Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells.
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.
Kamesaki
T
T
Department of Legal Medicine and Human Genetics, Jichi Medical School, Tochigi 329-0498, Japan. kmskt@jichi.ac.jp
Iwamoto
S
S
Kumada
M
M
Omi
T
T
Okuda
H
H
Tanaka
M
M
Takahashi
J
J
Obara
K
K
Seno
T
T
Tani
Y
Y
Kajii
E
E
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
D000911:Antibodies, Monoclonal; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; D049452:Green Fluorescent Proteins