Abstract: Objective This study aims to conduct a genotyping analysis of the RHCE blood-group system among regular blood donors in the Shandong Peninsula using molecular biology techniques. It evaluates the reliability of predicting C/c and E/e antigen phenotypes based on two single-nucleotide polymorphisms (SNPs) in the RHCE gene, namely c.307C>T (Exon 2) and c.676C>G (Exon 5). Additionally, it analyzes the genetic characteristics of D-phenotypes and individuals with weak RhCE antigen expression using third-generation single-molecule sequencing technology, with the goal of elucidating the regional distribution patterns of the RHCE gene and its variants. Methods A total of 13 691 regular blood donors from the Shandong Peninsula were selected as study subjects. The TaqMan-MGB genotyping method, combined with an improved U-shaped microplate method, was employed for typing the C, c, E, and e antigens of the RHCE gene. The differences between the results obtained by the two methods were compared. For the four rare D-phenotypic samples and two samples with weak RhCE antigen expression identified during screening, full-length haplotype sequencing was carried out using the third-generation sequencing platform to precisely identify variant genotypes. Results Both methods were used to test 9 411 donors. Discrepancies were mainly observed in C/c antigen typing, with 74 inconsistent specimens detected. Specifically, there were 31 cases where the serological Cc phenotype matched the cc genotype, 21 cases where the serological CC phenotype matched the cc genotype, and 22 cases where the serological cc phenotype matched the Cc genotype. E/e antigen typing showed high consistency, with only three discrepancies: two cases where the serological ee phenotype matched the Ee genotype and one case where the serological EE phenotype matched the Ee genotype. Third-generation sequencing revealed that among the four rare D- phenotypic samples and two samples with weak RhCE antigen expression, four D-silent alleles (RHCE*02N.07, RHCE*02N.08, RHCE*02N.14, c.940-3C>G) and two RHCE recombinant variant genes (RHCE-D(2)-CE-D(9)-CE, RHCE-D(4-5)-CE) were identified. Conclusion The TaqMan-MGB method using a single SNP site is relatively reliable for predicting E/e antigens, but for more accurate prediction of C/c antigens, additional SNPs should be included. The improved U-shaped microplate method can be applied for RhCE typing in blood collection and supply institutions. The RHCE gene is prone to structural variations such as recombination and inversion with the RHD gene, which are difficult to accurately detect using conventional methods. Third - generation single-molecule full-length sequencing provides a reliable approach for the precise identification of RHCE variant genotypes, offering valuable data support for regional precision transfusion and blood-group genetics research.

Key words: RHCE gene, Improved U-shaped microplate method, TaqMan-MGB, Single-molecule sequencing technology