* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Rearrangements of the Blood Group RhD Gene
Zinc finger nuclease wikipedia , lookup
X-inactivation wikipedia , lookup
Protein moonlighting wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Copy-number variation wikipedia , lookup
Metagenomics wikipedia , lookup
DNA vaccination wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Genome evolution wikipedia , lookup
Genetic engineering wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Gene expression programming wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Gene desert wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Genome (book) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Genome editing wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene therapy wikipedia , lookup
Gene nomenclature wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Point mutation wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Microevolution wikipedia , lookup
Helitron (biology) wikipedia , lookup
From www.bloodjournal.org by guest on June 15, 2017. For personal use only. Rearrangements of the Blood Group RhD Gene Associated With the DV' Category Phenotype By 1. Mouro, C. Le Van Kim, C. Rouillac, D.J. van Rhenen, P.Y. Le Pennec, p. Bailly, J.P. Canron, andy . Colin The Rh (Rhesus) blood group antigens, D, Cc, and Ee, are carried by three unglycosylated membrane proteins of the human erythrocytes encoded by t w o highly relatedgenes, D and CcEe. The major antigen, D, is a mosaic composedof at least ninedeterminants (epD1 through epD9). The lack of expression of some of these D epitopes at thesurface of variant red blood cells defines the so-called D category phenotypes. In thisreport, w e have determined the molecular basis of the D"' category phenotype characterized by the lack of epitopes D l , D2, D5, D6/7. and D8. Southern blot analysis and mRNA sequencing showed that the D"' phenotype is associated with two types ofrearrangement of the D gene. Of10 D"' genomes investigated, 8 exhibited a segmental DNA replacement (gene conversion) between the D fragment encompassing exons 4. 5, and 6 and the equivalent region of the Cc€e gene. In the two other variants, these threeexons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the variant RhD transcripts that are translated in 417 and 266 amino acid polypeptides, respectively. A heterogeneity of category D"' samples basedon variable reactivity of the red blood cells with anti-D antibodies was previously found t o be associated with the CDV'eor cDWE haplotypes. Interestingly, our present results indicated that thisserologic subdivision of the D"' category is correlated to two types of genomic rearrangements of the D gene. 0 1994 by The American Society of Hematology. T In this report, we have shownthat the structure of the D gene camed by 10 unrelated variants is stronglyaltered after two differentgenomic rearrangements. The sequence of the abnormal D protein encoded by the two types of DV' gene has been deduced from mRNA cloning and sequencing. HE RhD ANTIGEN is the major antigen of the RH blood group system. Its presence or its absence at the human red blood cell(RBC) surface determine the Rh-positive (85% of Caucasians) and Rh-negative (1 5% of Caucasians) phenotypes, respectively. However, both types of cells carry antigens of the C/c and E/e series, all definedby specific antibodies.' The D, C/c, and E/e antigens are encoded by a single RH locus located on chromosome 1p34-~36:~ which is composed oftwo homologous structural genes, D and CcEe, that have been recently cloned."6The genome of Rh-positive and Rh-negative individuals can be distinguished, becausethe former carry two genes(Dand CcEe) and the latter only one ( C C E ~The ) . ~D gene encodes a multispanning membrane protein of 417 amino acids and the CcEe gene encodes the Cc and Ee proteins, most likelyby a mechanism ofalternative splicing of a single tran~ript.',~ It is known that some Rh-positive individuals can produce anti-D antibodies in response to immunization by transfusion with D-positive bloodor by pregnancy witha Dpositive This is explained by assuming that the D antigen is a "mosaic" structure and that the RBCs from some D variant individuals may lackpart of this mosaic and become immunized to the D epitopes that they do not possess. Rh-positive individuals that make anti-D have been classified into six main different categories (D" through DV", D' being obsolete), each having a different abnormality in the D antigen.'"I3 Examination of these variants with a panel of human monoclonal antibodies led to the identification of nine different epitopes on the D antigen, termed epDl through epD9.I4-I6The