A novel RHCE*02 variant with a recombination of RHD exon 3 in a Chinese D+ voluntary blood donor with weak expression of e antigen

The Rh blood group system comprises two closely linked homologous genes, RHD (MIM: 111680) and RHCE (MIM: 111700), which encode the common red blood cell-specific Rh antigens. The Rh system is one of the most diverse and currently comprises 56 antigens, with the main antigens being RH1 (D), RH2 (C), RH3 (E), RH4 (c), and RH5 (e).1 To date, over 150 RHCE alleles have been identified through molecular analysis across various populations. Most rare RHCE phenotypes are often identified through the recognition of weak and partial expression of major RhCE antigens.2-4 For patients with Thalassemia, who frequently require blood transfusion, RH genetic polymorphisms that change the conformation of RH proteins in both patients and healthy blood donors contribute to compatibility issues. Aligning Sanger or NGS reads for RH, given RHD-RHCE homology, is challenging. Short reads hinder complete gene phasing and allele assignment, notably in samples with multiple polymorphisms.5 Sanger sequencing may not address the problems caused by RH recombination. PacBio sequencing is a single-molecule sequencing technology that is capable of reading long DNA fragments and reduces ambiguity and overcomes the inherent limitations of Sanger sequencing by using additional assembly markers. We report a novel RHCE allele in a 28-year-old Chinese donor through long-read sequencing on the Pacific Biosciences (PacBio) platform with weak expression of e antigen. Fresh blood from the first-time blood donor was typed in the Rh blood group system by (1) an automated microplate testing and gel card typing, (2) the final result was confirmed by test tube method. A discrepancy prompted an investigation into the donor's RHCE gene. Serologic testing for C, E, c, and e antigens was conducted using standard hemagglutination techniques on the Microlab STARlet BG (Hamilton, Switzerland) for automated testing and the test-tube method for tube testing. Both methods utilized commercially available reagents from Shanghai Hematology Co., Ltd. (Shanghai, China). RH Gel card typing (Bioxun, Changchun, China) was conducted following the provided instructions. Genomic DNA was extracted using the MolPure® Blood DNA Kit (Yeason, Shanghai, China) from peripheral blood following the manufacturer's guidelines. Fifteen primer sets were developed to comprehensively amplify both RHD and RHCE genes. Sequencing was performed using Sanger sequence and long-read techniques on the Pacific Biosciences (PacBio) platform. Data analysis was conducted using SMRT Link v10.1.0 software, with subreads processed to generate Circular Consensus Sequence (CCS) reads. The CCS fragments relevant to RHD and RHCE were batched and realigned to the reference genomic sequence (RHD (NG_007494.1) and RHCE (NG_009208.3) genes) using pbmm2. The donor, a 28-year-old woman from China, had RBCs with the RH: 1, 2, 3, −4, 5w phenotype. In automated microplate testing and gel card methods, there was no agglutination observed with IgM monoclonal anti-e. However, the tube test revealed a C+, E+, c−, e+w phenotype, as depicted in Figure S1. Sanger sequence results of RHCE showed eight nucleotide substitutions separately at c.48G>C and c.105C/T in exon 1, c.150C>T, c.178C>A, c.201A>G, c.203A>G, and c.307C>T in exon 2, and c.676G/C in exon 5, as shown in Figure S2. Considering that the RHCE*cE and RHCE*ce alleles differ only at c.676,6 and our sequencing results showed the c.676 was heterozygous mutation, we conclude that the RHCE genotype yielded a RHCE*Ce allele. The long-read sequencing results of RHCE and the amino acid sequence are presented in Table 1 and Figure S3. In SNV2, a RHCE-D recombination was first detected in RHCE*Ce allele backbone exons 3, similar to RHCE*03.32,7 resulting in a RH phenotype of cEw. Since this sample's phenotype is C+E+c-e+w, we speculate that the RHCE*Ce-D(3)-Ce may also result in reduced expression of the e antigen, just as RHCE*03.32 leads to reduced expression of the E antigen. RHCE*03.32 was previously described only as c.361A>T c.380C>T, c.383G>A, c.455C>A. But with the full-length RHCE sequence from the PacBio platform, we've confirmed that our sample aligns with RHCE*Ce-D(3)-Ce. Further, we can find the exact starting and ending regions of the reorganization, the starting region between c.148–807 to c.148–997 and the ending region between c.486+6289 to c.486+6696. We report a novel RHCE02 with a replacement of exon 3 with RHD, which was identified for the first time in the Chinese people. The RH type of the donor was C+E+c−e+w in serological methods. Long read sequencing revealed a RHCE*Ce-D(3)-Ce recombination, which may be related to the e weak phenotype. Overall, we recommend caution for newly discovered variant. This study reminds us that the phenotype does not always reflect the genotype and the importance of associating serologic and molecular methods for the detection of new RH alleles and ensuring patient safety. On the other hand, Sanger sequencing's short reads cannot effectively resolve issues arising from RH gene recombination, while long-read sequencing is demonstrating significant technical advantages in blood group genotype. The bioinformatics of these recombination alleles outstretched our knowledge of RHCE variants, which was pivotal for evaluating their impact to guide transfusion support and avoid immune-related blood transfusion reactions. The study was sponsored by Changzhou Institute of Technology (Grant Nos. CJ20245046 and CJ20241124). The authors have disclosed no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. FIGURE S1. RHCE typing results from gel card and tube test. The RH phenotype of the donor was not accurately identified using different reagents. While the RH phenotype appeared as D+ C+ E+ c- e- according to the gel card method (A), it appeared as C+ E+ c- ew+ using the tube-test method (B). FIGURE S2. Sanger sequencing results of RHCE exon 1–10. Sanger sequencing results showed the mutation separately at c.48G>C and c.105C/T in RHCE exon 1(A), c.150C>T, c.178C>A, c.201A>G, c.203A>G and c.307C>T in RHCE exon 2(B), c.676G/C in RHCE exon 5(C), and the positions were indicated with a box. FIGURE S3. PCR amplification scheme and the haplotype sequences of RHCE. (A)Two haplotype sequences of this sample. In the haplotype sequences, white bars indicate positions or insertions of one or more bases that differ from the RHCE reference sequence (NG_009208.3). (B) The initial sites of reorganization in allele 2 within intron 1. (C) Nucleotide variations within the exon 3 region. (D) The initial sites of reorganization in allele 2 within intron 3. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.