France Noizat‐Pirenne

The molecular backgrounds of variants encountered in Afro-Caribbean black individuals and associated with the production of clinically significant antibodies against high-incidence antigens (anti-RH18, anti-RH34) and against Rhe epitopes were determined. We showed that RH:−18 phenotypes are produced by 3 distinct RHCEalleles: ceEK carrying 48G>C (exon 1), 712A>G, 787A>G, 800T>A (exon 5); ceBI carrying 48G>C (exon 1), 712A>G (exon 5), 818C>T (exon 6), 1132C>G (exon 8); and the already knownceAR allele carrying 48G>C (exon 1), 712A>G, 733C>G, 787A>G, 800T>A (exon 5), and 916A>G (exon 6). The RH:−34 phenotype is produced by the (C)ce^s haplotype described previously and composed of a hybrid D-CE(3-8)-D gene with 4 extra mutations next to a ce^s allele (733C>G; exon 5) with an extra mutation in exon 7 (1006G>T). Partial Rhe with risk of immunization against lacking epitopes can be produced by the new ce^s allele carrying an extra mutation in exon 3 (340C>T) and by the ceMO allele described previously. A population of sickle cell disease patients was screened to estimate the incidence of these rare alleles, with the conclusion that a procedure is required to detect the associated phenotypes in black donors to ensure transfusion safety for patients. We also described a new variant [ce^s(748)] and variants carrying different altered alleles in nonimmunized patients and for whom the risk of immunization is discussed.

BACKGROUND: D(C)(e) and D(C)e haplotypes may be encountered in the white population. Few data are available on the molecular backgrounds responsible for depressed expression of C and e. STUDY DESIGN AND METHODS: Individuals of white origin carrying a D(C)(e) genotype resulting in depressed expression of C or both C and e were subdivided into two categories based on the RBC reactivity with the human sera Mol and Hor, which contain antibodies against low‐frequency antigens of the Rh (RH) system and other non‐Rh low‐frequency antigens. Neither Hor+, Mol+ nor Hor+, Mol– RBCs expressed the V (RH10), VS (RH20), and/or Rh32 (RH32) low‐frequency antigens. These results suggested that Hor+, Mol+ variants expressed Rh33 (RH33 or Har) and FPTT (RH50), whereas Hor+, Mol– variants might express an undefined low‐frequency antigen. Further serologic and molecular analyses were performed. RESULTS: Molecular analysis of Hor+, Mol+ variants revealed a hybrid gene structure RHCe‐D(5)‐Ce, in which exon 5 of RHCE (RHCe allele) was replaced by exon 5 of RHD (the so‐called RHCeVA allele). The presence of exon 5RHD resulted in several amino acid alterations predicted in the external loop 4 of the CeVA polypeptide. Molecular analysis of Hor+, Mol– variants revealed the presence of a new RHCe allele characterized by a single point mutation C340T within exon 3 (the so‐called RHCeMA allele), resulting in a R114W substitution predicted on the external loop 2 of the CeMA polypeptide. A serologic study showed a different pattern of reactivity with C and e MoAbs. CONCLUSION: Two types of mutations resulted in amino acid substitutions predicted in external loops 4 and 2, respectively, which altered both the C and e reactivity, and indicated conformation changes or defective interaction between nonadjacent loops of the Ce polypeptide. Serologic analysis showed that together with Hor and Mol sera testing, the use of different C and e MoAbs could help to identify these variants within the white population.

Six unrelated individuals of Afro‐Caribbean origin, whose red cells have a marked reduction of the Rhe antigen expression, have been identified. All exhibited the same serological profile with anti‐e monoclonal antibodies and lacked expression of the high frequency e‐related antigen hr^S. Transcripts and genomic analysis showed that these phenotypes resulted from the presence of two new RHCE alleles, ceMO and cEMI. The ceMO allele corresponded to a RHce gene carrying a G667T mutation (exon 5) and was detected at the homozygous state in sample 1 and at the heterozygous state in samples 2–6. The G667T mutation resulted in a Val223Phe substitution on the Rhce polypeptide, in close proximity to Ala226 (e‐antigen polymorphism), which might account for the altered expression of e. The ceMO allele is also associated with the lack of expression of the hr^S antigen. The absence of the hr^S antigen expression may have implications in transfusion as hr^S‐negative individuals may develop clinically significant antibodies. The cEMI allele corresponded to a silent RHE allele carrying a nine nucleotide deletion within exon 3 and was detected at the heterozygous state in sample 2. This deletion resulted in a shortened polypeptide of 414 residues (instead of 417) that was absent (or severely reduced) at the red cell surface, as the E antigen was undetectable using serology and Western blot analysis with anti‐E reagents. In DNA‐based polymerase chain reaction genotyping for RHE determination, the cEMI allele provided a false positive result as the cells carrying this allele are serologically phenotyped as E‐negative. The incidence of this allele in the Black population is unknown but, as shown already for D genotyping, one must exercise caution when genotyping is performed to detect the e/E polymorphism.