Clin Chem. 2025 Aug 8:hvaf090. doi: 10.1093/clinchem/hvaf090. Online ahead of print.
ABSTRACT
BACKGROUND: Implementation of blood group genotyping has offered substantial benefits in transfusion medicine. However, the complex molecular basis of Rh antigen expression and a high degree of sequence homology between RHD and RHCE have long limited the accuracy of blood group genotyping, highlighting the need for a more systematic characterization of existing molecular variations.
METHODS: We employed a custom target enrichment strategy to perform high-fidelity (HiFi) long-read sequencing of the RHD-RHCE region on chromosome 1 in samples obtained from 63 individuals. The resulting HiFi long-read sequences were aligned to the human reference genome GRCh38, variants were identified and phased, and allelic reference sequences were generated. Phylogenetic analyses were then performed to classify RHD-RHCE alleles and elucidate their evolutionary relationships.
RESULTS: Our approach enabled the phasing of heterozygous variants at distant loci, as well as precise characterization of tandem repeat variations and structural variants. Complete phase resolution was achieved in 76.2% of samples, yielding 96 allelic reference sequences spanning the entire RHD-RHCE region. Alleles within each phylogenetic clade exhibited a characteristic sequence pattern spanning RHD to RHCE.
CONCLUSIONS: Our findings revealed that the current Eurasian allelic pool originated from 2 distinct primordial lineages, with occasional interallelic recombination events shaping the present-day RHD-RHCE haplotype diversity. While most previous classification approaches have treated RHD and RHCE independently, our results support the notion that analyzing them as a single evolutionary unit may offer practical advantages for molecular typing approaches.
PMID:40795298 | DOI:10.1093/clinchem/hvaf090