Molecular biology study of RHD gene in Chinese Han voluntary blood donors with RhD-negative blood type

Abstract

Objective To explore the RHD gene structure of Chinese Han voluntary blood donors with RhD-negative blood type, and to conduct a molecular biological analysis of a newly discovered RHD gene mutation. Methods From 2016 to 2017, a total of 85 Chinese Han voluntary blood donors who participated in blood donation in Shandong Blood Center were selected as the subject by randomized digital table. Real-time fluorescence quantitative (RQ)-PCR method was used to detect exon 7 of RHD gene in DNA samples from 85 blood donors. The multiplex PCR-sequence specific primer (SSP) method was used to detect exons 3 to 7 and 9 of RHD gene in DNA samples from these 85 blood donors. The RHD gene in DNA samples from 30 blood donors whose DNA samples expressed RHD gene exon 7 were sequenced using a fluorescently labeled Sanger dideoxy chain termination method. The procedure of this study is in accordance with the requirements of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each blood donor. Results ① In 85 blood donors with RhD-negative blood type, DNA samples of 30 donors had exon 7 of RHD gene, and of 55 blood donors had deletion of exon 7 of RHD gene.② Among the 30 donors with RHD gene exon 7, the DNA samples of 29 donors had exons 3 to 7 and 9 of the RHD gene. Exon 3, 4, 5, 7 and 9 of RHD gene were present in DNA sample of one donor, but exon 6 was deleted. DNA samples from 55 donors without RHD gene exon 7, were deletion of RHD gene exons 3 to 7 and 9. ③ Among the 30 blood donors with RHD gene exon 7, 29 DNA samples had one c. 1227G>A point mutation on RHD gene exon 9. There was a non-sense mutation in c. 933C>A on RHD gene exon 6 in one blood donor′s DNA sample with deletion of the exon 6 of RHD gene. This mutation produced a stop codon, resulting in inconsistent multiplex PCR-SSP and DNA sequencing results of RHD gene in this blood donor. Conclusions The deletion of RHD gene is the main molecular mechanism that causes the RhD-negative phenotype of Chinese Han people. The RHD gene c. 1227G>A mutation is a major feature of Del phenotype. A new c. 933C>A mutation in the RHD gene results in a RhD-negative phenotype. Key words: Rh-Hr blood system; RhD-negativity; Red cells; Immunohematology; Immunogenetics