Novel regulatory variant in ABO intronic RUNX1 binding site inducing A₃phenotype

Background and Objectives Mixed-field agglutination in ABO phenotyping (A 3 , B 3 ) has been linked to genetically different blood cell populations like in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. Materials and Methods We used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A 3 B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. Results Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A -allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor’s mother, who shared the novel variant and the anti-A specific mixed-field agglutination. Conclusion We discovered a regulatory variant in the 8-bp RUNX1 motif of ABO , which extends current knowledge of three other variants affecting the same motif and also leading to A 3 or B 3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as emerging strategy for resolving cases with cryptic ABO phenotypes.