Genetic and bioinformatics analysis of an individual with Am phenotype due to variant of A-glycosyltransferase enzyme gene

Objective To investigate the serological characteristics and molecular mechanism of an individual with Am phenotype. Methods The sample with ABO blood group discrepancy was confirmed by serological techniques. The full coding and flanking regions of the ABO gene including intron 1 transcription factor binding site were identified through direct sequencing of PCR-amplified products. PCR products of exon 6-7 were validated to isolate the ABO gene haplotypes by cloning and sequencing individual colonies. Bioinformatics software was used to analyze the structure of the mutant protein. Results The serologic characteristics of ABO blood typing showed the rare Am phenotype. The c.467C/T and c.912C/A heterozygous sites in exon 7 were identified by direct sequencing analysis. Further TA cloning and sequencing revealed that the patient carried an ABO* O.01.01 allele and a novel ABO*A allele. The new allele sequence had one nucleotide alteration (C>A) at position 912 on the background of the ABO*A1.02 allele. The new allele sequence has been included in the GenBank database with the entry number JX489776. The c.912C>A mutation was predicted to be “probably damaging” and “deleterious” by PolyPhen2 and PROVEAN algorithms, respectively. The free energy change (ΔΔG) value predicted it to have a destabilizing effect on the GTA protein. Meanwhile, modeling of the 3D structure predicted that the p.S304R amino acid substitution may alter the hydrogen bond of the GTA protein. Conclusion The p.S304R substitution of α-1,3-N-acetylgalactosaminyltransferase gene may reduce the antigen expression owing to a greatly destabilizing effect on the structure and function of the GTA protein.