The Rh blood group system is highly polymorphic: 56 antigens described to-date.1 Antigen immunogenicity and prevalence, contribute to clinical relevance and especially in pregnancy. Here, we report a novel RHCE allele found in two unrelated probands: a 23-year-old pregnant woman of European descent in France (S1-A) whose red blood cells (RBCs) typed D + C-E + wc + e + serologically and a White donor in the USA (S2) whose RBCs typed C-E- serologically but predicted E-positive by genotyping. A sample from the father of S1-A, a healthy blood donor (S1-B), was also tested. For samples S1-A and S1-B, serologic typing was performed by automated column agglutination technology (CAT) with IH-1000 (Bio-Rad, Hercules, CA), Vision Max (Quidel Ortho, San Diego, CA) and Qwalys 3 Evo (Diagast, Loos, France) instruments and manual CAT with DG Gel Rh Pheno (Grifols, Barcelona, Spain). S2 was tested on automated PK7300 (Beckman Coulter, Brea, CA) with Diagast reagents and in tube with Bio-Rad reagents. Genomic DNA was extracted from whole blood. S1-A was tested by: in-house Real-Time PCR assays, RHCE BeadChip (BioArray Solutions/Werfen, Warren, NJ) and Sanger sequencing of RHCE exons 1–10 and RHD exon 3 on a 3500 Dx genetic analyzer, (Thermo Fisher Scientific, Waltham, MA). RHCE exon 3 of S1-B was Sanger sequenced. S2 was tested by: PreciseType HEA and RHCE BeadChip, Sanger sequencing of RHCE exons 1 to 10, targeted RH next-generation sequencing (NGS) (MiSeq Illumina, San Diego, CA), and RHCE*E-specific Sanger sequencing for phasing. By automation S1-A RBCs typed E + W with Anti-E on IH-1000, Vision Max, and Qwalys Evo, and E negative by manual testing. See Table 1 for anti-E testing on all probands. S1-A RBCs also typed D+, C–, c+ and e+ with no ambiguity. By real-time PCR, E (c.676C) and e (c.676G) were present (E/e). RHCE*cEIV,2 frequent weak E allele in individuals of African and European descent, was excluded. RHCE Beadchip results were consistent with these findings. By Sanger sequencing, c/c, and c.676C/G (E/e) were detected. A heterozygous c.341G>A change (rs1238030431) was found in RHCE exon 3 and no other changes in sequenced regions. S1-A RHD exon 3 was also sequenced and no changes from conventional were found. RBCs from S1-B typed E + e + with multiple anti-E; and sequencing of RHCE exon 3 found no changes from wild type. S2 RBCs typed E- negative by automation and by tube testing, but was predicted E+ by PreciseType HEA. RHCE BeadChip analysis showed cE/ce, with Sanger and NGS identifying c.341G>A in heterozygosity. c.341A was found in cis with c.676C (E) by E-specific sequencing. S2 RH NGS showed RHD*01 and RHD*01 N.01. The c.341G>A change is predicted to encode p.Arg114Gln, located in the 4th transmembrane domain of the RhCE protein, in close proximity with p.226Pro (Figure S1).3 The same variant is responsible for RHD*weak D type 25 and very different phenotypes for changes at amino acid 114 have been reported, suggesting conformational sensitivity in this region.4 RHCE*ce(341A) was described in 2009 by Hustinx et al.5 in a Caucasian patient with an e+W, JAL+ phenotype. The clinical significance of the variant was uncertain, in the absence of known immunization. RHCE*cE(341A) has been previously described, but with no associated serology.6 RBCs from S1-A and S2 were not available for JAL testing. In S1-A, the change was inferred to be on a RHCE*cE background based on the patient's E + w, e+ phenotype and also on the absence of the variant allele or a E + W RBC type in S1-B. In S2, RHCE*E-specific Sanger sequencing confirmed RHCE*cE(341A), explaining the discrepancy between phenotype and genotype. These cases highlight the value of having multiple samples to confirm serologic phenotype of novel variants. The sequence was deposited in Genbank (Accession number PX392379), and given the ISBT designation RHCE*03.37. The effect of c.341A on the expression of c could not be assessed, due to RHCE*ce in trans. Although no anti-D or anti-e have been reported associated with RHD*weak D type 25 or RHCE*ce341A, respectively, the change is predicted to be located in the transmembrane region of RhCE, in close proximity with p.226. For transfusion, E-negative RBC units should be considered if transfusion is necessary for female patients of childbearing age with RHCE*cE(341A) in the absence of a conventional RHCE*E in-trans. New York Blood Center RHCE website https://www.nybce.org/national-center-for-blood-group-genomics/rhce-table/ accessed January 11, 2026. RHeference https://www.rheference.org/ accessed January 11, 2026. ISBT Blood Group Database https://blooddatabase.isbtweb.org/ accessed January 11, 2026. AF, RV, LR, CH, BSS, RWV and SV collected and analyzed the data, AF and RV prepared the original draft, OC, RF, CR and performed serologic testing, GOG, AB and CT performed molecular testing; and all authors reviewed and edited the manuscript. 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. FIGURE S1: Illustration of the proximity between residue p.114 in blue and p.226 in red in 3 dimensions. Based on the cryo-electromiscroscopy structure by Vallese et al.,3 3D representation of the RhAG2RhCE trimer, shown from above. The RhAG subunits are shown in gray and RhCE in light green, with the residue p.114Gln of the novel variant in blue and p.226Pro typical of E antigen in red. 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