SEARCH:   Advanced

Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0298. This Article Abstract Full Text (PDF) All Versions of this Article: 2001-12-0298v1 100/3/1063    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Lucien, N. Articles by Bailly, P. Search for Related Content PubMed PubMed Citation Articles by Lucien, N. Articles by Bailly, P. Related Collections Red Cells Transfusion Medicine Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 August 2002, Vol. 100, No. 3, pp. 1063-1064 BRIEF REPORT Short deletion within the blood group Dombrock locus causing a Donull phenotype Nicole Lucien, Jean-Louis Celton, Pierre-Yves Le Pennec, Jean-Pierre Cartron, and Pascal Bailly From INSERM-U76, Institut National de la Transfusion Sanguine, Paris, and EFS Guyane, Cayenne, France.     Abstract Top Abstract Introduction Study design Results and discussion References A new alteration of the blood group DO*A allele was identified in a female Donull donor from Reunion Island with allo- anti-DO3 in her serum; her parents are consanguineous. Because the amplification of the DO transcript failed, each exon and intron-exon junction from the DO gene were examined. After polymerase chain reaction (PCR) amplification and sequencing, the only deviation from the wild-type DO*A allele sequence was an 8-nucleotide deletion (nt 343-350) within exon 2. This short deletion generates a premature stop codon and encodes a truncated protein lacking the predicted functional motif of the adenosine diphosphate-ribosyltransferase enzyme and the glycosyl-phosphatidylinositol anchor motif essential for RBC membrane attachment. An allele-specific PCR to detect the DO(8nt) deletion was developed. (Blood. 2002;100:1063-1064) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The antithetical Dombrock antigens (Doa/Dob) reside on a glycosyl-phosphatidylinositol (GPI)-anchored red blood cell (RBC) membrane glycoprotein that also carries 3 high-incidence antigens, Gregory (Gya), Holley (Hy), and Joseph (Joa).1,2 The Dombrock gene locus (DO) consists of 3 exons spanning 14 kb on the short arm of chromosome 12, which predicts a 314-amino acid polypeptide containing an arginine-glycine-aspartic acid (RGD) motif (DO*B allele product only) commonly involved in cell-to-cell interactions involving integrin binding and the GPI anchor motif.3 A search of the GenBank database indicated that the full sequence is identical to adenosine diphosphate (ADP)-ribosyltransferase 4 (ART-4; GenBank accession numbers NM-021071 and AF290204),4 but enzymatic activity has not been demonstrated yet in any cell type including erythroid cells. The polymorphisms associated with DO*A/DO*B alleles, the 2 C378T and T624C silent transitions, and the A793G transition resulting in an Asn265Asp amino-acid substitution located in the RGD motif have been reported.5,6 Recently, from unrelated donors with the rare Hy-negative phenotype [Do(ab+w), Gy(a+w), Hy, Jo(a)], it has been shown that both DO*HY1/DO*HY2 alleles derive from a DO*B background with a G323T transition, resulting in a Gly108Val substitution and T378C silent transition, but that they differ by a C898G transition resulting in a Leu300Val substitution.7 More recently, it was shown that the Joseph-negative phenotype [Do(a+wb), Gy(a+), Hy(+w), Jo(a)], results from a JO allele with a DO*A background but differs by a C350T transition, resulting in a Thr117Ile substitution and a C378T silent mutation.8 The rare Donull phenotype, in which the RBCs lack all 5 antigens, may arise from a single nucleotide mutation in the acceptor splice site of intron 1, causing the skipping of exon 29 or from the absence of GPI-anchored proteins on RBCs from patients with paroxysmal nocturnal hemoglobinuria.10 In all cases, negative and null phenotypes can develop anti-Dombrock antibodies that may cause severe hemolytic transfusion reactions.11,12 Here, we report a new molecular mechanism from a patient with a Donull phenotype, identified as a short deletion in exon 2 altering the reading frame of a DO*A allele.     