Identification of a novel variant (c.1-111A>G) located in GATA-1 motif of RHCE proximal promoter in two Chinese patients with the rare D-- phenotype

Background: D-- is a rare phenotype lacking the expression of the C, c, E, and e antigens and several high-frequency antigens on the red cells. Anti-Rh17 (Hr0) could be developed in individuals with the D-- phenotype to cause hemolytic transfusion reactions (HTR) and hemolytic disease of the fetus and newborn (HDFN). Nuleotide(s) change of the RHCE gene and RHCE-D-CE hybrid alleles are the common molecular basis of the D-- phenotype. Study Design and Methods: One D-- Chinese patient detected in routine RhD and RhCE serologic testing and another D-- Chinese patient identified with anti-Rh17 were recruited. Further RHD, RHCE, and RHAG whole gene sequences were analyzed using the PacBio sequencing. A dual-luciferase reporter assay was performed to verify the effect of the variant identified in the promoter of the RHCE gene on the transcriptional activity of the reporter gene in vitro. Results: A homozygous RHCE*Ce(1-111G)/Ce(1-111G) genotype and a heterozygous RHCE*CeN.08/Ce(1-111G) genotype carried one novel variant (c.1-111A>G) located in the GATA-1 motif of the RHCE proximal promoter was identified in two D-- patients, respectively. In the reporter assay, the luciferase transcriptional activity of the mutant RHCE promoter [c.1-111G] construct reduced from similar to 1.0 to 0.28 relative luciferase activity normalized to RHCE wild-type, with a similar to 72% reduction rate. Conclusion: The novel variant of the GATA-1 motif of the RHCE proximal promoter was identified to diminish the binding of the GATA-1 transcription factor and markedly down-regulate the transcription activity of the RHCE gene to abolish the expression of RhCE antigens, causing the rare D-- phenotype.