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From www.bloodjournal.org by guest on August 11, 2017. For personal use only. 1518 CORRESPONDENCE Lack of RHCE-Encoded Proteins in the D-- Phenotype May Result From Homologous Recombination Between the Two RH Genes To the Editor: by TheRh blood groupantigens D, C/c,andE/earecarried atleastthree red blood cellmembraneproteinsencodedbytwo homologousgenes, RHD and RHCE. Thesetwogenesarepresent thein Rh-positive only individuals, whereas RHCE encoding gene the Cc and Ee proteins is present in Rh-negative cells.' Rare homozygousindividualscarrying the D-- genecomplexarecharacterized by atotalabsence o f C/candE/eantigensand an excessrepresentation o f the D antigen.* The RH locus o f three D-- individuals From www.bloodjournal.org by guest on August 11, 2017. For personal use only. CORRESPONDENCE (French. Icelandic. and Italian donors) had been previously analyzed by Southern blot and Rh cDNAs sequencing.'" The results indicated that the lack of C/c and E/e antigens resulted from either a reduced transcriptional activity' or from an internal gene deletion','of the RHCE gene. We describe here a third mechanism leading to this phenotype by analyzing the RH locus of a D-- individual (LM) of Italian origin. We discovered that this donor was independently studied by Huang et al.' but our results are different. because we ascertained that this D-- gene complex most likely resulted from a homologous recombination event between RHD and RHCE genes. as indicated by sequence analysis and by a reverse transcriptasepolymerase chain reaction (RT-PCR) assay that specifically detected hybrid gene products (see below). Southern blot analysis with Rh cDNA and exon-specific probes showed an alteration of the RHCE gene of LM. because the RomHI and Hind111 restriction fragments carrying exons 4 through 6 and exons 5 through 7, respectively. were lacking (data not shown). Reticulocyte RNAs from LM were reverse transcribed to cDNA and amplified by PCR using a primer located in the S' untranslated region (S'UT; nt -19 to -2; + I representing A of the initiation ATG codon) common to the RHD and RHCE genes and a primer specific of the 3' untranslated sequence of the RHD (3'UTD: nt1,421 to 1.437) or RHCE (3'UTCE: nt 1.363 to 1.380) genes. respectively (Fig IA). The cDNA amplified between the S'UT and 3'UTD primers (1.45 kb) carried three base substitutions (G,&. T,,,,,C. and AII',IT) that were previously assigned to exons I and 9 of the RHCE gene. This cDNA was also identified by Huang et al.' Sequencing of the PCR product amplified between the S'UT and 3'UTCE primers ( I .40 kb) showed a hybrid cDNA composed of an RHD sequence transcribed from exons 1 to 9 and an RHCE sequence corresponding to exon IO. This cDNA was not identified by Huang et al.'Both cDNAs carried the G520A transition, resulting in a V174M substitution. These data indicated that the Rh RNAs from LM cells were transcribed from two rearranged RH genes (Fig IA). To identify the sequence upstream nt48 in the two cDNAs (1.45 and 1.40 kb), we have amplified by PCR a 815-bp fragment, including 676 bp upstream the intiation ATG codon, using a S' primer located in the promoter region (nt -676 to -659) and a 3' primer in exon I (nt 122 to 139). because several substitutions (nt -597. -554. -390, - 1 4 4 . and -132) allow us to differentiate the RHD from the RHCE promoter (our unpublished data). Sequencing analysis indicated that the 1.45-kb cDNA was transcribed from the RHCE promoter. whereas the I .40-kb cDNA was transcribed from the RHD promoter. These results suggest an unusual complexity of the RH locus from LM, which we assume as being composed of two hybrid genes. C€-D-C€-D (a similar complex hybrid between the genes encoding the erythrocyte glycophorins A and B has been described") and D-C€. The proteins encoded by these rearranged genes would carry D but not CcEe epitopes. thus explaning the serologic properties of the D-- complex (greater than the normal amount of D and a total absence of C c E e antigens). To confirm the presence of the D-C€hybrid transcript in LM reticulocytes. a hybrid RT-PCR using 5' primers located in exon 5 of RHD or RHCE sequence (E'D, nt 695 to 712: or E'CE, nt 650 to 667) and the 3'UTCE primer was setup (Fig IB). The primer pair E5D/3'UTCE amplified a 690-bp product from LM cDNA (as well as 556- and 403-bp isoforms). but it did not amplify any product from the cDNA of a normal Rh-positive (DCe) control (Fig IB). Conversely. the primer pair E5CE/3'UTCE amplified a 733-bp product (as well as several minor isofonns) from the RHCE gene of the Rh-positive (DCe) control, but it did not amplify any