RHD Genotyping in RhD-negative Korean Donors by Polymerase Chain Reaction: Sequence Specific Primers(PCR-SSP) Method

Kim, Young Ree; Kim, Young Tae; Kim, Sang Bae; Seo, Dong Hee; Hwang, Yoo Sung; Cho, Nam Sun; Cho, Yun Jung

Abstract

BACKGROUND: In Caucacians, almost all RhD-negatives have deletion in RHD gene and the RHD genotyping by PCR-SSP is a valuable tool. The aim of this study is to investigate the frequency of RHD gene deletion in RhD-negative Korean donors and evaluate the clinical usefulness of various RHD genotyping methods in Korean.
METHODS
Two hundred fifty RhD-positive blood obtained from Blood Transfusion Research Institute and 119 RhD-negative blood samples were obtained from Korea Red Cross Dong Bu Center. Phenotyping of RhD, RhC/c, and RhE/e antigen was performed using polyclonal and monoclonal antibodies(Dade AG, Switzerland). PCR-SSP was performed by primer sets, specific for exon 3, exon 4, exon 5, exon 6, exon 7, exon 9, and exon 10 of RHD gene and for exon 2, intron 4, and intron 4-exon 5 common to RHD and RHCE genes.
RESULTS
The phenotypes of 250 RhD-positives consisted of 106(42.4%) CCee, 93(37.2%) CcEe, 26(10.4%) ccEE, 21(8.4%) Ccee, 3(1.2%) ccEe, and 1(0.4%) ccee. In RhD-negative donors, 62(52.1%) were ccee, 37(31.1%) Ccee, 10(8.4%) ccEE, 6(5.0%) CcEe, 2(1.7%) ccEE, and 2(1.7%) CCee. Twenty-two out of 101 RhD-negatives showed no deletion in all used methods. Deletion frequency of RhD negatives varied according to the methods: 76(75.3%) in intron 4-exon 5 boundary; 74(73.3%) in intron 4; 72(71.3%) in exon 4; 67(66.3%) in exon 7; 63(62.4%) in exon 10; 9(8.9%) in exon 5; no deletion in exon 3, exon 6, and exon 9. Different RhD phenotypes also showed different RHD gene deletion frequency: 80-90% deletion in ccee; 70% in ccEe; 40-50% in Ccee and CcEe; no deletion in CCee phenotypes.
CONCLUSIONS
RHD gene deletion frequency varied according to the methods applied and individual's own RhD phenotypes. Therefore, RHD genotyping is not appropriate for a routine test in Blood Bank and individual variation should be considered in prenatal care of RhD-negative women.