Abstract: Objective To investigate the polymorphism of the RhD gene and its molecular mechanism in RhD-negative blood donors in Kunming region, and to provide data support for establishing a regional RHD gene database for blood donors. Methods A total of 218 RHD-negative samples were enrolled from November 2023 to August 2024. RhD negativity was confirmed by the Indirect Antiglobulin Test (IAT), and the RhCE phenotypes were identified using the saline test tube method. Genomic DNA was extracted from whole blood samples. RHD genotyping was performed via PCR-SSP method /SSP fluorescent PCR dye methods. For samples with undetermined genotypes, Sanger sequencing analysis of exons 1~10 of the RHD gene was conducted. Results A total of 179 cases (82.11%) were identified as serologically confirmed RhD-negative phenotypes, among which 154 cases (86.03%) carried the RHD*01N.01 genotype (complete RHD deletion) , with the predominant phenotype being ccee (87.01%). Twenty-five cases (13.97%) harbored non-functional RHD alleles, including RHD*01N.03 (20 cases), RHD*01N.16 (3 cases), RHD*01N.05 (1 cases), and RHD*01N.59 (1 cases). The main phenotype was Ccee (64%). Thirty-nine cases (15.89%) were classified as D variants, among which 34 cases belonged to the RHD*DEL1(c.1227G>A) type, all of which expressed the c antigen (comprising 27 Ccee and 7 CCee phenotypes). Four cases were identified as weak D/partial D types, including RHD* VI.3 (2 cases), RHD*weak D Type 71 (1 cases), and RHD*weak D Type108 (1 cases). Additionally, 1 case with the RHD*01/RHD*01N.01 genotype was detected, showing inconsistency between genotype and serological phenotype. The seropositivity rate of irregular antibodies (predominantly anti-D) in female blood donors with the RHD*01N.01 genotype was 9.84%. Conclusion The RhD gene exhibits significant polymorphism among initially screened RhD-negative donors in Kunming region. The proportion of true RhD-negative donors is higher than that in some other regions. The Asian-type DEL is the predominant D variant, though its overall proportion is relatively low. This study provides theoretical and data support for precision blood transfusion in individuals with RhD-negative and D variants.

Key words: RhD negative, D variants, Asian-type DEL, Gene polymorphism, Molecular mechanism