number of epitopes varies from one D category to another, from nine in D"' to three only in DV'. This explains why category VI is the one that most frequently fails to react with polyclonal anti-D. Indeed, only 15% of unselected polyclonalanti-D and35% of selected anti-D made by D-negative subjects reacted with DV'cells. This observation can leadto a confusion between the DV' and the D weak (or D') phenotypes, which correspond to qualitative and quantitative alteration of the D antigenicity, respectively. However,D weak individuals never produce anti-D antibodies, whereas DV'female can be immunized strongly enough to cause hemolytic disease of the n e ~ b o r n ,as ' ~often observed in case of incompatibilitiesbetween an RhD-positive fetus and an RhD-negative mother. Blood. Vol83, No 4 (February 15). 1994:pp 1 129-1135 MATERIALS AND METHODS Materials. Restrictionenzymes, bacterialalkaline phosphatase, and pUC vectors were from Appligene (Strasbourg, France). T4 polynucleotide kinase, DNA polymerase I Klenow fragment, and radiolabeled nucleotideswere from Amersham (Bucks, UK). Avian myeloblastosis virus (AMV) reverse transcriptase were obtained from Promega Biotec(Madison, WI) and Thermus aquaticus polymerase (Taq polymerase) was from Perkin-Elmer-Cetus(Norwalk, CT). Random priming labeling kits were from Boehringer Mannheim (Mannheim, Germany) and pUC sequencing kitswere from Pharmacia (Uppsala, Sweden). Blood samples. Blood samples from RhD-positive, RhD-negative, and rare DV'donors were collected on heparin or EDTA. Eight DV' samples (identification numbers 307, 509, 570, 643, 836, 848, 86 1, and 9 13) were provided by the Rode h i s Bloedbank ZuidLimburg(Maastritch,The Netherlands).The two other DV' samples (DEL and BOUF) were provided by Dr M. Beolet (CRTS Lille, France) and by the Centre National de Referencepour les Groupes Sanguins (CNRGS; Paris, France), respectively. Antibodies and agglutination techniques. Polyclonal antibodies for Rh blood group typings were from the CNRGS and human monoclonal anti-D antibodies have been described at the First" From the Unit6 INSERM U76, Institut National de Transfusion Sanguine, Paris, France;and the Rode KruisBloedbank Rotterdam, Rotterdam, TheNetherlands. Submitted June 7, 1993; accepted October 12,1993. Supported in part by the Institut National de la SantC et de la Recherche Mdicale, bythe Caisse Nationaled'Assurance Maladie des Travailleurs Salarib, and by NATOGrant No. 88/0556. Address reprint requests to YvesColin,PhD,INSERM U76, INTS, 6 rue Alexandre Cabanel, 75015 Paris, France. The publication costs ofthis article were defiayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section I734 solely to indicate this fact. 0I994 by The American Society of Hematology. 0006-4971/94/8304-0012$3.00/0 l129 From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 1130 MOURO ET AL and Second" International Workshop on Monoclonal Antibodies Against Human Red Cells and Related Antigens. Agglutination studies were performed by the antiglobulin test and the papain gel test according to the protocol of manufacturer (Diamed SA, Morat, Switzerland) with native or papain-treated cells according to published procedures." DNA probes. The full-length human RhIXb cDNAencoding the Cc/Ee proteins4 and the PCR-amplified exon 3-, exon 4-, exon 5-, exon 6-, and exon 7-specific fragments deduced from the CcEe gene structure analysis2' wereprepared as previously described' and labeled by the random priming method. Southern blot analysis. Human genomic DNA extracted from peripheral leukocytes" were digested with BamHI ( I O U/pg DNA), resolved by electrophoresis in 0.7% agarose gel, and transferred as described by Southern to a Zeta probe GT nylon membrane (BioRad, Richmond, CA). Hybridization with the DNA probes (3 X IO6 cpm/mL) was performed for 24 hours at 65°C in 7% sodium dodecyl sulfate (SDS), 500 mmol/L NaHPO,, 1 mmol/L EDTA. Final washes were performed at 65°C for 45 minutes in 5% SDS, 40 mmol/L NaHPO,, I mmol/L EDTA, and for 30 minutes in 1%) SDS, 40 mmol/L NaHP04, l mmol/L EDTA. Reverse transcription coupledwith polwnerase chain reaction (PCR)amplification. Total RNAs were extracted from I O mL of whole blood by the acid-phenol-guanidium method.22 RNA samples prepared from heparinized blood were treated with heparinase I to avoid further inhibition of the Taq polymerase.23RNA ( 1 pg) was firstincubated for 5 minutes at 70°C with random primers (0.5 pg) and then cooled to room temperature. Reverse transcription was performed at 42°C for 60 minutes in a reaction mixture (25 pL) containing 50 mmol/L Tris HCI (pH 8.3), 50 mmol/L KCI, I O mmol/L MgC12, 20 mmol/L DTT, 0.5 mmol/L spermidine, 1 mmol/L of each deoxynucleotide triphosphate (dNTPs), 25 U of ribonuclease inhibitor (RNasine), and 15 U of AMV reverse transcriptase. One-fifth of the cDNA products were subjected to PCR amplifi~ation~~ in 50 mmol/L KCI, IO mmol/L Tris (pH 8.3). 