Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles
Monique Silvy
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorSophie Simon
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJulia Gouvitsos
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJulie Di Cristofaro
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorVirginie Ferrera
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJacques Chiaroni
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorPascal Bailly
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorMonique Silvy
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorSophie Simon
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJulia Gouvitsos
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJulie Di Cristofaro
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorVirginie Ferrera
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorJacques Chiaroni
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorPascal Bailly
From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
Search for more papers by this authorThis study was supported by Établissement Français du Sang, Paris, France (all co-authors).
Abstract
BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians.
STUDY DESIGN AND METHODS: Preliminary profiling was carried out on single-nucleotide polymorphisms (SNPs) associated with 13 prevalent RHD alleles, that is, weak D Types 1, 2, 3, 4.0, 4.0.1, 4.1, 4.2, 5, 11, 15, and 17; RHD(IVS3+1g>a); and RHD(K409K). Multiplex PCR was used to amplify six RHD regions encompassing 14 SNPs. Identification was obtained by incorporation of the complementary dye single base at the 3′-end of each probe-primer. A prospective analysis was then carried out on 152 blood samples from patients (n = 53) and donors (n = 88) with equivocal RhD serology and pregnant women (n = 11).
RESULTS: After validation, our SNaPshot assay allowed direct genotyping of 82.9% of samples overall and 100% of samples harboring weak D Types 1, 2, 3, and 4.1 alleles. In the remaining 17.1% of samples overall, sequence investigation allowed accurate genotyping. In addition, four novel RHD alleles were identified, that is, RHD(S256P), RHD(L390L), RHD(F410V), and RHD(IVS4-2a>g).
CONCLUSION: The SNaPshot assay described herein is a helpful supplementary tool for resolving doubtful RhD serology. By allowing accurate identification of weak D and DEL alleles this assay should allow better management of the donors and the patients genotyped weak D Types 1, 2, 3, and 4.1 who can receive D+ blood units.
REFERENCES
- 1 Race RR, Sanger R. Blood groups in man. 6th ed. Oxford: Blackwell; 1975.
- 2 Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol 2007; 44: 42-50.
- 3 Ye L, Yue D, Wo D Ding X, Guo S, Li Q, Guo Z, Zhu Z. Molecular bases of unexpressed RHD alleles in Chinese D– persons. Transfusion 2009; 49: 1655-60.
- 4 Singleton BK, Green CA, Avent ND Martin PG, Smart E, Daka A, Narter-Olaga EG, Hawthorne LM, Daniels G. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood 2000; 95: 12-18.
- 5 Faas BH, Beckers EA, Wildoer P Ligthart PC, Overbeeke MA, Zondervan HA, von dem Borne AE, van der Schoot CE. Molecular background of VS and weak C expression in blacks. Transfusion 1997; 37: 38-44.
- 6 Daniels GL, Faas BH, Green CA Smart E, Maaskant-van Wijk PA, Avent ND, Zondervan HA, von dem Borne AE, van der Schoot CE. The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis. Transfusion 1998; 38: 951-8.
- 7 Touinssi M, Chapel-Fernandes S, Granier T Bokilo A, Bailly P, Chiaroni J. Molecular analysis of inactive and active RHD alleles in native Congolese cohorts. Transfusion 2009; 49: 1353-60.
- 8 Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000; 95: 375-87.
- 9 Flegel WA. The genetics of the Rhesus blood group system. Blood Transfus 2007; 5: 50-7.
- 10 Colin Y, Cherif-Zahar B, Le Van Kim C Raynal V, Van Huffel V, Cartron JP. Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 1991; 78: 2747-52.
- 11 Mourant AE, Kopec AC, Domaniewska-Sobczak K. The distribution of the human blood groups and other polymorphisms. London: Oxford University Press; 1976.
- 12 Wagner FF, Kasulke D, Kerowgan M, Flegel WA. Frequencies of the blood groups ABO, Rhesus, D category VI, Kell, and of clinically relevant high-frequency antigens in south-western Germany. Infusionsther Transfusionsmed 1995; 22: 285-90.
- 13 Muller TH, Wagner FF, Trockenbacher A Eicher NI, Flegel WA, Schönitzer D, Schunter F, Gassner C. PCR screening for common weak D types shows different distributions in three Central European populations. Transfusion 2001; 41: 45-52.
- 14 Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY Roussel M, Patereau C, Noizat-Pirenne F. RhD variants in Caucasians: consequences for checking clinically relevant alleles. Transfusion 2004; 44: 1282-6.
- 15 Wagner FF, Gassner C, Muller TH Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood 1999; 93: 385-93.
- 16 Okuda H, Kawano M, Iwamoto S Tanaka M, Seno T, Okubo Y, Kajii E. The RHD gene is highly detectable in RhD-negative Japanese donors. J Clin Invest 1997; 100: 373-9.
- 17 Shao CP, Maas JH, Su YQ Köhler M, Legler TJ. Molecular background of Rh D-positive, D-negative, D(el) and weak D phenotypes in Chinese. Vox Sang 2002; 83: 156-61.
- 18 Li Q, Hou L, Guo ZH Ye LY, Yue DQ, Zhu ZY. Molecular basis of the RHD gene in blood donors with DEL phenotypes in Shanghai. Vox Sang 2009; 97: 139-46.
- 19 Denomme GA, Wagner FF, Fernandes BJ Li W, Flegel WA. Partial D, weak D types, and novel RHD alleles among 33864 multiethnic patients: implications for anti-D alloimmunization and prevention. Transfusion 2005; 45: 1554-60.
