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


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.


CONCLUSIONS
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.
Laboratoire d'Hématologie Moléculaire, Établissement Français du Sang Alpes Méditerranée, UMR 6578, Université Aix-Marseille, Marseille, France.
Monique
M
Silvy
Sylvie
S
Chapel-Fernandes
Isabelle
I
Callebaut
Sophie
S
Beley
Cécile
C
Durousseau
Sophie
S
Simon
Pierre
P
Lauroua
Nadine
N
Dubosc-Marchenay
Catherine
C
Babault
Chantal
C
Mouchet
Virginie
V
Ferrera
Jacques
J
Chiaroni
Pascal
P
Bailly
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D018604:Epitope Mapping; D056726:Genetic Association Studies; D005838:Genotype; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide / Q000502:physiology; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000235:genetics; D012204:Rh-Hr Blood-Group System / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D050505:Mutant Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen