Weak D alleles in Japanese: a c.960G>A silent mutation in exon 7 of the RHD gene that affects D expression.
OBJECTIVE
The molecular basis of the weak D phenotype has been investigated for many years, and more than 80 different alleles producing weak D phenotypes have been identified. Most alleles producing weak D phenotypes have a single missense mutation in exons corresponding to a transmembrane domain of the RhD polypeptide. We report here RHD alleles with single nucleotide mutations in Japanese accounting for weak expression of D antigen.


METHODS
Seventy-five blood samples with a weak D phenotype were detected from 763 408 blood donors by standard serological methods. Forty-five of the 75 blood donors were available for RHD gene analysis by PCR and sequencing using genomic DNA and reticulocyte mRNA. Real-time PCR was performed to estimate the relative amounts of the RHD transcripts.


RESULTS
We detected 16 different RHD alleles in the 45 individuals with weak D by nucleotide sequencing; 12 were newly identified. Thirty-two of the 45 individuals had an RHD allele with a single missense mutation, while the other 13 individuals had RHD with a c.960G>A silent mutation in exon 7. Red blood cells of these 13 individuals showed direct agglutination with anti-D at a strength of 3+ or less. Semi-quantitative analysis of the RHD transcripts by real-time PCR revealed that the cDNA samples with the c.960G>A mutation showed a significant increment of exon 7 skipping compared with the common RHD.


CONCLUSIONS
Reduced expression of D antigen is caused not only by missense mutation of the RHD gene, but also by silent mutation that may affect splicing.
Japanese Red Cross Central Blood Institute, Tokyo, Japan.
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K
Ogasawara
Japanese Red Cross Central Blood Institute, Tokyo, Japan.
K
K
Sasaki
Japanese Red Cross Central Blood Institute, Tokyo, Japan.
K
K
Isa
Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Tokyo, Japan.
H
H
Tsuneyama
Japanese Red Cross Kanto-Koshinetsu Block Blood Center, Tokyo, Japan.
M
M
Uchikawa
Japanese Red Cross Central Blood Institute, Tokyo, Japan.
M
M
Satake
Japanese Red Cross Central Blood Institute, Tokyo, Japan.
K
K
Tadokoro
D000483:Alleles; D005091:Exons; D006801:Humans; D020125:Mutation, Missense; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000069456:Silent Mutation
D012333:RNA, Messenger; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen
RHD gene; Rh blood group; exonic splicing regulator; silent mutation; weak D