- 20 Kormoczi GF, Gassner C, Shao CP Uchikawa M, Legler TJ. A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization. Transfusion 2005; 45: 1561-7.
- 21 Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Secondary anti-D immunization by Del red blood cells. Transfusion 2005; 45: 1581-4.
- 22 Kumpel B. Are weak D RBCs really immunogenic? [Letter]. Transfusion 2006; 46: 1061-2. discussion 2-6.
- 23 Kim KH, Kim KE, Woo KS Han JY, Kim JM, Park KU. Primary anti-D immunization by DEL red blood cells. Korean J Lab Med 2009; 29: 361-5.
- 24 Wagner FF, Frohmajer A, Ladewig B Eicher NI, Lonicer CB, Müller TH, Siegel MH, Flegel WA. Weak D alleles express distinct phenotypes. Blood 2000; 95: 2699-708.
- 25 Flegel WA. How I manage donors and patients with a weak D phenotype. Curr Opin Hematol 2006; 13: 476-83.
- 26 Noizat-Pirenne F, Verdier M, Lejealle A Mercadier A, Bonin P, Peltier-Pujol F, Fialaire-Legendre A, Tournamille C, Bierling P, Ansart-Pirenne H. Weak D phenotypes and transfusion safety: where do we stand in daily practice? Transfusion 2007; 47: 1616-20.
- 27 Denomme GA, Flegel WA. Applying molecular immunohematology discoveries to standards of practice in blood banks: now is the time. Transfusion 2008; 48: 2461-75.
- 28 Flegel WA, von Zabern I, Wagner FF. Six years' experience performing RHD genotyping to confirm D– red blood cell units in Germany for preventing anti-D immunizations. Transfusion 2009; 49: 465-71.
- 29 Le Marechal C, Guerry C, Benech C Burlot L, Cavelier B, Porra V, Delamaire M, Férec C, Chen JM. Identification of 12 novel RHD alleles in western France by denaturing high-performance liquid chromatography analysis. Transfusion 2007; 47: 858-63.
- 30 Chen Q, Flegel WA. Random survey for RHD alleles among D+ European persons. Transfusion 2005; 45: 1183-91.
- 31 Polin H, Danzer M, Hofer K Gassner W, Gabriel C. Effective molecular RHD typing strategy for blood donations. Transfusion 2007; 47: 1350-5.
- 32 Mezokh N, Song Y, Moulds JM, Hashmi PG. Reliable identification of diverse rhd and rhce variants in african american donors by beadchip analysis. [abstract]. Transfusion 2009; 49: SP177.
- 33 Palacajornsuk P, Halter C, Isakova V Tarnawski M, Farmar J, Reid ME, Chaudhuri A. Detection of blood group genes using multiplex SNaPshot method. Transfusion 2009; 49: 740-9.
- 34 Di Cristofaro J, Silvy M, Chiaroni J, Bailly P. Single PCR multiplex snapshot reaction for detection of eleven blood group nucleotide polymorphisms: optimization, validation and one-year of routine clinical use. Journal Molecular Diagnostics 2010; 12: 453-60.
- 35 Gassner C, Doescher A, Drnovsek TD Rozman P, Eicher NI, Legler TJ, Lukin S, Garritsen H, Kleinrath T, Egger B, Ehling R, Körmöczi GF, Kilga-Nogler S, Schoenitzer D, Petershofen EK. Presence of RHD in serologically D−, C/E+ individuals: a European multicenter study. Transfusion 2005; 45: 527-38.
- 36 Polin H, Danzer M, Gaszner W Broda D, St-Louis M, Pröll J, Hofer K, Gabriel C. Identification of RHD alleles with the potential of anti-D immunization among seemingly D– blood donors in Upper Austria. Transfusion 2009; 49: 676-81.
- 37 Legler TJ, Maas JH, Kohler M Wagner T, Daniels GL, Perco P, Panzer S. RHD sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis. Transfus Med 2001; 11: 383-8.
- 38 Chiaroni J, Touinssi M, Frassati C Degioanni A, Gibert M, Reviron D, Mercier P, Boëtsch G. Genetic characterization of the population of Grande Comore Island (Njazidja) according to major blood groups. Hum Biol 2004; 76: 527-41.
- 39 Hemker MB, Ligthart PC, Berger L van Rhenen DJ, van der Schoot CE, Wijk PA. DAR, a new RhD variant involving exons 4, 5, and 7, often in linkage with ceAR, a new Rhce variant frequently found in African blacks. Blood 1999; 94: 4337-42.
- 40 Maaskant-van Wijk PA, Faas BH, de Ruijter JA Overbeeke MA, von dem Borne AE, van Rhenen DJ, van der Schoot CE. Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons. Transfusion 1998; 38: 1015-21.
- 41 Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol 2006; 13: 4-12.
- 42 Shao CP, Xiong W, Zhou YY. Multiple isoforms excluding normal RhD mRNA detected in Rh blood group Del phenotype with RHD 1227A allele. Transfus Apher Sci 2006; 34: 145-52.
- 43 Vege S, Copeland TR, Nickle PA, Westhoff CM. RHD exon consensus splice-site changes, 334A>G and 1228T>G, associated with weak D expression. [abstract]. Transfusion 2009; 49: SP172.
- 44 Nicolas V, Mouro-Chanteloup I, Lopez C Gane P, Gimm A, Mohandas N, Cartron JP, Le Van Kim C, Colin Y. Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells. Transfus Clin Biol 2006; 13: 23-8.
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