In this study, we investigated the molecular basis of the D-negative phenotype, including the DEL type, and determined the distribution and prevalence of other blood group genotypes in the Korean population. The performance of the MLPA assay has not been validated in the East Asian population with serologically D-negative phenotype. Furthermore, unlike the most widely used comprehensive blood group genotyping platforms such as the PreciseType HEA Molecular Beadchip (Immucor, Warren, NJ, USA) and the Progenika ID CORE XT (Progenika Biopharma-Grifols, Bizkaia, Spain), the MLPA assay can detect four frequent DEL alleles. Compared with previously proposed strategies, the MLPA assay has more advantages, as the presence, absence, and copy number of 48 blood group alleles and 112 variant alleles can be determined simultaneously. Recently, a multiple ligation-dependent probe amplification (MLPA) assay has been developed to detect RHD variants and zygosity, as well as the 17 other blood group systems. The proposed methods use RHD-specific multiplex PCR for the detection of total- or partial deletion and are followed by PCR with sequence-specific priming (PCR-SSP) or exon 9 sequencing to detect DEL alleles. Several RHD genotyping strategies have been proposed specifically for East Asians. Therefore, the accurate determination of D variant (including DEL type) is important to protect D-negative individuals from inadvertent alloimmunization to the D antigen. However, Seo et al reported that 20 individuals with DEL type (n=16, 14.5%), weak D (n=2, 1.8%), and partial D (n=2, 1.8%) were classified as D-negative among 110 RhD-Negative Blood Club members. Owing to the very low frequency of the D-negative phenotype (0.15%) in Korea, ‘RhD-Negative Blood Club’ was organized to assist the supply of D-negative blood products since 1973. In East Asians, 15–20% of serologically D-negative individuals are DEL type, and the RHD(1227G>A) allele is the most common. In Caucasians, the frequency of DEL type among serologically D-negative individuals is 1/1,000–1/3,000, and RHD(885G>T) and RHD(IVS3+1G>A) are the most common DEL alleles. Currently, about 30 RHD alleles associated with DEL type have been reported, showing differences in distribution based on ethnicity. Several cases of anti-D alloimmunization resulting from RBC transfusion of DEL type have been reported. In addition, DEL type can cause anti-D alloimmunization despite small amounts of D antigen on red blood cell (RBC) surfaces. DEL type is a very weak D variant that cannot be detected by routine serological tests and presents as a D-negative phenotype unless adsorption and elution studies are performed. The Rh blood group D antigen is highly immunogenic in the human blood group system, and anti-D alloimmunization can cause severe hemolytic transfusion reactions and hemolytic disease in the fetus and newborn. Keywords: Rh-Hr blood-group system, Genotype, Multiplex ligation-dependent probe amplification The use of MLPA assay on serologically D-negative individuals may help improve transfusion safety by preventing anti-D alloimmunization. MLPA assay correctly determined RHD genotype, including RHD-CE-D hybrid alleles or RHD(1227G>A) allele, and other clinically relevant blood group genotypes in D-negative Koreans. Genotyping results and allele distribution of the other 17 blood group systems were consistent with previous reports on the East Asian population. The RhCE phenotypes of RHD(1227G>A) alleles were composed of 14 Cce and 3 CcEe, and all 60 cases of the ce phenotype were revealed to have a total deletion of the RHD. The other 17 cases (17.9%) had an RHD(1227G>A) allele, which was further confirmed by sequencing analysis. Out of 95 cases, total deletion of the RHD was observed in 74 cases (77.9%) and four cases (4.2%) had an RHD-CE-D hybrid allele. The MLPA results were verified by multiplex PCR for the RHD promoter, exons 4, 7, and 10 and by direct sequencing of RHD exon 9. Methodsīlood group genotyping using the MLPA assay and RhCE phenotyping were performed on randomly selected 95 D-negative red blood cell products. We investigated the molecular basis of serologically D-negative phenotypes, including the DEL type, and the distribution of other blood group systems in the Korean population using the recently developed multiplex ligation-dependent probe amplification (MLPA) assay. The DEL blood type, a very weak D variant, is a major concern in the field of transfusion medicine because of its potential to cause anti-D alloimmunization.
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