Study design Top Abstract Introduction Study design Results and discussion References Reagents Expand High Fidelity and primers were from Boehringer-Mannheim/Roche Diagnostics (Mannheim, Germany) and Genset (Paris, France), respectively. Nucleotide sequences were determined with BigDye Terminator Cycle Sequencing ready Reaction Kit and analyzed on an ABI-PRISM 310 Genetic Analyser (PE Applied Biosystem, Foster City, CA). Blood samples from the family of the Donull donor were collected after informed consent. Genomic DNA analysis Polymerase chain reaction (PCR) from 500 ng leukocyte genomic DNA extracted with the Wizard Genomic DNA Purification kit (Promega, Madison, WI) was performed to amplify each exon and intron-exon junction of the DO gene. For the first exon, PCR between primers SP-A (position 250 to 229, upstream of the initiation codon) and AS-B (position 67 to 50, downstream of exon 1) was performed under stringent conditions (94°C for 2 minutes [1 cycle]; 94°C for 30 seconds, 58°C for 30 seconds, 68°C for 1 minute [30 cycles]; 68°C for 7 minutes [1 cycle]) using Expand High Fidelity in a total volume of 50 µL. The same conditions were used to amplify the second and third exons with primers (1) SP-C (position 80 to 58, upstream of exon 2) and SP-D (position 55 to 35, downstream of exon 2) and (2) SP-E (position 41 to 20, upstream of exon 3) and SP-F (position 1054 to 1034). Then the final PCR products were subcloned into pcR2-1 vector from Invitrogen (Groningen, The Netherlands) and sequenced on both strands. All position primers refer to the first nucleotide of the initiation codon of the DOK1 clone3 and the BAC-clone (GenBank accession numbers AF290204 and AC007655, respectively). PCR genotyping To identify the Do(8nt) variant, allele-specific PCR reactions were performed between the following sets of primers within exon 2: PCR-1 between the primers SP-1 (position 165-182) and AS-1 (position 363-343) specific to the wild-type DO alleles and PCR-2 between the primers SP-1 and AS-2 (position 363-337, deleted of 350 to 343 nucleotides) specific of the DO*A(8nt) allele, under the same conditions as above except that annealing was performed at 56°C. Expected PCR products were electrophoresed on a 3% (wt/vol) Metaphor gel (BMA, Rockland, ME) and were stained with ethidium bromide before analysis.     Results and discussion Top Abstract Introduction Study design Results and discussion References The healthy female donor with a Donull phenotype was identified at the Reunion Island Blood Service (St Denis, France) and was typed as Do(ab), Gy(a) with routine reagents (not shown) at the CNRGS (Paris, France) and is most likely also Hy() and Jo(a) because her serum contains an anti-DO3 antibody reacting with Hy() and Jo(a) cells that are not Donull. Two sisters of the propositus were phenotyped as Do(ab+). PCR-genotyping6 indicated that the propositus and her sisters were homozygous for the DO*A and DO*B alleles, respectively. To determine the molecular defect occurring in the propositus, genomic DNA was analyzed because PCR amplification of the full-length DO cDNA failed repeatedly. PCR products for each of the 3 Dombrock exons, including intron-exon junctions, were amplified, subcloned, and sequenced as described in "Study design." Sequence analysis revealed that the only deviation from the wild-type DO*A allele sequence was an 8-bp deletion (ATGACTAC) within exon 2 behind nucleotide 342 (Figure 1B). This short deletion causes a frameshift that generates a premature stop codon 17 amino acids further (Figure 1A). Thus, the resultant truncated protein without the predicted functional motifs of the ADP-ribosyltransferase enzyme and the GPI-anchor motif explains the loss of Dombrock blood group antigens on erythrocytes from this patient. To confirm this finding and to screen the DO(8nt) deletion in random blood donors, an allele-specific PCR assay was developed (described in "Study design"). As expected, a 199-bp PCR product was detected only in the Do(a+b+) control as in the 2 sisters' Do(ab+) samples, whereas a 191-bp PCR product was obtained only in the propositus demonstrating the homozygosity of the short deletion in this Donull donor. Neither of the sisters carried the DO(8nt) deletion because both inherited the normal DO*B allele from their heterozygous parents. In conclusion, a novel silent DO*A allele is described, but its frequency and geographic distribution are unknown. This second alteration of the DO