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The main RhCE antigens, C, c, E and e, are differentially encoded by the four reference RHCE alleles: RHCE*Ce, RHCE*ce, RHCE*CE and RHCE*cE. Expression of these antigens may be regulated at both the qualitative and quantitative levels by additional rare variants, which are organized and described within the International Society of Blood Transfusion (ISBT) Blood Group Database, resulting in partial and weak antigen expression, respectively. In this study, we describe a new splicing variant of the RHCE gene discovered for a donor of European ancestry who was referred to our laboratory for a very weak C phenotype. His red blood cells (RBCs) typed D+, weakly C+, E−, c+, e+ (RH:1,w2,-3,4,5). Automated serologic typing for C was performed by conventional hemagglutination techniques deployed on blood donation qualification platforms, that is, automated microplate testing (PK7400 instrument (Beckman Coulter, Villepinte, France) with P3x25513 G8 + MS24 or DGCO2 anti-C, Diagast, Loos, France), gel testing (ERYTRA with MS24, Grifols, Barcelona, Spain; and IH1000 with MS24 and MS273 + P3x25513G8 anti-C, Bio-Rad—Hercules, CA, USA). Genomic DNA was isolated from whole blood by using an automated method (EZ2 Connect, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Genotyping was performed for the DNA sample using the HEA BeadChip array (BioArray Solutions/Immucor-Werfen, Warren, New Jersey, USA) and Sanger sequencing (RHCE exons 1 to 10 and flanking regions—3500 Dx genetic analyzer, Thermo Fisher, Les Ulis, France), as previously described.1, 2 The effect of the variant was predicted in silico using SpliceAI3 and SPiP4 and reporter minigene splicing assay was performed to further investigate the effect of the variant in an in vitro model.5 The donor's RBCs were non-reactive with DGCO2, ambiguous with P3x25513 G8 + MS24, non-reactive with MS273 + P3x25513 G8, and very weakly positive with MS24. These observations suggested a massive weakening of RH2 (C) antigen expression. RHCE Beadchip analysis identified heterozygous positions in exons 1 and 2 defining both the RHCE*C and RHCE*c alleles and homozygosity for RHCE*e in exon 5; while sequencing analyses have also enabled the identification of a novel, heterozygous c.149-8C>G single nucleotide variant (SNV; unknown in dbSNP and gnomAD v4.1) in RHCE intron 1 close to the dinucleotide acceptor splice site. Because of the weak C phenotype, this variant was preferentially assigned to the RHCE*Ce allele: RHCE*02(149-8C>G). In silico investigation predicted the creation of a novel acceptor splice site (Figure 1A), which is located seven base pairs (bp) upstream of and is thought to strongly challenge the constitutive site (SpliceAI score: 0.88 “acceptor loss”; SPiP v2.1 score: 0.842 “Alter by SPiCE”). Functional disruption of this latter site was confirmed in vitro by the minigene splicing assay (Figure 1B). Indeed, in our model, a putative transcript, which is 7-bp longer than in the wild-type condition (Figure 1B, full red vs. full black arrowheads, respectively), is not supposed to result in the translation of a normal protein expression due to the frame shift (sequencing data not shown), while a “full-length” product is not generated. This result seems to be in slight contradiction with the very weak C antigen expression observed in the donor. However, although potent for assessing the qualitative alteration due to variants, it is worth mentioning that evidently the minigene model does not perfectly mimic the physiological situations, notably at the quantitative level. Therefore, it cannot be ruled out that a minute amount of the “normal” transcript, which is sufficient for the translation of a “normal” RhCE protein, may be produced in vivo, thus accounting for the weak expression of RH2. Also, transcriptional frameshifting or alternate translational frameshifting can explain the weak detection of the RH2 antigen.6 Unfortunately, a fresh sample could not be obtained in order to characterize formally the RHCE mRNA products in the donor. In this work, we describe a novel variant RHCE*Ce allele designated RHCE*02(149-8C>G) that disrupts the expression of the C antigen in a donor of European descent. The novel RHCE*02(149-8C>G) allele has been provisionally assigned RHCE*02.52 designation by ISBT and sequence was deposited in Genbank under accession number PX849539. ISBT Blood Group Database (RH blood group system): http://blooddatabase.isbtweb.org/system/RH (accessed on January 1, 2026). Open access publication funding provided by COUPERIN CY26. The authors have disclosed no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.