3 mmol/L MgC12, 0.001% (wt/vol) gelatin, 0.2 mmol/L of the four dNTPs, 50 pmol of each primer, 2.5 U of Taq polymerase. Oligonucleotide sequences, deduced from the human RhDcDNA clone: were 5'TGT CGG TGC TGA TCT CA3' (sense primer, exon 3 specific) and 5'GGC TCC GACGGT ATCY (antisense primer,exon 7 specific).Thirty cycles of amplification were performed in a PerkinElmer Cetus thermal cycler under the following conditions: denaturation for I minute at 94"C, primer annealing at 48°C for I minute, and extension at 72°C for I minute. Amplified cDNA products were purified on agarose gels, phosphorylated with the polynucleotide kinase, and then subcloned in pUC18 vectors. DNA sequencing. Inserts fromrecombinant pUCl8 vectors were sequenced on both strands by the dideoxy chain termination method25with a Pharmacia T7 sequencing kit. Membrane proteins analysis. For immunostaining analysis, SDS-lysates from RBC membrane preparations prepared as described26were separated by polyacrylamide gradient gel (10% to 20%) electrophoresis (BioRad), transferred to nitrocellulose sheets, and incubatedas described" with a rabbit polyclonal antibody raised against asynthetic Rh peptide (MPC8 reacting with the COOH-terminal region of the Rh proteins).** Bound antibodies were detected with alkaline phosphatase-labeled goat antirabbit IgG diluted (1:800) followed by revelation with the alkaline-phosphatase conjugate substrate kit (BioRad Laboratories, Rockville Center, NY). RESULTS Immunochemical characterization of the D"' category cells. Ten unrelated RBC samples classified as DV' in pre- Table 1. D Epitopes on D"' Category Cells Analyzed With Human MoAbs Epitopes epD 1 epD2 epD3 epD4 epD5 epD6/7 epD8 epD9 NS NS D"' Cells ~ - + + ~ - + - .I-* MoAb-D NT 23\1\13 NT 1 ow3 6W5 26\1\16 105 NA 89 113 D'" RBCs are reactive (+) or unreactive (-) with anti-D MoAbs (MoAbD) according to the presence or the absence of D epitopes'6,'eon their membrane. Abbreviations: NS, not suitablefor epitope determination; NT, not tested; NA, not available. Antibody 1 13 reacts with all DV'samples, except DEL and 3 0 7 . liminary investigations were collected and tested simultaneously withpolyclonal and monoclonal anti-D antibodies. All cells gavepositive reactions with the selected polyclonal IgG anti-D, but the agglutination titers in the antiglobulin tests performed at low ionic strength were significatively lower than those given by common D-positive samples (titers 8 to 32 v 2,048). Subsequently, human monoclonal anti-Ds antibodies (anti-D MoAbs), definedat the First and the Second International Workshop on Monoclonal Antibodies Against Human Red Cellsand Related Antigens,I8.l9 were used in the papain-gel test (Diamed). MoAbs 23W3, 6W5, and 26W6, which recognize epitopes D2, D5, D6/7, and D8, respectively, and MoAb 89, which does not react with DV' cells, were unreactive with the 10 D variant cells under study. On the otherhand, MoAb 10W3, which recognizes epitope D4, gave positivereactions of similar intensity as compared with the polyclonal antibodies. These results clearly established that all the 10 D variants cells belongto the DV' category (Table 1). Moreover, these cells were not reactive with two anti-Ds made by other DV'individuals and some individuals included in this study havedeveloped anti-D after immunization. Antibody 1 13 was peculiar because it reacted (although weakly) in the antiglobulin test and the papain gel test with allthe DV'samples, except DEL and 307, a behavior indicating heterogeneity inside DV' samples, as previously reported.29 Full RBC phenotyping with anti-Rh reagents (anti-C, -c, -E, and -e) indicated that DEL and 307wereDV'ccEe, whereas the other samples were Dv'Ccee. Thus, all samples are heterozygous for D with the mostlikely genotypes cDV'E/cde (DEL and 307) and CDV'e/cde (others), respectively. DNAstudies have confirmed these expectations (see below). Southern blot analysis ojthe RH locus in D" phenotypes. Genomic DNA from the I O DV'individuals were digested with BamHI restriction enzyme and subjected to Southern blot analysiswith the RhIXb cDNA probe. DNA from RhD-positive (DCCee) and RhD-negative (ddccee) donors From www.bloodjournal.org by guest on June 15, 2017. For personal use only. REARRANGEMENTS OF THE RH D" 1131 GENE m U m 0 : : a n A A B Fig 1. Southem blot analysis of D"' genomic DNAs and comparison with RhD-positive and RhD-negative genomes. DNAs from two D"' individuals and from RhD-positive (DCCee) and