locus suggests molecular heterogeneity of the Donull phenotype. View larger version (38K): [in this window] [in a new window]   Figure 1. Genomic DNA analysis. (A) Schematic representation of DO alleles from a control Dombrock-positive donor [DO*A or DO*B allele] and from the Donull donor [DO*A(8nt)]. Exon 2 limits are indicated by filled rectangles. The 8-nucleotide deletion within exon 2 of the variant is schematically illustrated. Reading frame and premature stop codon are shown in the DO*A(8nt) allele. Primers used for allele-specific PCR and the expected PCR product sizes are indicated. (B) Partial sequence diagram from a common donor and from the Donull variant, DO(8nt). The nucleotide deletion is underlined with a dotted line. (C) Gel analysis of PCR products. For PCR-1, a 191-bp PCR product was amplified only from the Donull variant, whereas for PCR-2, 199-bp products were generated only from a control Do(a+b+) sample and the 2 sisters (S1, S2) genotyped as homozygous for the DO*B allele. Sizes of fragments (bp) are given on the right.     Footnotes Submitted December 20, 2001; accepted March 9, 2002. Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0298. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Jean-Pierre Cartron, Unité INSERM U76 INTS, 6, rue Alexandre Cabanel, 75015 Paris, France; e-mail: cartron{at}idf.inserm.fr.     References Top Abstract Introduction Study design Results and discussion References 1. Banks JA, Hemming N, Poole J. Evidence that the Gya, Hy and Joa antigens belong to the Dombrock blood group system. Vox Sang. 1995;68:177-182[Medline] [Order article via Infotrieve]. 2. Rao N, Udani M, Nelson J, Reid ME, Telen MJ. Investigations using a novel monoclonal antibody to the glycosylphosphatidylinositol-anchored protein that carries Gregory, Holley, and Dombrock blood group antigens. Transfusion. 1995;35:459-464[CrossRef][Medline] [Order article via Infotrieve]. 3. Gubin AN, Njoroge JM, Wojda U, et al. Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family. Blood. 2000;96:2621-2627[Abstract/Free Full Text]. 4. Koch-Nolte F, Haag F, Braren R, et al. Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins. Genomics. 1997;39:370-376[CrossRef][Medline] [Order article via Infotrieve]. 5. Wu GG, Jin SZ, Deng ZH, Zhao TM. Polymerase chain reaction with sequence-specific primers-based genotyping of the human Dombrock blood group DO1 and DO2 alleles and the DO gene frequencies in Chinese blood donors. Vox Sang. 2001;81:49-51[Medline] [Order article via Infotrieve]. 6. Rios M, Hue-Roye K, Lee AH, Chiofolo JT, Miller JL, Reid ME. DNA analysis for the Dombrock polymorphism. Transfusion. 2001;41:1143-1146[Medline] [Order article via Infotrieve]. 7. Rios M, Hue-Roye K, Yen R, Reid ME. Molecular basis of the Gy(a+w) Hy() phenotype. Transfus Clin Biol. 2001;8(suppl 1):14S. 8. Rios M, Hue-Roye K, Oyen R, Miller JL, Reid ME. Molecular basis of the Joseph-negative phenotype. Transfusion. 2001;41(suppl):14S. 9. Rios M, Hue-Roye K, Storry JR, Lee T, Miller JL, Reid ME. Molecular basis of the Dombrock null phenotype. Transfusion. 2001;41:1405-1407[Medline] [Order article via Infotrieve]. 10. Telen MJ, Rosse WF, Parker CJ, Moulds MK, Moulds JJ. Evidence that several high-frequency human blood group antigens reside on phosphatidylinositol-linked erythrocyte membrane proteins. Blood. 1990;75:1404-1407[Abstract/Free Full Text]. 11. Strupp A, Cash K, Uehlinger J. Difficulties in identifying antibodies in the Dombrock blood group system in multiply alloimmunized patients. Transfusion. 1998;38:1022-1025[CrossRef][Medline] [Order article via Infotrieve]. 12. Halverson G, Shanahan E, Santiago I, et al. The first reported case of anti-Dob causing an acute hemolytic transfusion reaction. Vox Sang. 1994;66:206-209[Medline] [Order article via Infotrieve]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This Article Abstract Full Text (PDF) All Versions of this Article: 2001-12-0298v1 100/3/1063    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Lucien, N. Articles by Bailly, P. Search for Related Content PubMed PubMed Citation Articles by Lucien, N. Articles by Bailly, P. Related Collections Red Cells Transfusion Medicine Brief Reports Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020