Haplotype-level evidence supporting a possible role of RHce(48C)– RHD exon two recombination in the origin of the RHC alleles in the Chinese Han population

Background The RH blood group is the most polymorphic and immunogenic blood group. RHc(48C) alleles carry c.48G>C on a c-positive background, resulting in c.48C at position 48, the same base present in RHC alleles. While this phenomenon has been observed in the Chinese Han population, the carrier frequency, full-length sequence, and genetic background of the RHc(48C ) allele have not been thoroughly studied. Patients and Methods Serological samples with DCcee, DccEe, and Dccee phenotypes were used for RHc(48C) allele screening. RHCE genotyping by PCR-SSP was conducted to detect key mutations in 83 Chinese patients. RHCE , RHD , and RHAG analysis was performed via PacBio sequencing. 3D models of wild-type and mutant Rhce were generated using SWISS-MODEL, and structural changes were predicted using DynaMut and Missense3D. Additionally, PCR-SSP was performed on 316 Chinese Han samples to determine the RHc(48C) carrier frequency. Results RHc(48C) was detected in 11/40 DCcee, 15/38 DccEe, and 4/5 Dccee samples. PacBio sequencing revealed two RHce(48C) haplotypes, which resembled RHC but lacked the RHD -shared segment present in RHC . In silico analysis predicted that the p.W16C substitution reduces the cavity volume by 80.784 Å 3 and forms a new hydrogen bond. The carrier frequency of RHc(48C) was 2.5% (8/316); all eight carriers were heterozygous, corresponding to an allele frequency of 1.27% (8/632). Conclusion The two RHce(48C) haplotypes identified by long-read sequencing showed SNV patterns closely related to those of RHC alleles, but they lacked the RHD -shared segment found in RHC alleles. This pattern suggests that nonreciprocal recombination between RHce(48C) and RHD exon 2 may have contributed to the origin of RHC .