Analysis of red blood cell RhAG protein, Rh D, and Rh CE antigens expression in carriers of RHAG* 808A: a common variant in the Chinese population

[Objective] To investigate the impact of RHAG* 808A variant, commonly identified in the Chinese population, on RhAG protein, RhD and RhCE antigens expression through in vivo and in vitro expression analysis. [Methods] A missense mutation of RHAG gene (c. 808G>A, p. Val270Ile) with high frequency was found in KMxD database. Bioinformatics analysis was performed using Polyphen-2 and Provean software. High resolution melting (HRM) method was utilized to screen for the variant carriers in the blood donors. The expression of RhAG protein, RhD and RhCE antigens on the surface of red cells of variant carriers were detected via flow cytometry. Wild-type and mutant vectors of RHAG were constructed and transfected into HEK 293T cells for in vitro expression analysis. Then, the expression of RhAG protein, RhD and RhCE antigens were analyzed by flow cytometry. [Results] Polyphen-2 and Provean software suggested that the amino acid change (p. Val270Ile) of RhAG protein may be harmful or neutral respectively. Among the 999 blood donors from Guangzhou Blood Center, 4 homozygous carriers and 99 heterozygous carriers of RHAG* 808A mutant allele were identified. The frequency of this allele was 5.4% (107/1 998). No significant differences in RhAG protein, RhD and RhCE antigens expression level was identified between the homozygous carriers, heterozygous carriers of RHAG* 808A variant allele and the wild-type individuals. In vitro analysis for antigen expression study obtained the similar results. [Conclusion] The RHAG* 808A variant allele commonly identified in the Chinese population has no effect on the expression of RhAG protein, RhD and RhCE antigens, so the variant should be a population polymorphism site.