Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture.

ABSTRACT

BACKGROUND: RH1 is one of the most clinically important blood group antigens in the field of transfusion and prevention of fetomaternal incompatibilities. New variant RHD alleles are regularly identified and their characterization is essential to ensuring patient safety. STUDY DESIGN AND METHODS: Blood samples with uncertain RhD phenotypes not resolved by our first-line SNaPshot assay were sequenced for all 10 RHD exons. RHD zygosity was investigated. Flow cytometry was performed to determine RhD antigen density and epitope pattern. RESULTS: Seven novel RHD alleles were identified. Six, that is, RHD(T55P), RHD(A85G), RHD(G132R), RHD(G132E), RHD(D403V), and DAR(T203A), resulted from nucleotide polymorphisms. The seventh, that is, RHD(S182WfsX46), resulted from a 4-bp deletion that led to a reading frame shift and the appearance of a premature stop codon. Study of RhD expression of the first five alleles at hemizygous state showed greatly reduced antigen densities ranging from 50 to 618 antigens per red blood cell (RBC). DAR(T203A) was classified as a partial D antigen with a weakened reactivity profile similar to that of DAR. As expected, no D antigen was detected on RBCs carrying the RHD(S182WfsX46) allele. In parallel, RhD expression of RHD(G336R)/weak D type 58, RHD(F410V), and suspected RHD(1-9)-CE was determined to be less than or equal to 50 antigens per RBC. RhAG/RhD(2) trimer model supports the observed phenotypes. CONCLUSION: Although the frequency of the new RHD alleles presented herein is low, their phenotypic and genotypic description adds to the repertoire of reported RHD alleles. These data can be useful for optimization of molecular screening tools.