A variant RhAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes

BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c-E–e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.