[Genetic sequencing and analysis of a patient with ABO blood group Bw28 subtype]

To investigate the gene sequence, protein structure, and impact of the variant on glycosyltransferase B (GTB) structural stability in a patient with a ABO*Bw.28 subtype.

A 33-year-old man with mixed hemorrhoids, confirmed as ABO*Bw.28 by genetic sequencing at the Second Affiliated Hospital of Wenzhou Medical University on December 8, 2022 was selected as study subject. ABO phenotyping was performed using tube test, microcolumn gel assay, and absorption-elution test. Genetic testing was conducted by PCR-direct sequencing, with haplotype confirmation by TOPO-TA cloning. Homology modeling was performed using PyMOL, and 100 ns molecular dynamics simulations of wild-type and mutant GTB were conducted using GROMACS to assess root-mean-square deviation (RMSD), radius of gyration (Rg), solventaccessible surface area (SASA), hydrogen bonds, and binding free energy. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2021-K-289-01).

Serological tests revealed characteristics of Bw subtype. ABO gene sequencing identified a c.541T>C variant in exon 7, resulting in p.Trp181Arg substitution. Haplotype analysis confirmed presence of the ABO*Bw.28 allele. Molecular modeling showed that the Trp181Arg substitution largely abolished hydrophobic interactions with the UDP-galactose (UDP-Gal) ligand and reduced the number of active site hydrogen bonds (wildtype: 2 ~ 8; variant: 1 ~ 6). Molecular dynamics simulation suggested increased binding free energy from 11.586 kcal/mol (wild type) to 4.680 kcal/mol (variant), indicating a disrupted local hydrogen bond network and reduced conformational stability and substrate binding affinity.

This study has identified a case with a c.541T>C (p.Trp181Arg) ABO variant. The variant may disrupt the hydrophobic interactions and local hydrogen bond network, reducing GTB catalytic domain stability and B antigen expression, thus providing a molecular basis for the Bw.28 subtype.