A novel RHCE*01.48 allele associated with weak clone‐dependent E‐like reactivity: Serologic, molecular, and computational analysis

BACKGROUND: The polymorphic nature of the RH blood group system frequently causes serologic discrepancies relevant to transfusion practice. We describe a novel RHCE variant associated with weak, clone-dependent E-like reactivity and explore its molecular and structural correlates. METHODS: Serologic investigation was performed by standard tube testing using multiple commercial monoclonal anti-E reagents. Molecular characterization utilized Illumina next-generation sequencing (NGS). Computational analyses included I-TASSER-based model generation, integration into the 7UZQ cryogenic electron microscopy (cryo-EM) erythrocyte membrane scaffold, FoldX energy calculations, three-dimensional (3D)-Surfer surface comparison, CASTp cavity analysis, and membrane-orientation simulations. RESULTS: Donor red blood cells showed discrepant reactivity with anti-E reagents, demonstrating weak positive reactions (1+) with clones GAMA402, MS-260, and DEM1, but no reactivity with clone 906 or single-source anti-E plasma. NGS identified a novel c.680T>C variant in one of two RHCE*01 alleles on a c.676G homozygous background, predicting a p.Leu227Pro substitution. The allele was provisionally designated RHCE*01.48 by the International Society of Blood Transfusion (ISBT) (GenBank: PZ350914). The amino acid change p.Leu227Pro is immediately adjacent to the canonical residue 226 E/e determinant and modeling identified localized differences in surface topology, cavity architecture, and solvent exposure relative to the reference ce model. CONCLUSION: RHCE*01.48 is a novel RHCE allele associated with weak, clone-dependent E-like reactivity. Structural modeling generated a plausible hypothesis that localized conformational differences near the canonical E/e determinant may contribute to the observed serologic phenotype. Recognition of such variants may help resolve genotype-phenotype discrepancies and support accurate Rh typing.