Kamesaki T, Iwamoto S, Kumada M, Omi T, Okuda H, Tanaka M, Takahashi J, Obara K, Seno T, Tani Y, Kajii E
Department of Legal Medicine and Human Genetics, Jichi Medical School, Tochigi 329-0498, Japan.
Vox Sang. 2001 Nov;81(4):254-8. doi: 10.1046/j.1423-0410.2001.00118.x.
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.
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.
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.
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.
在161例来自高加索人的弱D样本中检测到的突变已被分为16种类型。由于使用单克隆抗-D抗体（mAb）的流式细胞术显示弱D红细胞呈现类型特异性抗原密度，因此这些跨膜区域的突变被赋予了弱D表型。本研究试图证实或反驳这一分类。
我们使用聚合酶链反应（PCR）从4例日本弱D样本中扩增DNA，并对扩增后的DNA进行直接测序。利用定点诱变技术，我们构建了3种在K562细胞中表达突变型RHD的载体——G212C、V270G（弱D1型）和G358A（2型）。使用mAb通过流式细胞术检测RhD抗原的表达。
在1例日本弱D样本中检测到一个导致氨基酸残基212处发生转换（甘氨酸突变为半胱氨酸）的新突变。用突变型RhD cDNA转导的K562细胞与mAb以类型特异性方式发生弱反应。
跨膜区域的突变——G212C（新的弱D型）、V270G（弱D1型）和G358A（2型）——对mAb识别的D表位有明显影响。本研究结果提供了直接证据，证明这些突变可解释弱D表型。
