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

Abstract

Abstract 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.