Advertisement

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

  • Chang-Lin Wu
    Affiliations
    Department of Blood Transfusion, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China
    Search for articles by this author
  • Pin Yi
    Affiliations
    Department of Blood Transfusion, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China
    Search for articles by this author
  • Chao-Peng Shao
    Correspondence
    Corresponding author at: The First Affiliated Hospital of Shenzhen University School of Medicine, Shenzhen Second People's Hospital, Shungang West Road No. 3002 Futian, Shenzhen, 518035, China.
    Affiliations
    Department of Blood Transfusion, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, China
    Search for articles by this author
Published:October 17, 2021DOI:https://doi.org/10.1016/j.transci.2021.103295

      Keywords

      There are nearly forty-three RBC blood group systems until 2021 year, which are named formally by ISBT, The RH locus is the most polymorphic of those encoding over 400 antigens. Rh blood group antigens are carried on two similar membrane proteins encoded by homologous RHD and RHCE genes. RHD encodes D and several partial d-associated low-prevalence antigens, whereas nearly 50 different antigens, in addition to the five major Rh antigens (D, C, E, c, e), are encoded by RHCE genes. Besides, RHCE genes mutations or polymorphisms may arise from nucleotide changes or gene rearrangements and encode variant proteins with qualitatively or quantitatively altered expression of RH antigens [
      • Daniels G.
      Human blood groups.
      ,
      • Shao C.P.
      • Qin J.J.
      • Sun G.D.
      • et al.
      An analysis of RHD zygosity of Rh(d)-positive Chinese Han population.
      ].

      1. Case Study and aims

      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.
      Fig. 1
      Fig. 1Results of RhCcEe phenotype and genotype. RhCcEe phenotype was unidentified accurately by microcolumn gel card using different regents for the patient. As the RhCcEe phenotype showed C + c+wE+we+ (weakened expression of c and E antigens) by one regent (A, Chuangqun,bode co.), but the RhCcEe phenotype showed C + c+wE-e + by the other regent (B,Sanquin reagents, Cellind direct Type,K7012). However, the RhCcEe genotype was CcEe as the C showed by PCR-SSP.

      2. Materials and methods

      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.

      3. Results

      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.
      Fig. 2
      Fig. 2Alignment of the Exon 7 sequence of the novel allele. This novel allele showed one nucleotide difference with RHCE*cE at position 986 A > G by sequencing [Color figure can be viewed at https://blast.ncbi.nlm.nih.gov/Blast.cgi].

      4. Discussion and conclusions

      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 [
      • Lyu Hongjuan
      • Jiao Shuxian
      • Ma Wenhui
      • et al.
      A novel RHD allele caused by c.739 G& C mutation was identified in a Chinese individual.
      ]. 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 [
      • Hue-Roye Kim
      • Hipsky Christine Halter
      • Randall W.A.
      Novel RHCE*ce 48C, 733G allele with nucleotide 941C in exon 7, encodes an altered red cell e antigen.
      ].
      Fig. 3
      Fig. 3RhCE protein homology model based on Swissmodel on line. RhCE protein model was established according to the highest similarity of amino acid sequences in the database: 3hd6.1. A Ammonium transporter RH type C Crystal Structure of the Human Rhesus Glycoprotein RHCG.
      The 329th amino acid substitution, a change from histidine(His,H)to arginine(Arg, R), had taken place and resulted in the altered expression of RHCE antigens qualitatively or quantitatively [Wild type RhCE protein model can be viewed at https://swissmodel.expasyorg/interactive./SwrX7e/models/report.html; Mutation type weak-cE protein model can be viewed at https://swissmodel.expasy.org/interactive/RNpXS6/models/].

      Source of funds

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

      Declaration of Competing Interest

      The authors have disclosed no conflicts of interest.

      References

        • Daniels G.
        Human blood groups.
        3rd ed. Wiley-Blackwell, Oxford2013
        • Shao C.P.
        • Qin J.J.
        • Sun G.D.
        • et al.
        An analysis of RHD zygosity of Rh(d)-positive Chinese Han population.
        Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2011; 28: 29-32
        • Lyu Hongjuan
        • Jiao Shuxian
        • Ma Wenhui
        • et al.
        A novel RHD allele caused by c.739 G& C mutation was identified in a Chinese individual.
        Transfusion. 2020; : 1-2
        • Hue-Roye Kim
        • Hipsky Christine Halter
        • Randall W.A.
        Novel RHCE*ce 48C, 733G allele with nucleotide 941C in exon 7, encodes an altered red cell e antigen.
        Transfusion. 2011; 51: 32-35