Abstract
BACKGROUND: The D-- phenotype is an extremely rare RhCE variant characterized by the complete absence of RhCE antigens in the Rh blood group system and is associated with a highly complex molecular mechanism. In this study, several D-- individuals with discrepancies between phenotypic and genotypic results were analyzed, and a complex RH gene inversion-recombination variant with a novel breakpoint was identified via multi-platform analyses. MATERIALS AND METHODS: Six D-- individuals from the Chinese population were collected. The full-length sequences of the RHD, RHCE, and RHAG genes were amplified, and long-read haplotype sequencing was performed using PacBio technology. Complex structural variations in the RH gene were detected through Bionano Optical Genome Mapping (OGM). Lastly, fusion regions in the RHCE haplotype were amplified and sequenced using PacBio technology and PCR-SBT. RESULTS: PacBio third-generation haplotype sequencing (TGS) suggested the presence of potential structural variants. Subsequently, Bionano optical genome mapping (OGM) identified a complex gene inversion and recombination structural variant, designated RHCE*Ce(1-2)-D(3-10)-TMEM50A-Ce(10-8)-Ce(3-10). Moreover, a novel fusion breakpoint was validated by PacBio haplotype sequencing and PCR sequencing-based typing (PCR-SBT). The structural inversion, which originated within intron 7 of the RHCE gene at chr1: 25377650, was rejoined to intron 2 at chr1: 25404500, forming a novel breakpoint. Finally, all D-- individuals in this study harbored this complex structural variation with an identical breakpoint position. CONCLUSION: The RH gene inversion and recombination may represent a prevalent molecular basis for the D-- phenotype in the Chinese population. These findings expand our understanding of the molecular mechanisms underlying the Rh blood group system and hold significant implications for transfusion safety in individuals with this rare D-- phenotype.