ABO genotyping with next-generation sequencing to resolve heterogeneity in donors with serology discrepancies.

BACKGROUND: ABO subtypes are characterized by the alteration of antigens present and their expression levels on red blood cells and many are linked to genetic changes in the ABO gene. Weakened expression of antigens should be identified to prevent transfusion reactions or ABO-incompatible transplantations. Genotyping can be applied to identify subtypes to complement serologic testing. Next-generation sequencing (NGS) has shown to provide sensitive and accurate genotyping results as well as valuable cis/trans information. Here we took advantage of NGS and applied it to resolve serology discrepancies in ABO typing.

STUDY DESIGN AND METHODS: In this study, we customized capture probes targeting the entire ABO gene and sequenced on MiSeq Illumina. The subtype-causing variants were identified, and cis/trans association to ABO alleles was determined. The results from NGS, serology, and Sanger sequencing were compared.

RESULTS: Four control samples typed A, B, O, and AB were correctly genotyped. Of 24 serologically discrepant samples, subtype-causing variations were found in 20 cases, with two unresolved and two identified as weakening of ABO antibody in reverse. The types of variations include 17 known subtype alleles, one novel variant, one novel large deletion, and one microchimerism. Haplotypes encompassing Exons 6 and 7 of ABO were reconstructed in 17 of the 20 samples.

CONCLUSION: This study demonstrated a full coverage of ABO by capture-based panel, phasing analysis with NGS in ABO genotyping resolved heterogeneity with novel allele and microchimerism findings. This approach provided a more precise method for subtyping and thereby leading to safer transfusion.