title: Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.
abstract: OBJECTIVE
  Mutations detected in 161 weak D samples from Caucasians have been classified into 16 types. Because flow cytometry using monoclonal anti-D antibodies (mAbs) has shown that weak D red cells display type-specific antigen density, these mutations in transmembranous regions have been assigned weak D phenotypes. The present study attempts to confirm or refute this assignment.
  
  
  METHODS
  We amplified DNA from four Japanese weak D samples using the polymerase chain reaction (PCR), and directly sequenced the amplified DNA. Using site-directed mutagenesis, we constructed three vectors expressing mutant RHDs-- G212C, V270G (weak D type 1) and G358A (type 2)--in K562 cells. The expression of RhD antigens was examined by flow cytometry using mAbs.
  
  
  RESULTS
  A new mutation resulting in a conversion at amino acid residue 212 (Gly to Cys) was detected in a Japanese weak D sample. K562 cells transduced with mutant RhD cDNA reacted weakly in a type-specific manner with mAbs.
  
  
  CONCLUSIONS
  The mutations--G212C (new weak D type), V270G (weak D type 1) and G358A (type 2)-- in transmembranous regions had obvious effects on the D epitopes recognized by mAbs. The results of this study provide direct evidence that these mutations can account for weak D phenotypes.
authors.affiliation: Department of Legal Medicine and Human Genetics, Jichi Medical School, Tochigi 329-0498, Japan. kmskt@jichi.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Kamesaki
authors.forename: S
authors.initials: S
authors.lastname: Iwamoto
authors.forename: M
authors.initials: M
authors.lastname: Kumada
authors.forename: T
authors.initials: T
authors.lastname: Omi
authors.forename: H
authors.initials: H
authors.lastname: Okuda
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.forename: J
authors.initials: J
authors.lastname: Takahashi
authors.forename: K
authors.initials: K
authors.lastname: Obara
authors.forename: T
authors.initials: T
authors.lastname: Seno
authors.forename: Y
authors.initials: Y
authors.lastname: Tani
authors.forename: E
authors.initials: E
authors.lastname: Kajii
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000235:genetics* / Q000276:immunology*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; D049452:Green Fluorescent Proteins