RhD-negative (ddccee) donors were digested with the restriction enzyme BamHl and hybridized on aSouthern blot with the RhlXb cDNA (A) and with the exon 4-specific probe (B). Rehybridizationof the same blot withexons 5- or 6-specific probes gave the same pattern. Of the 10 D"' DNA samples studied, 8exhibited the restriction pattern of sample 848 and 2 had the patternof DEL (not shown). were included as control. As shown in Fig 1A, two types of genomic polymorphisms were shown when the hybridization patterns of the DNAs fromD"' and RhD-positive individuals were compared. In eight D"' variants, represented by sample 848, a 4.5-kb BamHl fragment wasmissing, whereas an additional fragment of 5.1 kb appeared (Fig1A). In the two other D"' genomes, representedby sample DEL, the 4.5-kb BamHI fragment was also missing, but no abnormal band was detected. Previous Southern blot analyses have indicated that the 4.5-kb BamHI fragment is specific for the RhD gene, becauseit is present inthe D-positive genomes and absentin the D-negative DNAs' (Fig1A). To better characterizethe 4.5-kb polymorphic fragment, the same blot was successively rehybridized with several exon-specificprobesdeducedfrom CcEe gene structure analysis (see Materialsand Methods). Because of the colinearity and of the high degree of identity(96%)between the D and non-D cDNAs,~it has been assumedthat the intron/ exon organization of the D and CcEe genes is most likely identicalZoand, therefore, the exonic probesused are able to detect equivalent coding regions of both the D and CcEe genes. Results from partial sequence analysis the of D gene support this hypothesis (our unpublished results).The 4.5kb BamHl fragment was detected inthe RhD-positive con- trol DNA by probes specific of exons 4, 5, and 6 (Fig 1 B). These probes also showed a band of 5.3 kb in both the RhDpositive and RhD-negative genomes, which corresponded to the CcEe gene fragment encompassing exons 4,5, and 6. In the DEL sample, only the CcEe 5.3-kb fragment was detected, suggesting that the restriction fragment carrying exons 4,5, and 6 of the D gene is deleted. In the 848 sample, the CcEe 5.3-kb fragmentwas shown togetherwith the abnormal 5. l-kb fragment, suggesting either that the fragment carrying exons4,5, and 6 of the D gene is rearranged or that one of the BamHI sites generating this restriction fragment is polymorphic. Ithasbeenpreviouslyshown that hybridization with exon-specific probes allows us to determine the homozygous or heterozygous status for the D gene bycomparing the intensities of the CcEe-and D-specific signals.30 Using either exon 4 or exon 5 or exon 6 as probes, the CcEe- and Dspecific fragments were detected withthe same intensity in the homozygous D-positivecontrol DNA (two copies of the D gene and two copies of the CcEe gene), whereasa 1 :2 gene dosage effect was observed in the 848 DNA sample (FigI B), indicating the D heterozygosity of this donor. Similarly, hybridizationperformed withexon 7 probe (not shown) showed that the nine other D"' variants were heterozygous for the D gene, as expected (see above). Analysis gf the Rh transcripts in DV'phenotypes. Total RNAs extracted from peripheral blood of two D"'individuals (samples 848and DEL) and of RhD-positive and RhD- + 701 bp + 248 bp Fig 2. PCR amplification of the D"' transcripts and comparison with the amplified products of RhD-positiveand RhD-negativetranscripts. Total RNAs extracted from theperipheral blood of the two types of D"' individuals (DEL and 848 samples) and of RhD-positive and RhD-negative donors were reverse transcribed. The cDNAs were subjected to a PCR amplification between an oligonucleotide common to D and CcEe cDNAs in the exon 3 and a D-specific oligonucleotide in the exon 7 (see Materials and Methods). The PCR products were resolvedon an agarose gel. From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 1132 MOURO ET AL E4 D DvI(848) Dvl(DEL) D Dvl(848) Dvl(DEL) 210 TATIPSLSAM R-----"-- E5 220 LGALFLWMFW -_________ ****************** 230 PSFNSALLRS --V----"- ********** 240 PIERKNAVFN --Q"--"- ********** E5 E 6 2 250 60 280 270 TYYAVAVSVVTAISGSSLAHPQGKISKTYVHSAVLAGGVA ----L""- _ _ - _ _ _ _ --R---"-__ ********** R********* ********** __________ ********** L"_ E6 D Dvl (848) Dvl(DEL) 290 310 300 VGTSCHLIPSPWLAMVLGLVAFLISVGGAKYLPGCCNRV--""C-"1"" " " " " " " " " " " ********** ********** ********** *** (417 GCCNRV- - - ( 4 1 7 GCCNRV---(266 aa) aa) aa) Fig 3. Amino acid sequence comparison ofD, DV'848, and D'" DEL encoded proteins in the affected region. The D proteinwas deduced from the recently cloned RhD cDNA.' Identical positions noted are as dashes.The bars abovethe protein sequence delineate the amino acids