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Related Collections Brief Reports Transfusion Medicine Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 February 2002, Vol. 99, No. 3, pp. 1079-1081 BRIEF REPORT Partial deletion in the JK locus causing a Jknull phenotype Nicole Lucien, Jacques Chiaroni, Jean-Pierre Cartron, and Pascal Bailly From INSERM-U76, Institut National de la Transfusion Sanguine, Paris, France, and EFS Alpes-Provence, Marseille, France.     Abstract Top Abstract Introduction Study design Results and discussion References A new alteration of the blood group JK*A allele was identified in a Jknull patient from Tunisia with an allo-anti-Jk3 in her serum. Southern blot and exon mapping analyses revealed an internal deletion within the Kidd (JK) locus encompassing exons 4 and 5. Sequence analysis of the Jk transcript showed that exons 4 and 5 were missing but were replaced by a 136-base-pair (bp) intron 3 sequence located 315 bp and 179 bp upstream from exon 4. This sequence is flanked by typical donor-acceptor cryptic splice sites used in the mutant but not in the normal JK gene. Because the translation initiation codon is located in exon 4, the Jk protein is not produced. (Blood. 2002;99:1079-1081) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The urea transporter of human erythrocytes (hUT-B1) is encoded by the Kidd (JK) locus, which spans 30 kb of DNA on chromosome 18q12-q21 and is organized into 11 exons.1,2 The JK*A/JK*B polymorphism arises from an Asp280Asn substitution on the Jk/hUT-B1 polypeptide.3 Red blood cells (RBCs) lacking Kidd antigens define a rare phenotype called Jk(ab) or Jknull, the frequency of which is increased in some populations.4 This phenotype may result from homozygous inheritance of a silent allele at the JK locus or, less often, from a dominant inhibitor gene not linked to the JK locus.4 Persons with the Jknull phenotype should be detected because anti-Jk3 antibody can develop after immunization by transfusion or pregnancy, and this antibody may cause immediate and delayed hemolytic transfusion reactions. Jknull RBCs have reduced urea permeability,5 but the Jk deficiency is not associated with any obvious clinical syndrome except for a urine concentration defect6 that probably results from the absence of Jk/hUT-B1 protein expressed on endothelial cells of the vasa recta of kidney.7,8 The silent-type Jknull may arise by at least 3 distinct mechanisms: (1) splice-site mutations in JK*B alleles, causing the skipping of either exon 61,9-11 or exon 71; (2) missense mutation in the JK*B allele resulting in a Ser291P substitution9,10; and (3) nonsense mutation in a JK*A allele resulting in a Tyr194Stop substitution.12 We now report a fourth mechanism, a deletion removing exons 4 and 5 of a JK*A allele.     Study design Top Abstract Introduction Study design Results and discussion References Reagents Expand High Fidelity, Expand Long Template PCR, and Titan One tube RT-PCR systems were from Boehringer-Mannheim/Roche Diagnostics (Mannheim, Germany). Nucleotide sequences were determined with ThermoSequenase sequencing kit from Amersham Pharmacia Biotech (Bucks, United Kingdom) using 5'(Cy5)-primers (Genset, Paris, France). Affinity-purified rabbit antibodies directed against the N-terminal and the C-terminal regions of the Kidd/hUT-B1 protein were as described.1,7,9 Amplification by reverse transcription coupled with polymerase chain reaction For primer designation, position +1 refers to the first nucleotide of the initiation codon of the JK gene (GenBank accession number Y19039). Total blood RNA extracted by the acid-phenol-guanidinium method13 was used for the first PCR in Titan One tube reverse transcription-polymerase chain reaction (RT-PCR) (50°C for 30 minutes [1 cycle], 94°C for 2 minutes [1 cycle], 94°C for 30 seconds, 64°C for 30 seconds, 68°C for 2 minutes [30 cycles], 68°C for 7 minutes [1 cycle]) between primers SP-1 (positions 41 to 22, exon 3) and AS-2 (position 1260-1237) according to the manufacturer's instructions. The second PCR was performed with one twentieth-fifth of the first reaction in the same conditions with primers SP-1 and AS-3 (position 1234-1211, exon 11) and Expand High Fidelity system. Genomic DNA analysis PCR reactions (v = 50 µL) contained 500 ng leukocyte DNA extracted with the Wizard Genomic DNA Purification kit from Promega (Madison, WI). A first PCR between primers SP-4 (5'-ggtagcattacagacactgatggc-3', position 207-184 upstream exon 4) and AS-5 (position 470-446) encompassing the internal deletion was performed under stringent conditions (93°C for 2 minutes [1 cycle], 93°C for 10 seconds, 66°C for 30 seconds, 68°C for 5 minutes [10 cycles], 93°C for 10 seconds, 66°C for 30 seconds, 68°C for 5 minutes plus 20 sec/cycle [25 cycles], 68°C for 7 minutes [1 cycle]) using Expand Long Template PCR. The second PCR was performed with one fiftieth of the first reaction using primers SP-4 and AS-6 (position 445-421, exon 6) under the same conditions except for the annealing temperature (62°C). PCR products were subcloned and sequenced.     