Detection of RHD 1227G>A and 1222T>C Using PCR-Restriction Fragment Length Polymorphism

Background: DEL is an RhD variant that cannot be detected by routine serologic tests because of the extremely low expression of the RhD antigen. Detecting the common genotypes of RHD 1227G ＞ A and 1222T ＞ C in Korean DEL is important for safe and efficient blood transfusions. Therefore, in this study, a PCR-restriction enzyme fragment polymorphism (RFLP) method was applied to detect RHD 1227G ＞ A and 1222T ＞ C. Methods: DNA extracted from the blood of each segment of 56 units of RhD-negative red blood cell were used. The promoter, exon 7 and exon 9 of RHD , and exon 9 of RHCE were amplified. The PCR products of RHD exon 7, RHD exon 9, and RHCE exon 9 were treated with the restriction enzymes HpyAV and MspI, and the RFLP patterns were observed by electrophoresis. The results of PCR-RFLP of RHD exon 9 were confirmed by PCR-direct sequencing. Results: RHCE exon 9 was amplified in all 56 DNAs. RHD promoters, exon 7, and exon 9 were all amplified in 10 samples, RHD promoter, exon 7, and exon 9 were not amplified in 38 samples, and RHD promoter only was amplified in eight samples. As a result of the RHD exon 9 PCR-RFLP performed on 10 samples with all targets amplified, 10 samples were determined to be 9 samples with 1227G ＞ A and 1 sample with 1222T ＞ C. The PCR-RFLP result and the sequencing result were 100% identical. Conclusion: PCR-RFLP using HpyAV and MspI is a reliable and applicable method for detecting RHD 1227G>A and 1222T ＞ C in serologically RhD negative samples. (Korean J Blood Transfus 2021;32:28-34) binding sites indicate ranges of primer binding sequences according to reference sequences (accession No. RHD NG_007494.1 and RHCE NG_009208.3). Abbreviations: Ta, annealing temperature; P, PCR; R, restriction fragment length polymorphism; S, sequencing.