A novel intronic variant <i>ABO</i> * <i>AW</i> allele resulting in weak A expression

While the International Society of Blood Transfusion (ISBT) currently recognizes over 250 distinct ABO alleles, only four intronic variants have been designated with allele names associated with weak A phenotype.1 With rapid advances in molecular techniques, such as next-generation sequencing, the identification of additional molecular variants associated with Aweak phenotype has accelerated especially in coding regions. In this report, we describe a novel intronic alteration associated with weak A antigen expression. Serologic ABO typing was performed on four different patients using monoclonal reagents: anti-A (clone Brima-I; Seraclone® A003), anti-B (clones ES4, LB-2; Seraclone® B005), and anti-A,B (clones Birma-I, ES4, ES15); and A1, A2, and B cells (Immucor, Inc., Norcross, GA, USA) along with anti-A1 lectin (Dolichos biflorus; Ortho Clinical Diagnostics, Inc., Raritan, NJ, USA). Serologic testing for Sample 4 was conducted at an outside institution. Genomic DNA was extracted from EDTA-anticoagulated blood samples using the EZ1&2 DNA Blood 350uL Kit (Qiagen, Hilden, Germany). ABO genotyping was performed by Sanger sequencing of exons 2 through 7, including adjacent intronic flanking regions, using the SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Also, Sanger sequencing of proximal promoter, exon 1, and intron 1 enhancer were performed and analyzed with SeqScape software (SeqScape v4, Thermo Fisher Scientific, Waltham, MA, USA). The ABO*A1.01 (NG_006669.1) served as the reference sequence. To phase the novel intronic variant, allele-specific PCR (AS-PCR) leveraging c.261G>delG was used to amplify non-O.01 and O.01 alleles, followed by Sanger sequencing (Samples 1, 2, and 4). For Sample 3 lacking ABO*O.01, Sanger sequencing was performed with primer utilizing ABO*O.02-associated c.526C>G variant. The novel variant was assessed with in silico predictor, SpliceAI. This study was reviewed and approved by the WCG Institutional Review Board. From January 2024 to August 2025, four patients with weak A phenotype were identified and found to be heterozygous for ABO*A(375-14A) allele by ABO sequencing. Table 1 summarizes their clinical and laboratory characteristics. All patients were of European descent with no reported history of transfusion. Three females and one male were included, with an age range of 23–58 years. All samples showed ABO discrepancies with weak A expression and mixed field (mf) on forward typing, while reverse typing clearly indicated group A. Samples 1 and 4 were submitted for prenatal workup and demonstrated weak A antigen expression with mf reactions. Anti-A1 lectin testing for Sample 1 was negative. Molecular testing results for both samples revealed ABO*O.01.01 and ABO*A allele with the novel intron 6 variant c.375-14C>A. Sample 2, submitted for preoperative evaluation prior to myomectomy, showed weak positive mf reaction with anti-A and weak-1+ mf reaction with anti-A,B. Sequencing identified variants associated with the ABO*O.02.01 and ABO*A alleles, and the novel variant, c.375-14C>A. Sample 3 from a patient undergoing preoperative testing for L4–L5 lumbar fusion showed weak A expression with mf reactions to anti-A and anti-A,B. Anti-A1 lectin testing was negative. Genotyping revealed the novel alteration in noncoding region, c.375-14C>A, along with variants consistent with the ABO*O.01.02 and ABO*A alleles. For all four samples, sequencing of AS-PCR products phased the variant c.375-14A to ABO*A allele. SpliceAI showed the Δ scores of 0.31 and 0.20 for predicting the splice acceptor site loss and gain, respectively. Both scores were equal or greater than 0.20 (high recall) but below the recommended threshold of 0.50.2 We report a novel ABO*AW allele defined by c.375-14A (GenBank accession number: OK048724), identified in four patients, all of whom exhibited weak A antigen expression with mf reaction on forward typing. This variant is not consistent with the conventional ABO*A1.01 allele, and the allele can be provisionally described as ABO*A(375-14A). Although this intronic variant was presented in two separate poster presentations in 2022,3, 4 each described only a single case, suggesting a possible link between the variant and weakened A antigen expression. To our knowledge, no peer-reviewed publication has yet characterized this variant in detail. The majority of ABO allele designations by ISBT are attributed to exonic changes, with only a few intronic variants in noncoding regions recognized, believed to affect splicing.1 The rare intronic variants associated with Aweak phenotype listed on the ISBT website are located in introns 2, 4, or 6, all involving canonical splice site alterations within five base pairs of the nearest exon. Although several new alleles associated with Aweak phenotype have been reported,5 only one involves an intronic variant, located in the intron 6 splice acceptor region, just five base pairs from the exon. In contrast, the c.375-14A variant we report is in intron 6 and notably lies significantly farther from the exon–intron boundary than previously designated intronic variants, with in silico analysis suggesting a potential impact on splicing, consistent with weak A phenotype. While not considered a deep intronic variant by conventional criteria, its relative distance from the splice junction compared to other ISBT-listed alleles raises the question of whether its location has additional functional or clinical relevance. The authors have nothing to report. The authors have disclosed no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.