Molecular genetics and clinical applications for RH

Abstract

Rhesus is the clinically most important protein-based blood group system. It represents the largest number of antigens and the most complex genetics of the 30 known blood group systems. The RHD and RHCE genes are strongly homologous. Some genetic complexity is explained by their close chromosomal proximity and unusual orientation, with their tail ends facing each other. The antigens are expressed by the RhD and the RhCE proteins. Rhesus exemplifies the correlation of genotype and phenotype, facilitating the understanding of general genetic mechanisms. For clinical purposes, genetic diagnostics of Rhesus antigens will improve the cost-effective development of transfusion medicine.

Figure 1. Duplication of the RH gene and loss of the RHD gene

The ancestral configuration is shown as represented by the RH gene locus in mouse. The single RH gene is in close proximity to the three genes SMP1, P29-associated protein (P), and NPD014 (N). A duplication event introduced a second RH gene in reverse orientation between N and SMP1. At the two break points in front and behind the RHD gene, DNA segments of approximately 9,000 base pairs (bp) occur. Both DNA segments are flanking the RHD gene and dubbed ”upstream Rhesus box“ and ”downstream Rhesus box“. In the RHD positive haplotype, the RHD gene may have been lost by a recombination event (see Figure 3).

Figure 2. RHD deletion

An unequal crossing over event between an upstream Rhesus box and a downstream Rhesus box caused the RHD deletion. If one of the two crossed-over chromosomal threads are resolved, an RH gene locus results that lacks the RHD gene completely and harbors a hybrid Rhesus box.

Figure 3. RHD/RHCE hairpin formation

The schematic diagram depicts the mechanism of gene conversion at the Rhesus gene locus on one chromosome. (i) The RHD and RHCE genes are inversely orientated, which is typical for clustered genes. (ii) A hairpin formation of the chromosome would generate the close proximity of homologous segments in identical orientation. This structural feature is generally instrumental in gene conversion events in cis. (iii) Resolving the hairpin yields an RHD-CE-D hybrid gene structure, many of which have been observed to date at the RH gene locus. The RHD-CE(4–7)-D hybrid exon structure shown here is an example. Modified from Wagner et al., licensee BioMed Central Ltd. Reprinted with permission.

Figure 4. Model of Rhesus proteins in the red blood cell membrane

Both Rhesus proteins comprise 417 amino acids, shown here as circles. Mature proteins in the membrane lack the first amino acid. The amino acid substitutions that distinguish the RhCE from the RhD protein are marked in yellow, with the 4 amino acids that code for the C antigen in green and the one that codes for the E antigen in black. The single amino acids substitutions which code for partial D are in blue, and those that code for weak D are in red. The mutations that had been identified at the Ulm Institute since 1999 are in light blue and orange. The extracellular Rh vestibule is depicted by the inverted black arc and bordered in part by amino acids of loops 3 and 4. The nine exon boundaries in the RHD cDNA, as reflected in the amino acid sequence, are indicated by black bars.