Two genes (RHD, RHCE) in close proximity on chromosome 1 encode the erythrocyte Rh proteins, RhD and RhCE; one carries the D antigen, and the other carries CE antigens in various combinations (ce, Ce, cE, or CE), ().16-19
The genes each have ten exons, are 97% identical, and arose via gene duplication. RhD and RhCE proteins differ by 32-35 of 416 amino acids ( shown as circles on RhD). This is in contrast to most blood group antigens that are encoded by single genes with alleles that differ by only one or a few amino acids. Individuals who lack RhD protein, “Rh or D negative”, most often have a complete deletion of the RHD gene (). An important consideration in the immunogenicity of a protein is the degree of foreignness to the host. The large number of amino acid changes explains why exposure to RhD can result in a potent immune response in a D-negative individual.
Fig. 1 A). Diagram of the RHD and RHCE locus. The two RH genes have opposite orientation, with the 3′ends facing each other. Rh negative Caucasian individuals have a complete deletion of RHD. B). Rh proteins in the RBC membrane. The RhD and RhCE proteins (more ...)
RHCE, expressed in all but rare D- - individuals, encodes both C/c and E/e antigens on a single protein. C and c antigens differ by four amino acids, but only the amino acid change Ser103Pro is extracellular (). The E and e antigens differ by one amino acid, Pro226Ala, located on the fourth extracellular loop of the protein.
The RH genes and proteins detailed in and are typical for the majority of individuals, and commercial antibody reagents detect expression of these “conventional” D, C, c, E and e antigens shown. The proximity of the two RH genes, and their inverted orientation (), augments opportunity for genetic exchange.20
Many RH genes carry point mutations, or have rearrangements and exchanges between RHD and RHCE that result from gene conversion events. The latter encode hybrid proteins that have RhCE-specific amino acids in RhD, or RhD-specific residues in RhCE. These can generate new antigens in the Rh blood group system, and alter or weaken expression of the conventional antigens.
Fig. 4 Diagram of the RHD and RHCE genes. The ten RHD exons are shown as white boxes, and the RHCE as grey. A). RHD and RHCE genes responsible for the common D, C, c, E and e antigen polymorphisms. B). Altered RHD and C). Altered RHCE genes indicating the changes (more ...)
Numerous mutations in RHD (greater than 100) are currently known. Most encode single amino acid changes, and many of these simply alter the quantity of RhD protein in the membrane, while others alter RhD membrane topology and D-epitopes. The latter are responsible for the enigmatic individual with D-positive RBCs who presents with anti-D following transfusion or pregnancy.
Mutations in both RHD and RHCE are found in Blacks, and other ethnically diverse groups, and alter D, C, or e antigen expression (detailed below). The incidence of variant RH genes in patients with sickle cell disease underlies the complex incompatibilities that can arise following transfusion in this patient population. Commercial typing reagents are not available to detect red cells that express variant Rh proteins, and individuals at risk go undetected until they produce antibodies reactive with all, or most, conventional Rh antigens. Finding compatible blood then becomes a serious and potentially life-threatening problem. Understanding of RH gene variation can now be applied to assess alloimmunization risk in SCD, because patients who inherit variant RH genes can now be identified at the genomic level.