encoded by exons3 through 7 (E3 through E7). Amino acid sequencefrom position 163 to position 3 1 3 of the D"' 848 protein corresponds to the amino acid sequence of the Ce or ce encodedp r ~ t e i n .In ~ ,D"' ~ DEL, amino acids1 6 3 to 3 1 3 encoded by exons 4,5, and 6 are deleted (*) and, therefore, amino acid 1 6 2 is linkedto amino acid3 14 inthe new protein variant. negative donors were converted to cDNAs and enzymatically amplified. The PCR amplification was performed between a first primer corresponding to an exon 3 sequence common to the D and non-D cDNAs and a second primer designed froma D-specific sequencein exon 7 (see Materials and Methods).6As expected, no amplification product was obtained with the RhD-negative cDNA, whereas a 701-bp fragment was amplified fromthe RhD-positive cDNAtemplate (Fig2). Amplification performed with the transcripts from the DEL donor yielded a PCR product of 248 bp (Fig 2). Sequence analysis indicated that this cDNA fragment lacked nucleotides 487 to 939 of the normal RhD cDNA (+ 1 taken as the first residue of the initiator methionine.6Based on the analysis ofthe R h gene@)organization (Chirif-Zahar et alZo and our unpublished data) we deduced that the 5' and 3' boundaries of the deletion in the transcription product corresponded to the exon 3lintron 3 and intron6/exon l transitions, respectively. This result, together with the Southern analysis described above, showed that the genomic region encompassing exons 4, 5 , and 6 of the D gene is deleted in donor DEL. Becauseintrons 3 and 6 interrupt the D coding sequence after the third and before the first nucleotide of a codon triplet, respectively,the reading frame of the mRNA resulting fromthe fusion of exons 3 and 7 in the DEL gene was not changed. Therefore, the deduced DV' (DEL)-encoded protein represents a shortened form of the normal D polypeptide composed of 266 amino acids that lacks residues 163 to 3 13 (Fig 3). The size ofthe PCR products obtained after amplification of the D transcript from the D"' (848) and the RhD-positive donors were identical (70 l bp; Fig 2). Sequence comparison between these products showed 17 nucleotide substitutions all located within the regions transcribed from exons 4, 5 , and 6. No other polymorphisms were identified in the remaining coding region of the 848 cDNA (not shown). Interestingly, allthe substitutions between 848 and the normal D cDNAs corresponded to that previously identified between the D and non-D transcript^.^.^ These findings, together with the Southern blot analysis, indicated that the variant 848 is characterized by a rearranged RH locus in which the region carrying exons 4, 5 , and 6 of the D gene has been replaced by an equivalent fragment of the CcEe gene. The deduced protein encoded by this hybrid D-CcEe-D gene isa 4 17 amino acid polypeptide that differs fromthe normal D polypeptide by 15 amino acid substitutions (Fig 3). Western blot analysis of the R h polypeptides in D" phenotypes. The calculated molecular mass of the normal D and variant D"' (DEL) polypeptides being significatively different (45.5 and 29.2 kD, respectively), a comparative Western blot analysis was performed with a polyclonal antibody raised against a synthetic peptide derived from the COOH sequence common to the D and non-D polypep tides.** Surprisingly, evenafter migration in gradient poly- From www.bloodjournal.org by guest on June 15, 2017. For personal use only. REARRANGEMENTS OF THE RH D"' GENE 1133 (calculated Mr,45.5 kD). However,the shortened polypep tide could not be separated from the normal Rh polypep tides expressed in common phenotypes, which all migrate with an apparent Mr of 32 kD on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).This is most likely explained because these proteins are highly hydrophobic molecules that exhibit an abnormal behavior in SDS-PAGE."' Thisobservation may explain why the Cc and Ee proteins that are thought to be translatedfromdifferentsplicingisoform transcripts of the CcEe gene, one of which precisely lacks exons 4, 5, and 6,8*9 could not be distinguished from each other by SDS gel electrophoresis. In the type I1 variants, representedby the 8 other DV'samples, a different rearrangementof the D gene was identified, because exons 4, 5, and 6 were present but camed by a restriction fragmentof abnormal size. cDNA sequence analysis indicatedthat the region ofthe DV'transcript encoded by Fig 4. lmmunostaining of the RBC membrane proteins from these three exonscorresponded to that of the non-D DCCee, D"' 848, and D"' DEL donors. Total membrane proteins (60 mRNAs, and more precisely to the transcription productof pg) separated on a polyacrylamide gradient (1 gel 0%to 20%)were the Ce or ce alleles accordingto the presence of an Alanine transferred to nitrocellulose sheets and incubated with a rabbit anti-Rh protein antibody (MPC8: 1/4,000). After washing, the niresidue at the E/e-associated polymorphic position 226.9 trocellulose membrane was incubated with a goat antirabbit IgG These results strongly suggested that the DNA segment of conjugated to alkaline phosphatase. Antibodies bound to the Rh the D gene encompassing exons 4, 5, and 6 has been reproteinswere visualized by an alkaline-phosphatase conjugate subplaced inthese variants by the equivalent region ofthe CcEe strate kit. The arrow on the right side indicates the migration of the gene. Becausethe D and CcEegenes are sequence related?*' detectable in Rh polypeptides.(*) Undefined molecular species not Rh-null sampleswith the MPC8 polyclonal antibody(P. Bailly. perit is assumedthat such a DNA transfer occurred after chrosonal communication). mosomal misalignment between the two Rh genes during meiosis (Fig 5B). Whether the resulting hybrid"D-CcEe-D" gene isthe product of an intergenic double crossing-over or acrylamide gels (1 0% to 20%), no difference in the electroofa combination ofgene conversion between the CcEegene phoreticmobility ofDELversusnormal D membrane (as donor) and the D gene (as acceptor) and branch migraproteins could be detected (Fig 4). tion cannot be determined because the second product of these gene rearrangements is not available.However, gene DISCUSSION conversion providesa simpler modeland is thus commonly favored when,as in the present case,the length of the region We have analyzedthe organization and expression of the RH blood group locus in 10 variants of the DV' category affected is ofthe order of a few kilo base^."^ Homologous rephenotype, which represents the most frequently detected combination through a mechanism of gene conversion has variant of the D categories."*'*Our results clearly estabbeen widely reported ina number of multigene familiesin lished that the DV' phenotype can be associated withat least humans, includingglobin,33haptoglobin,"* Ig,34.35major two typesof genomic rearrangementsof the D gene, called histocompatibility complex,36 complement ~ystem,~' and here typesI and 11. glycophorin gene c l u ~ t e r s . ' ~Analysis * ~ ~ of the evolution of the mammalian &like globin genes indicated that segmental In the type I variants, represented by 2 of the 10 samples investigated, Southern blot analysis indicated that the D DNA replacement, by transfemng several base substitugene fragment encompassing exons 4,5, and 6 was deleted. tions in a single event,can lead to wrong phylogenetic relaSequence analysisof the Rh mRNA transcribed from one tionships of specie^.^.^' Therefore, the present description ofthe type I variantsconfirmedthis result and indicated that of such a rearrangement inthe Rh gene cluster shouldgive the breakpoints of the deletion were located withinintrons rise to special caution during the phylogenetic analysis of 3 and 6 of the D gene. We assumed that, as frequently dethe RH locus. scribed in case of partial gene deletion, this defective D gene It is noteworthy that the DV'-associated gene remost likely results from an unequal crossing over caused by arrangements of types I and I1 both involve exons4,5, and recombination between homologous intronic regions (Fig 6. This result suggests that the D-specific amino acids en5A). However, these putative repeated sequences have not coded by these three exons ( 15 residues; see Fig 3) might be yet been characterized. Accordingto this model, the recip critical for the reactivity with the different MoAbs characrocal product of the recombination event canbe predicted terizing epitopesD I , D2, D5,D6/7, and D8 that are camed to be an abnormal D gene with a duplication of exons 4,5, by the normal D but not by the D"' protein.I5.l6 Conversely, and 6 that remains to be identified.The deduced translation the remaining part of the D"' protein still cames epitopes product of the deleted D" gene is a 266 amino acid protein D3, D4, and D9, which, however, have not yet been prethat lacks 15 1 amino acids (calculated Mr, 29.2 kD) as comcisely mapped. pared withthe 4 I7 amino acids ofthe normal D polypeptide The D"' polypeptides encodedby the type I and type I1 From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 1134 MOURO ET AL D gene CcEe gene A 16 + D"' type I (DEL) D gene 13 CcEe gene E B E l E2 E3 E4 E5 E6 E7 E8 + D"' type It (848) Fig 5. Schematic representation showing possible genetic rearrangements resulting in D"' genes. The high degreeof similarity between that the D and the &€e genes have a similar organization? Exons (El the CcEe gene andthe D gene' aswell as preliminary studies suggest through E l 0)from CcEe and D genes are represented by open and dotted boxes, respectively (not to scale). 