Korean J Blood Transfus.  2021 Apr;32(1):28-34. 10.17945/kjbt.2021.32.1.2 8.

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

• Kim W
• Park G

• KMID: 2515261
• DOI: http://doi.org/10.17945/kjbt.2021.32.1.2 8

Abstract

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.

Keyword

RHD; DEL; Genotyping; RFLP