Molecular characterization of RhD-negative Indians: development of RHD genotyping strategies: Indian RHD typing strategy

Background - Accurate determination of RhD antigen status is clinically essential. Due to the limitations of serological testing, molecular methods are required for reliable RHD typing. RHD genotyping strategy depends on understanding the molecular background underlying the RhD-negative phenotype in a population. This study aimed to characterize the molecular basis of RhD negativity in the Indian population and to develop an appropriate DNA-based diagnostic strategy for accurate RHD typing. Materials and methods - A total of 3,023 serologically RhD-negative individuals from across India were typed for D, C, c, E, and e antigens. Molecular analysis was performed using an indigenously developed RHD-specific multiplex PCR for detecting weak D type 150, quantitative multiplex PCR of short fluorescent fragments (QMPSF) for screening RHD-CE-D hybrid alleles, and Sanger sequencing for unresolved cases. Results - RhD variants were identified in 1.16% (No.=35) of apparently RhD-negative individuals, all of whom expressed C and/or E antigens. QMPSF analysis confirmed complete RHD gene deletion in 96.9% (2,929/3,023) of RhD-negative samples. RHD-CE-D hybrid alleles (RHD-CE (3-9)-D, RHD-CE (3-8)-D, and RHD-CE(4-9)-D) and single-nucleotide variants were detected in 1.74% (No.=52) and 0.23% (No.=7), respectively. Discussion - This study outlines the molecular mechanisms underlying the RhD-negative phenotype in the Indian population. Complete RHD gene deletion was identified as the predominant mechanism, followed by the presence of RHD-CE-D hybrid alleles. Based on these findings, three population-specific molecular strategies are proposed to enable accurate determination of RhD status in Indians.