Abstract: Objective To investigate the serological and molecular biological characteristics of two ABO subtypes, ABO*A3.07/B.01 and ABO*BEL.03/O.01.01, analyze associated alterations in protein structure, and validate the accuracy of predicted protein structures using RamAchandran plots. Methods Blood samples from two donors were initially screened using serological techniques and detection of blood group substances in saliva. Genotyping was performed using fluorescent quantitative polymerase chain reaction (PCR), followed by sequencing of all exons of the ABO gene. Three-dimensional protein structure models were generated using AlphaFold 3.0. Comparative structural analysis was conducted using PyMOL software, and model reliability was assessed via RamAchandran plot evaluation. Results Donor 1: Serological forward typing showed weak agglutination with anti-A and no agglutination with anti-A1. Genotyping identified the subtype as ABO*A3.07/B.01. Sequence analysis identified two specific mutations, c.467C>T and c.745C>T compared to the standard sequence, resulting in amino acid changes p.Pro156Leu and p.Arg249Trp, respectively. Structural modeling indicated a reduction in hydrogen bonding following the substitution at position 249. Donor 2: Forward typing appeared as type O, while reverse typing showed weak agglutination with B cells. Genotyping identified the subtype as ABO*BEL.03/O.01. Direct sequencing detected a single mutation at c.502C>T, resulting in amino acid change p.Arg168Trp. Structural modeling indicated a reduction in hydrogen bonding following the substitution at position 168. Ramachandran plot validation confirmed the high quality of the predicted protein structure models for both subtypes. Conclusion Molecular analysis confirmed the two rare ABO subtypes as ABO*A3.07/B.01 and ABO*BEL.03/O.01.01, which were the first reported cases of blood donor population in Jinan region.

Key words: RamAchandran plots, ABO blood group subtypes, Protein structure, Voluntary blood donor