13 and I6 denote introns3 and 6, respectively. The given position ofthe D and CcEe genes onthe chromosome isarbiirary. (A) Intragenic unequal crossingover occurring 6 of the other chromosome giving rise to a new chromosome carryinga deletion of exons 4,5, within intron 3 of one chromosome and intron and 6. as observedin D"' DEL. (B) lntergenic double crossing over or gene conversion occurring between a D and a Cc€e gene giving rise to a hybrid D-&€e-D gene, as observedin D"' 848. Brackets indicatethat the genetic rearrangementmay have occurredbetween a D-positive chromosome and a D-positive orD-negative a chromosome. Reciprocal gene rearrangements generated by these recombination events are not shown (see text). variants might well be discriminated by immunologic methods because the deletion and conversion process generated unusual D proteins characterizedby a new amino acid junction or peptide insertion, respectively. As a matter of fact, some heterogeneity among DV' phenotypes has been suspectedserologically. Indeed, it has been reported that cDV'E/cde but not CDV'e/cde samples failed to react with one of two monoclonal anti-G antibodies and that CDV'e/ cde but not cDV'E/cde cells expressa "new" low frequency antigen inherited as a mendelian character.42It was found also that CDV'e/cde cells often gave stronger reactions than cDV'E/cde cells with some anti-D MoAbs.'' Interestingly, these subdivisions of the DV' category appear to be correlated at the molecular levelwith the twotypes ofrearrangement of the D gene, becausewe found here that the cDV'E and CDV'e haplotypes are associated with the deleted" (type I) or the "converted" (type 11) forms of the D gene, respectively.Our results strongly suggest that the serologic heterogeneity of the DV' phenotype described above is based on different primary, and most likely secondary, structures of the DV*polypeptides expressed at the surface of the variant RBCs. Further investigationsof partial D variants should help to delineate the structure of the D epitopes on D polypeptides and to determine if some play a predominant role inthe high immunogenicity ofthe D antigen. ACKNOWLEDGMENT We thank Dr Baya Cherif-Zahar for providing data on the structure of the Rh gene beforepublication, Dr Marylise Beolet (CRTS Lille, France) for the gift of DEL sample, Drs Pascal Bailly and Patricia Hermand for providingthe MPC8 antibody, and Marie-The&se Klein(CNRGS, Paris) for technical assistance. REFERENCES 1. Race RR, SangerR:Blood Groups in Man.Oxford, UK, Blackwell Scientific, 1975 2. Marsh WL, Chaganti RSK, Gardner FG, Mayer K, Nowell PC, German J: Mapping human autosomes: Evidence supporting assignment of Rhesus to the short arm of chromosome no 1. Science 184:966, 1974 3. Chirif-Zahar B, Matt& MG, Le Van KimC, Bailly P, Cartron J P Localization of the human Rh blood group gene structure to chromosome regionlp34-p36.1 by in situ hybridization. Hum Genet 86:398, 1991 4. Chirif-Zahar B, Bloy C , Le Van Kim C, Blanchard D, Bailly P, Hermand P, Salmon C,Cartron JP, Colin Y: Molecular cloning and protein structure of a human blood group Rh polypeptide. Proc Natl Acad Sci USA 87:6243, 1990 5. Avent ND, Ridgwell K, Tanner MJA, Anstee DJ: cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-groupantigen expression.Biochem J 271:82 1, 1990 6. Le Van Kim C , Mouro I, Chirif-Zahar B, Raynal V, Cherrier C, Cartron JP, Colin Y: Molecular cloningand primary structure of the human blood group RhD polypeptide. Proc Natl Acad Sci USA 89:10925,1992 7. Colin Y, Chdrif-Zahar B, Le Van Kim C, Raynal V,Van Huffel V, Cartron JP: Genetic basis ofthe RhD-positive and RhDnegative bloodgroup polymorphism.Blood 78:2747, 1991 8. Le Van Kim C, Chirif-Zahar B, Raynal V, Mouro I, Lopez From www.bloodjournal.org by guest on June 15, 2017. For personal use only. REARRANGEMENTS OF THE RH D"' GENE M, Cartron JP, Colin Y: Multiple Rh messenger RNA isoformsare produced by alternative splicing. Blood8 0 1074, 1992 9. Mouro I, Colin Y, CheriEZahar B, Cartron JP, Le Van Kim C: Molecular genetic basisof the human RH blood group system. Nature Genet 5:62, 1993 10. Mollison P, Engelfriet P,Contreras M: Blood Transfusion in Clinical Medicine. Oxford,UK, Blackwell Scientific, 1992 11. Tippett P, Sanger R: Observationson the subdivision of the Rh antigen. Vox Sang 7:9, 1962 12. Tippett P, Sanger R Further observations on the subdivisions of the Rh antigen. Ant1 Lab 23:476, 1977 13. Tippett P Subdivisions of the Rh(D) antigen. Med Lab Sci 45:88, 1988 14. LomasC, Tippett P, Thompson