title: Establishment of a medium-throughput approach for the genotyping of RHD variants and report of nine novel rare alleles.
abstract: BACKGROUND
  The routinely used serologic methods are robust in accurately typing standard D- or D+ blood. However, they result in discrepancy in weak or partial D blood, which requires genetic analysis. We have previously used denaturing high-performance liquid chromatography (DHPLC) to screen the entire RHD-coding sequence. However, DHPLC is technically challenging, labor-intensive, and time-consuming. To overcome these inconveniences, we sought to develop a new two-step approach.
  
  
  METHODS
  A total of 430 blood samples with D phenotype ambiguity were recruited for this study. The three most frequent weak D alleles (i.e., weak D, Type 1; weak D, Type 2; and weak D, Type 3), which altogether account for 60% to 90% of the atypical RHD alleles in the Caucasian population, were first identified by Tm-shift genotyping. The remaining unidentified samples were then subjected to a single-tube multiplex polymerase chain reaction (PCR) amplification of all 10 RHD exons followed by direct sequencing.
  
  
  RESULTS
  Optimal conditions for efficient and reliable identification of the three most common weak D variants by Tm-shift genotyping were established. All 10 RHD exons were successfully amplified in a single-multiplex PCR procedure. Employment of the two-step analysis identified RHD variants in 91.6% of the 430 studied samples. Two of the nine previously undescribed variants, c.335G>T and c.939G>A, were found to cause aberrant mRNA splicing by means of a splicing minigene assay.
  
  
  CONCLUSIONS
  The new two-step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium-throughput approach for RHD genotyping.
authors.affiliation: Etablissement Français du Sang (EFS)-Bretagne, Brest, France. Yann.Fichou@efs.sante.fr
authors.forename: Yann
authors.initials: Y
authors.lastname: Fichou
authors.forename: Cédric
authors.initials: C
authors.lastname: Le Maréchal
authors.forename: Déborah
authors.initials: D
authors.lastname: Jamet
authors.forename: Laurence
authors.initials: L
authors.lastname: Bryckaert
authors.forename: Chandran
authors.initials: C
authors.lastname: Ka
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Audrézet
authors.forename: Gérald
authors.initials: G
authors.lastname: Le Gac
authors.forename: Isabelle
authors.initials: I
authors.lastname: Dupont
authors.forename: Jian-Min
authors.initials: JM
authors.lastname: Chen
authors.forename: Claude
authors.initials: C
authors.lastname: Férec
mesh_terms: D000483:Alleles; D001483:Base Sequence; D015331:Cohort Studies; D017930:Genes, Reporter; D005819:Genetic Markers; D060005:Genotyping Techniques; D006801:Humans; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011446:Prospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D005819:Genetic Markers; D012204:Rh-Hr Blood-Group System