title: Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles.
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.
  
  
  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).
  
  
  CONCLUSIONS
  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.
authors: Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, France.
authors: Monique
authors: M
authors: Silvy
authors: Sophie
authors: S
authors: Simon
authors: Julia
authors: J
authors: Gouvitsos
authors: Julie
authors: J
authors: Di Cristofaro
authors: Virginie
authors: V
authors: Ferrera
authors: Jacques
authors: J
authors: Chiaroni
authors: Pascal
authors: P
authors: Bailly
mesh_terms: D000372:Agglutination Tests; D000483:Alleles; D019943:Amino Acid Substitution; D001782:Blood Donors; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D016384:Consensus Sequence / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005602:France; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011446:Prospective Studies; D022821:RNA Splice Sites / Q000235:genetics; D012204:Rh-Hr Blood-Group System / Q000737:chemistry / Q000235:genetics*
chemical_list: D022821:RNA Splice Sites; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen