Rhnulldisease 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. 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.