A novel RHCE*cE allele identified in a Chinese individual, encodes weakened expression of c and E antigens

RhCcEe phenotype on sample from a group A Rh D + female patient with bone tumors revealed the following results: C + c+^wE+^we+ [Fig. 1A]. A follow-up sample was requested before her blood transfusion, we used another reagent to test her RhCcEe phenotype, red blood cells (RBCs) typed C + c+^wE-e+ [Fig. 1B], c antigen showed stratified visual, E antigen showed negative [Fig. 1B]. The aim of this study was to investigate the molecular basis of the RHCE gene and to further characterize the c and E antigens serologically.

The patient’s RhCcEe phenotypes were tested with microcolumn gel card (Changchun bode biotechnology co., China; Sanquin reagents B.V, Cellbind Direct Type, Holland). Serum antibody was tested by IAT assay. Genomic DNA was extracted from the blood sample(Invitrogen, lot: 10,974,020) and was subjected to the following molecular tests: sequence specific primers polymerase chain reaction (PCR-SSP) and real-time fluorescence quantitative PCR were performed for RHCE genes(ABI7500, Applied Biosystems 3500). RHCE exons 1 through 10 were sequenced afterward by exon-specific amplification and analyzed using a program for sequence assembly and mutation detection (BioEidt sequence Alignment Eiditor). RhCE protein model was established and analyzed according to the highest similarity of amino acid sequences on Swissmodel on line.

Results of microcolumn gel card with anti-C, anti-c, anti-E and anti-e revealed weakened expression of c and E antigens with one reagent, while tests with the same method revealed E antigen non-reactivity with another reagent that contained IgM-anti-C(K1195,lot8000253348), IgM-anti-c(K1196,lot8000255063), IgM-anti-E(K1191,lot8000258024), IgM-anti-e(K1197,lot8000451611)[Figure1A/B]. IAT results showed no agglutination, and there were no unexpected antibodies in the patient serum. PCR-SSP method revealed an apparent RHCE*CcEe genotype, which is the same as the result of the real-time fluorescence quantitative PCR assay at the second time, consistent with the C + c+^wE+^we + phenotype. Sequence analysis revealed heterozygosity for a novel mutation, c.986 A > G, in Exon7, which encodes a change from histidine(His, H) to arginine(Arg, R)[Figure1C/D, Fig. 2]. The sequence was deposited in GenBank (KX767850), but which still was not assigned the number RHCE allele by the ISBT Working Party for Red Cell Immunogenetics and Blood Group Terminology. This amino acid change is have not reported in the dbSNP and ExAc databases.(Exome Aggregation Consortium; http://exac.broadinstitute.org/, https://www.ncbi.nlm.nih.gov/snp/?term=rhce/). From the surface image of the molecule, it can be clearly seen that the variations in the properties, sizes and configurations of the residues cause significant changes in the shape of the surface structure of the helix by RhCE protein homology model based on Swissmodel on line.

We describe a novel RHCE*cE 986A > G allele that gives rise to an altered expression of both the c and the E antigens in Chinese individual. Residue 329 is associated with the start of the sixth transmembrane helix and it is likely that a change from histidine to arginine could affect how the protein is inserted into the RBC membrane, however, this remains unproven in the absence of expression studies. The amino acid polymorphisms responsible for c and E antigens are encoded by Exon 2 and Exon 5, respectively []. But due to point mutation in exon 7 and the 329th amino acid substitution, RHCE molecular structure and the possible effects of the amino-acid residue variations were modeled and analyzed with Swissmodel software. The residue variations are very likely to change the peptide binding properties as well as antibody binding characteristics of the RHCE antigens [Fig. 3]. The failure of the RBCs to react with IgM-anti-E(K1191,lot8000258024), together with the weak reactivity using with one of the two monoclonal reagents including anti-c and anti-E, suggests that the c, E antigens expressed by this allele are partial antigens. This raises the question of whether this patient is at risk of producing anti-c or anti-E if challenged. DNA analysis of RHCE revealed heterozygosity for RHCE*C/c and RHCE*E/e as well as c.986A > G. While further samples could not be obtained for additional serologic testing, RHCE*cE-specific sequence analysis confirmed that c.986 G was carried on this allele. We conclude that an RHCE*cE allele containing the change c.986A > G gives rise to c and E antigens that are both qualitatively and quantitatively altered and that individuals with this allele risk being not only mistyped for RhCcEe antigens but at risk of producing alloanti-c or alloanti-E [].

Granted by Guangdong Natural Science Foudation (2020A1515011520) and Shenzhen Natural Science Foudation (JCYJ20190806164818079, JCYJ20180228162956597)