Results and discussion Top Abstract Introduction Study design Results and discussion References The patient with a Jknull phenotype identified at the EFS Alpes-Provence (Marseille, France) was typed Jk(ab) with routine reagents (not shown). Western blot analysis with affinity-purified antibodies directed against the N-terminal and C-terminal of the Jk/hUT-B1 polypeptide showed that RBCs from the propositus lacked the Jk membrane protein of 45 to 69 kd (Figure 1A), and genotyping3 indicated homozygosity for a JK*A allele (not shown). To characterize the molecular defect occurring in the silent JK*A allele, genomic DNA from the propositus was digested with SacI and was analyzed by Southern blot. Hybridization with a cDNA probe encoding the Jk/hUT-B1 protein revealed the lack of a 7-kb fragment that normally contains exons 4 and 5 (Figure 1B). To confirm this finding, total RNA from the Jknull blood was used as a template to amplify the Jk-cDNA by heminested PCR using primer pairs located in exons 3 and 11 (see "Study design"). A 1049-kb fragment (vs a 1275-kb fragment for Jk(a+b+) controls) was obtained, and sequence analysis confirmed that exons 4 and 5 were missing (Jk[4,5] mutant) but were replaced by a 136-bp sequence called E* on Figure 1C. Sequence comparison indicated that the 136-bp sequence was identical to an intronic sequence located 315 bp and 179 bp upstream from exon 4. This sequence is flanked by typical donor-acceptor cryptic splice sites used in the mutant but not in the normal JK gene. In vitro transcription-translation assays showed that no protein could be produced from the Jk(4,5) cDNA, as expected from the loss of the translation initiation codon normally present in exon 4 (not shown). View larger version (46K): [in this window] [in a new window]   Figure 1. RBC membrane proteins, genomic DNA, and Jk transcript from the Jknull and control Jk(a+b+) donors. (A) Immunoblot analysis. RBC membrane proteins (40 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies against the N-terminal or C-terminal of the Jk/hUT-B1 protein.9 (B) Southern blot analysis: Genomic DNA (15 µg/lane) was digested with SacI (10 U/µg), resolved by agarose gel electrophoresis, transferred to a nylon membrane, and hybridized with a [32P]-labeled cDNA probe (exons 4 to 11).2 Size of DNA markers (kb) and exons in each fragment are given in the left and right margins, respectively. (C) Partial sequence analysis. Jknull transcript sequence from Alf-Express DNA sequencer (Amersham Pharmacia, Uppsala, Sweden). E*, intronic sequence found in the Jk(4,5) transcript from the propositus but not in the Jk(a+b+) control. Genomic sequences were deposited to the European Molecular Biology Laboratory (EMBL) database under the accession numbers AJ316564 and AJ316565. To better characterize the internal deletion and to locate the breakpoints, a genomic fragment was PCR-amplified using primer pairs located in E* and exon 6 (Figure 2). The size difference of the PCR-1 products amplified from the Jk(a+b+) sample (4.3 kb) and the Jknull sample (2.7 kb) suggested a deletion of approximately 1.6 kb (Figure 2). After sequence analysis, the 5' and 3' breakpoints were localized 131 base pair (bp) upstream from exon 4 and 575 bp downstream from exon 5, respectively. A PCR spanning the breakpoint may be used to discriminate this novel silent JK*A allele from other Jk-deficient alleles, including that recently found in an English family.14 View larger version (46K): [in this window] [in a new window]   Figure 2. Internal deletion mapping. Schematic representation of JK alleles from the Jknull donor [JK*A(4,5)] and from a control Jk-positive donor [JK*A or JK*B allele]. Exons are indicated by rectangles. Filled and open symbols correspond to coding and