KM, MelamedMD, Hughes-JonesN C Demonstration of seven epitopes on the Rh antigen D using human monoclonal anti-D antibodies and red cells from D categories.Vox Sang 57:26I, 1989 15. Lomas C, McCollK, Tippett P Further complexities ofthe Rh antigen D disclosed by testing category D" cells with monoclonal anti-D. Transfusion Med 3:67, 1993 16. Tippett P: Serologically defined Rh determinants. J Immunogenet 17:247, 1990 17. Lacey P: An unexpected case of severe hemolytic disease of the newborn due to anti-D. Transfusion 18:642, 1978(abstr) 18. Tippett P Co-ordinator's report on group 3: Monoclonal Rh antibodies: Serologicaland biological studies. RevFr Transfus Immuno Hematol31:249, 1988 19. Tippett P, Moore S: Monoclonal antibodies against Rh and Rh related antigens.J lmmunogenet 17:309, 1990 20. Chirif-Zahar B, Le Van Kim C , Rouillac C, RaynalV, Cartron JP, Colin Y: Organization of the gene encoding the human blood group RhCcEe antigens and characterizationofthe promoter region. Genomics (in press) 2 1. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning (ed 2). ColdSpring Harbor, NY, Cold SpringHarbor Laboratory, 1989 22. Chomczynski P, SacchiN: Single-stepmethod of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156,I987 23. Izraeli S, Pfleiderer C, Lion T: Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Res 19:605 1, 1991 24. Saiki RK, Gelfand DH, Stoffel S, ScharfSJ, Higuchi R, Horn GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487,1988 25. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl AcadSciUSA 74:5463, 1977 26. Steck TL, Kant JA: Preparation of impermeable ghosts and inside-out vesicules fromhuman erythrocyte membranes. Methods Enzymol3 I :172, 1974 1135 27. Towbin H, Stachelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamidegels to nitrocellulose sheets. Proc Natl Acad Sci USA 76:4350, 1979 28. Hermand P, Mouro I, Huet M, BloyC, Suyama K, Goldstein J, Cartron JP, Bailly P Immunochemical characterization of Rh proteins with antibodies raised againstsyntheticpeptides. Blood 82: 669, 1993 29. Ernst M, Weber-Muscan M-T,Lenhard V, Sonneborn HH: Human monoclonal antibodies for the differentiation of Rh-D categories. lnfusionstherapie 19:9, 1992 30. Chirif-Zahar B, Raynal V, Le Van Kim C,DAmbrosio AM, Bailly P, Cartron JP, Colin Y Structure and expression ofthe RH locus in the Rh-deficiency syndrome. Blood 82:656, 1993 3 1. Agre P, Cartron J P Molecular biology of Rh antigens. Blood 78551, 1991 32. Maeda N, Smithies 0 The evolution of multigene families: Human haptoglobin genes. Annu RevGenet 20:8 l, 1986 33. Slightom JL, Blechl AE, Smithies 0 Human fetal G-y- and Ay-globin genes:Complete nucleotide sequences suggest that DNA can be exchanged between these duplicatedgenes. Cell2 1 :627,1980 34. Bentley DL, Rabbitts TH: Evolution of immunoglobulin V genes: Evidence indicating that recently duplicated human V, se1, 1983 quences have diverged by gene conversion. Cell1832: 35. Krawinkel U, ZoebeleinG, Bothwell ALM: Palindromic sequences are associated with sites of DNA breakageduring gene conversion. NucleicAcid Res 14:3871, 1986 36. Ehrlich HA, Gyllensten UB: Shared epitopes among HLA class I1 alleles: Gene conversion, common ancestry and balancing selection. Immunol Today 12:41 1, 1991 37. Braun L, Schneider PM, Giles CM, Bertrams J, Rittner C Null alleles of human complement C4: Evidence for pseudogenes at the C4A locus and for gene conversionat the C4B locus. J Exp Med 171:129, 1990 38. Huang C-H, Kikuchi M, McCreary J, Blumenfeld0 0 : Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity of human (GYP) locus. J Biol Chem 267: 3336, 1992 39. Huang C-H, Skov F, Daniels G, Tippett P, Blumenfeld 00 Molecular analysis human of glycophorin MiIX gene shows a silent segment transfer and untemplated mutation resuJing from gene conversion via sequence repeats. Blood 80:2379, 1992 40. Hardison RC, Margot JB: Rabbit globin pseudogene (p82 is a hybrid of 8- and @-globin gene sequences. Mol Biol Evol 1:302, 1984 41. Hardies SC,Edge11 MH, Hutchinson CA: Evolution of the mammalian P-globin genecluster. J Biol Chem 259:3748, 1984 42. Lomas C, Mougey R: Rh antigen D Variable expression in DV' phenotypes;a possible subdivisionof category VI by a low frequency antigen. Transfusion 29: 14, 1989(abstr, suppl) From www.bloodjournal.org by guest on June 15, 2017. For personal use only. 1994 83: 1129-1135 Rearrangements of the blood group RhD gene associated with the DVI category phenotype I Mouro, C Le Van Kim, C Rouillac, DJ van Rhenen, PY Le Pennec, P Bailly, JP Cartron and Y Colin Updated information and services can be found at: http://www.bloodjournal.org/content/83/4/1129.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.