Evaluation of rare and novel splicing variants in the RH system

BACKGROUND: Single nucleotide variants near intron/exon junctions can affect RNA splicing, resulting in weak or loss of blood group antigen expression. We investigated 11 samples with no causative variants found in RH coding exons: three with weak RhD phenotypes and 8 with discordance between phenotypes and DNA prediction for RhCE. METHODS: Serologic and DNA testing was performed by standard methods. RH exons 1-10 and flanking introns were amplified and Sanger sequenced. Variants were evaluated using an in vitro splicing minigene assay. RESULTS: Three weak D phenotypes were associated with intron 1, RHD*149-1G>A, or intron 3, RHD*487-3C>A or RHD*487-3C>G, variants. Splicing assay confirmed activation of a cryptic acceptor site for RHD*149-1A, and both RHD*487-3A and -3G resulted in mis-splicing with minor amounts of normal transcripts. Among eight RHCE discrepancies: three had an intron 4 variant, RHCE*Ce c.635-9G>A, causing a 7-bp insertion, frameshift/stop; five had other intron variants, RHCE*cE c.336-2A>G, RHCE*ce c.486+1G>A, RHCE*ce c.1154-1G>A, RHCE*Ce c.939+1G>A, showing complete splicing disruption, and RHCE*Ce c.148+5G>T with no splicing disruption detectable. CONCLUSIONS: Alleles studied here had intron variants associated with weak or absence of detectable antigen expression. Five are null alleles, RHCE*Ce635-9A, RHCE*ce486+1A, RHCE*ce1154-1A, RHCE*Ce148+5T, and RHCE*Ce939+1A, while RHCE*cE336-2G is associated with E antigen detected by adsorption/elution. Splicing assays support RHD*149-1A may encode an altered RhD, while the low level of normal splicing shown for RHD*487-3A and RHD*487-3G suggests very weak expression but the absence of alloimmunization risk. The minigene splicing assay offers insight into the effect of intron variants on antigen expression.