Identification of a novel <i>B</i> allele with a c.140_143del on the <i>ABO*B.01</i> allele

The ABO blood group system is crucial for both transfusion and transplantation. However, discrepancies between forward and reverse typing results can occur during serological testing, often due to genetic variations.1 Recent discoveries of novel ABO alleles through long-read sequencing are enhancing the characterization of A/B subtypes and helping to clarify ambiguities within the ABO blood group.2 This report highlights a novel B allele marked by a nucleotide deletion (c.140_143del, p.Gly47ValfsTer76) in exon 3 of the ABO*B.01 allele, identified in a 31-year-old Chinese female with weak expression of antigen B. Serologic investigations, including forward and reverse typing tests, were performed using standard methods to identify the ABO phenotype. For forward typing, anti-A, anti-B, anti-AB, and anti-H antibodies from Shanghai Hemo-pharmaceutical Biological Company were employed in the serologic tests. In reverse typing, known A1, B, and O phenotype cells (also from Shanghai Hemo-pharmaceutical Biological Company) were used to determine the antibodies present in the serum. The absorption and elution test was conducted to detect the B antigen, as described in the AABB technical manual.3 Genomic DNA was extracted from peripheral blood using the MagNA Pure LC DNA Isolation Kit I (Roche). To sequence the ABO gene haplotypes, which encompass over 23 kb of regulatory regions, overlapping fragments were sequenced with PacBio technology on a Sequel II/IIe system.4 Subsequently, the Flye assembler was employed to generate haplotype FASTA sequences for both alleles in the study samples. Haplotype sequences were compared to the reference allele ABO*A1.01 (GenBank: NG_006669.1) to identify variations. The serological and molecular findings are summarized in Table 1. The proband, a blood donor at our center, underwent further ABO gene sequencing due to discrepancies between the forward and reverse typing results (Table 1). The forward typing indicated an A-like phenotype, whereas the reverse typing suggested an AB profile. Additionally, the adsorption and elution test confirmed the presence of B antigen on the proband's red blood cells. Third-generation long-read sequencing revealed that the proband is a heterozygote with one ABO*A1.02 allele and a novel B allele (Table 1). The novel B allele contains the following variants: c.140_143del (p.Gly47ValfsTer76), c.297A>G, c.526C>G (p.Arg176Gly), c.657C>T (p.Gly235Ser), c.703G>A, c.796C>A (p.Leu266Met), c.803G>C (p.Gly268Ala), and c.930G>A. Notably, the deletion of TGGA at c.140–143 in exon 3 of the ABO*B.01 allele results in a frameshift, leading to a premature stop codon at c.230–232. This truncates the protein to only 75 amino acids. The sequence of this novel allele has been submitted to GenBank and assigned the accession number MW960366. In summary, the genetic analysis identified a novel B allele with a c.140_143del (p.Gly47ValfsTer76) variant in the ABO*B.01 allele, which is associated with weak B antigen expression. This frameshift mutation introduces a premature stop codon, theoretically truncating the B-glycosyltransferase and ablating critical catalytic residues (positions 176, 235, 266, and 268) essential for enzymatic activity. Notably, serological testing via adsorption-elution confirmed residual B antigen expression, suggesting potential functionality of the truncated protein. This paradox mirrors findings from our prior study, where a c.36_39delG variant in the ABO*A1.02 allele similarly resulted in weak A antigen detection despite predicted non-functional glycosyltransferases.5 Both cases share a similar pattern: the identified variants are predicted to produce truncated and non-functional glycosyltransferases, yet the corresponding antigens were still detected, albeit at weaker levels. We hypothesize that mechanisms such as leaky translation (yielding trace full-length enzyme) or alternative splicing (circumventing the frameshift) may underlie the observed antigen expression, though experimental validation is warranted. These cases underscore the complexity of ABO genotype–phenotype correlations and emphasize the necessity of integrating serological, molecular, and functional analyses to resolve such discrepancies. This work was supported by the Basic and Applied Basic Research Foundation of Guangdong Province (No. 2022A1515110195), the Natural Science Foundation of Shenzhen Municipality (No. JCYJ20230807154000002), the Shenzhen Key Medical Discipline Construction Fund (No. SZXK070), and the Sanming Project of Medicine in Shenzhen (No. SZSM202311032). The authors have disclosed no conflicts of interest. Figure S1. The observed sequencing pattern of the ABO*A1.02 and the novel B alleles identified through long-read sequencing. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.