noncoding sequences, respectively. Primers used for PCR-1 are indicated. S, SacI. (right) Gel analysis of PCR-1 products. The junction sequence found in the Jknull donor and the 5' and 3' breakpoints of the deletion are indicated (bottom). Sequence alignment of the wild type (wt) and deleted DNA from the Jknull donor around the deletion breakpoint showing the small direct repeats (shadowed). The mechanism responsible for the deletion is unknown, and there are no typical sequence motifs around the deletion breakpoint. However, the deletion breakpoint is flanked by small direct repeats (Figure 2), suggesting, as found in mitochondrial DNA,15 that recombination or slipped mispairing may cause the deletion.     Footnotes Submitted June 19, 2001; accepted September 24, 2001. 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, INSERM-U76, Institut National de la Transfusion Sanguine, 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. Olives B, Mattei MG, Huet M, et al. Kidd blood group and urea transport function of human erythrocytes are carried by the same protein. J Biol Chem. 1995;270:15607-15610[Abstract/Free Full Text]. 2. Lucien N, Sidoux-Walter F, Olives B, et al. Characterization of the gene encoding the human Kidd blood group/urea transporter protein: evidence for splice site mutations in Jknull individuals. J Biol Chem. 1998;273:12973-12980[Abstract/Free Full Text]. 3. Olives B, Merriman M, Bailly P, et al. The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility. Hum Mol Genet. 1997;6:1017-1020[Abstract/Free Full Text]. 4. Fröhlich O, Macey RI, Edwards-Moulds J, Gargus JJ, Gunn RB. Urea transport deficiency in Jk(ab) erythrocytes. Am J Physiol. 1991;260:C778-C783[Abstract/Free Full Text]. 5. Race RR, Sange R. Blood Groups in Man. 6th ed. Oxford, England: Blackwell Scientific; 1975:364-378. 6. Sands JM, Gargus JJ, Frohlich O, Gunn RB, Kokko JP. Urinary concentrating ability in patients with Jk(ab) blood type who lack carrier-mediated urea transport. J Am Soc Nephrol. 1992;2:1689-1696[Abstract]. 7. Xu Y, Olives B, Bailly P, et al. Endothelial cells of the kidney vasa recta express the urea transporter HUT11. Kidney Int. 1997;51:138-146[Medline] [Order article via Infotrieve]. 8. Promeneur D, Rousselet G, Bankir L, et al. Evidence for distinct vascular and tubular urea transporters in the rat kidney. J Am Soc Nephrol. 1996;7:852-860[Abstract]. 9. Sidoux-Walter F, Lucien N, Nissinen R, et al. Molecular heterogeneity of the Jknull phenotype: expression analysis of the Jk(S291P) mutation found in Finns. Blood. 2000;96:1566-1573[Abstract/Free Full Text]. 10. Irshaid NM, Henry SM, Olsson ML. Genomic characterization of the Kidd blood group gene: different molecular basis of the Jk(ab) phenotype in Polynesians and Finns. Transfusion. 2000;40:69-74[CrossRef][Medline] [Order article via Infotrieve]. 11. Ekman GC, Hessner MJ. Screening of six racial groups for the intron 5 GA 3' splice acceptor mutation responsible for the Polynesian Kidd(ab) phenotype: the null mutation is not always associated with the JKB allele. Transfusion. 2000;40:888-889[CrossRef][Medline] [Order article via Infotrieve]. 12. Irshaid NM, Hustinx H, Olsson ML. A novel molecular basis of the Jk(ab) phenotype in a Swiss family [abstract]. Vox Sanguinis. 2000;78(suppl 1):O019[CrossRef]. 13. Lozano ME, Grau O, Romanowski V. Isolation of RNA from whole blood for reliable use in RT-PCR amplification [abstract]. Trends Genet. 1993;9:296[CrossRef][Medline] [Order article via Infotrieve]. 14. Irshaid NM, Eicher N, Poole J, Hustinx H, Olsson ML. Molecular basis of the Jknull phenotype in non-Finnish Europeans [abstract]. Transfusion. 2000;40(suppl 10):S118. 15. Mita S, Rizzuto R, Moraes CT, et al. Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucleic Acids Res. 1990;18:561-567[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: N. Lucien, F. Sidoux-Walter, N. Roudier, P. Ripoche, M. Huet, M.-M. Trinh-Trang-Tan, J.-P. Cartron, and P. Bailly Antigenic and Functional Properties of the Human Red Blood Cell Urea Transporter hUT-B1 J. Biol. Chem., September 6, 2002; 277(37): 34101 - 34108. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) 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 HighWire 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 Brief Reports Transfusion Medicine Social Bookmarking                   What's this?

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