product from LM cDNA (Fig IB). Therefore, the hybrid RT-PCR method appears to be useful to in Rh variants. Accorddetect hybrid gene structure(s) present ingly, we found that the E5D/3'UTCE primer pair gave positive 1519 EsCE / 3'UTCE E5D I3'UTCE B Fig 1. RH locus and detection of hybrid transcripts in Rh variants by RT-PCR analysis. (A) Schematic structure of RH haplotypes from Rh-positive (DCe) and Rh-negative (dce) individuals compared with RH haplotypes from variants with a total or partial defect of Clc andlor Ele antigen expression. RHD and RHCE sequences are indicated by solid and open boxes, respectively. Small boxes upstream each gene correspond to therespective promoter. Numbers above gene boxes refer to exon regions of the RHD (solid symbols) or RHCE (open symbols) genes involved in the rearrangements. Arrows indicate primers used in the RT-PCR assays. Fulllength D and non-D cDNAs were amplified with primers 5'UT (arrow heads, common to RHD and RHCE genes), 3'UTD (solid arrow, D-specific), and 3'UTCE (open arrow, CE-specific) and sequenced (see text). (B) Detection of hybrid Rh transcripts by RTPCR analysis. PCR (30 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minutel were performed with the primer pairs E5CE/3'UTCE and E5D/3'UTCE, which were designed to amplify 733- and 690-bp fragments from theCE and hybrid D-CE transcripts, respectively. Primer ESCE(open arrow, sense primer) is located in exon 5 and is CE specific, whereas primer E5D (solid arrow, sense primer) is located in exon 5 and is D-specific. The primer 3'UTCE (open arrow) is CE-specific and was used in both assays as antisense primer. PCR products amplified from DCe, D-- (LM), D-- (Gou), DC- (Bol), and D . ' ( D a v )were resolved on 2% agarose gel and visualized under UV light after staining with ethidium bromide. Sizes of the CE (left) and hybrid D-CE (right) amplification products are indicated. The 556- and 403-bp fragments are isoforms lacking exon 7 and exons 7 through 9, respectively. signals(Fig I B ) with the cDNAsprepared from homozygous DC- (Bol.) and D - - ( D a v . ) individuals,whichcarrya hybrid CE-D(e.rort 4 to 9)-CE gene' and a D(e.ror? /-6)-CE(e.rorl7 - I O ) hybrid gene (our unpublished data), respectively. However. no signal was detected with the D-- sample (Gou) that carries an intact RHCE gene.' On the other hand. the E'CE13'UTCE primer pair could amplify a 733-bp RHCE gene product from the D-sample. but therewasnoamplilication with the DC- and D * samples (Fig I B). In conclusion. our results show the heterogeneity of the phenotypes with a total absence of RhCclEe antigen expression. They also show that the hybrid RT-PCR assay is useful to detect hybrid gene structures. suggesting that this method can be generalized to identify gene rearrangementsin many examples of R h variants. From www.bloodjournal.org by guest on August 11, 2017. For personal use only. CORRESPONDENCE 1520 ACKNOWLEDGMENT We thank Dr Georgio Reali (Immunoematologia e Transfusionale, Genova, Italy) andDr Marcela Contreras (North London Blood Transfusion Service, London, UK) for the generous gift of D-(LM) and D (Dav) samples, respectively. Baya Cherif-Zahar Virginie Raynal Jean-Pierre Cartron INSERM U76 Institur National de Iu Transfusion Sanguine Paris,France REFERENCES 1. Cartron JP, Agre P: Rh blood group antigens: Protein and gene structure. Semin Hematol 30:193, 1993 2. Race RR, Sanger R: Blood Groups in Man. Oxford, UK, Blackwell Scientific, 1975 3. Cherif-Zahar B, Raynal V, D’Ambrosio AM, Cartron JP, Colin Y: Molecular analysis of the structure and expression oftheRH locus in individuals with D--, DC-, and DC”- gene complexes. Blood 84:4354, 1994 4. Blunt T, Steers F, Daniels G, Carritt B: Lack of RHClE expression in the Rhesus D-- phenotype is the result of a gene deletion. Ann Hum Genet 58:19, 1994 5. Huang CH, Reid ME, Chen Y: Identification of a partial internal deletion in the RH locus causing the human erythrocytes D-phenotype. Blood 86:784, 1995 6. Huang CH, Lomas C, Daniels G, Blumenfeld 00:Glycophorin He (St;‘) of the humanredblood cell membrane is encoded by a complex hybrid gene resulting from two recombinational events. Blood 83:3369, 1994 From www.bloodjournal.org by guest on August 11, 2017. For personal use only. 1996 88: 1518-1520 Lack of RHCE-encoded proteins in the D--phenotype may result from homologous recombination between the two RH genes [letter; comment] B Cherif-Zahar, V Raynal and JP Cartron Updated information and services can be found at: http://www.bloodjournal.org/content/88/4/1518.citation.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. 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