Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles

Authors

  • Monique Silvy,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Sophie Simon,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Julia Gouvitsos,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Julie Di Cristofaro,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Virginie Ferrera,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Jacques Chiaroni,

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • Pascal Bailly

    1. From the Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
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  • This study was supported by Établissement Français du Sang, Paris, France (all co-authors).

Pascal Bailly, Laboratoire d'Hématologie Moléculaire, EFS Alpes Méditerranée, UMR 6578, Université de la Méditerranée, 207 Boulevard Sainte Marguerite, 13009, Marseille, France; e-mail: pascal.bailly@efs.sante.fr.

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.

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