Molecular characterization of weak D phenotypes by site-directed mutagenesis and expression of mutant Rh-green fluorescence protein fusions in K562 cells

Background and Objectives Mutations detected in 161 weak D samples from Cau casians have been classified into 16 types. Because flow cytometry using mo noclonal anti-D antibodies (mAbs) has shown that weak D red cells display t ype-specific antigen density, these mutations in transmembranous regions ha ve been assigned weak D phenotypes. The present study attempts to confirm o r refute this assignment. Materials and Methods We amplified DNA from four Japanese weak D samples us ing the polymerase chain reaction (PCR), and directly sequenced the amplifi ed DNA. Using site-directed mutagenesis, we constructed three vectors expre ssing mutant RHDs - G212C, V270G (weak D type 1) and G358A (type 2) - in K5 62 cells. The expression of RhD antigens was examined by flow cytometry usi ng 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 transduce d 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 evi dence that these mutations can account for weak D phenotypes.