Two novel variant allele of the RHD gene detected in two Chinese blood donors with the RHD negative phenotype.

Background: The D antigen is the most clinically important antigen in the Rh system due to its high immunogenicity and being the main cause of hemolytic disease of the foetus and newborns (HDFN). High polymorphism of the RHD gene implicates that new RHD variant alleles may be regularly identified.

Study design and methods: D antigen status was examined using saline tubes with IgM anti-D (clone RUM-1). If a negative result was observed, indirect antiglobulin test (IAT) and adsorption-elution test were performed with four monoclonal anti-D reagents. A D-screen identification kit was used for partial D identification. All 10 exons of the RHD gene plus flanking intronic regions were amplified and sequenced.

Results: The serological investigation showed clearly negative results with all anti-D reagents used when testing against both probands RBCs. The sequencing results of probands revealed c.802-1G>C mutation in proband 1 and c.1154-2A>T mutation in proband 2. These mutations resulted in the change of 3' splice site in the intron-exon junction of intron 5 or intron 8 of the RHD gene from AG to AC or TG, abolishing the splicing effect.

Conclusions: We identified two novel splicing variants resulting from c.802-1G>C and c.1154-2A>T mutations in the RHD gene. Both mutations may abolish the splicing effect of the involved exons, leading to the D-negative phenotype.