A Novel RHCE_*ce Variant Allele Is Highly Frequent in Chinese D-Negative Individuals and Results in C-Expression

Background: The RHCE allele is highly polymorphic and many variants have been described, especially in individuals of African origin. Donors carrying these variants can be falsely typed and elicit transfusion reactions, and patients carrying such a variant may be at risk to develop allo-antibodies in response to mismatched transfusions. Not much is known about the frequency of RHCE variants in Chinese populations, whereas in China genotyping assays are increasingly applied for typing of blood donors and patients.

Methods: Standard column agglutination was used to serologically type for C/c and E/e expression in 200 serologically D-negative and 200 serologically D-positive Chinese donors. The RH Multiplex Ligation-dependent Probe Amplification (MLPA) genotyping assay was used for genotyping the RHCE status (Transfusion 2013;53:1559). In donors with discrepant results of genotyping and phenotyping all 10 exons of RHCE were amplified and directly sequenced. A lentivirus containing the novel RHce variant was created to transduce human erythroblasts cultured from peripheral blood from 3 ccDee and 3 CCDee donors as previously described (Haematologica 2010;95:1594). FACS analysis was used to assess the c- and C-expression caused by the variant.

Results: In 6 out of 200 Chinese D-negative donors the results of the RH-MLPA indicated the presence of only one copy of exon 2 of the CE*c-allele, and no copy of exon 2 of the CE*C-allele, whereas these donors were serologically typed as Cc. Sequencing of all 10 RHCE exons revealed a novel RHCE*ce allele defined by 308C>T (p.103Pro>Leu) mutation next to a normal RHCE*ce allele. The variant allele was not found in the 200 Chinese D-positive donors, indicating the linkage of this new variant RHCE*ce allele with the D-negative haplotype. Wild type Rhce cDNA was mutated to create the RHce*308C>T mutation and subsequently cloned into a lentiviral vector. Transduction of human ccDee erythroblasts with this vector resulted in C expression, whereas virtually no c-expression was induced by transduction of human CCDee erythroblasts as assessed by FACS analysis with the monoclonals MS33, MS35 and MS42 to detect c expression and monoclonals MS24 and MS273 to detect C.

Discussion: A new RHCE variant (RHCE*ce308T) is identified, which is present in 3% of D-negative Chinese individuals. The 308C>T mutation in the triplet encoding the 103Pro results in the expression of 103Leu. Position 103 is one of the 4 aminoacid differences between the c- and C-carrying polypeptides (Pro and Ser, respectively). The Pro>Leu mutation in the novel variant leads to C-expression and loss or strongly diminished c-expression. Most Rhc-genotyping assays target the c-specific-307C nucleotide and most RhC-genotyping assays target the C-specific-intron 2, which are respectively present and absent in this variant allele. Therefore, when individuals carrying this allele are genotyped, the predictive phenotype will be falsely C-negative.

Conclusion: RHC-genotyping assays applied in Chinese populations, should be adapted to recognize the presence of this new RHCE*ce308T allele to prevent C-mismatched transfusions.