A novel missense variant c. 674T >C (p. Leu225Pro ) underlies the A el phenotype in a Chinese blood donor

The ABO system is the foremost blood group system in clinical transfusion medicine. Hence, accurate typing is of paramount importance for patient safety.1 However, the ABO gene exhibits significant polymorphism in both its coding and regulatory regions, resulting in a wide variety of subtypes that complicate this process. Ael, an extremely weak A subtype, cannot be distinguished from blood group O through routine serological tests unless adsorption and elution experiments are performed. In this study, a novel A allele was identified in a Chinese blood donor exhibiting the Ael phenotype, which was characterized by a nucleotide change (c.674T>C) in Exon 7 of the ABO gene. ABO serological typing was performed using standard techniques in accordance with the AABB Technical Manual. The reagents employed included monoclonal anti-A, anti-B, and anti-H antibodies, as well as A1 and B reagent red blood cells (Shanghai Hemopharmaceutical Biological Company, Shanghai, China). Adsorption-elution experiments were conducted using human polyclonal anti-A antibodies (Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China). Agglutination reactions were graded and interpreted as negative (0), weakly positive (1+ or 2+), or positive (3+ or 4+). Genomic DNA was isolated from Ethylenediaminetetraacetic Acid (EDTA)-anticoagulated peripheral blood using the TIANGEN Blood DNA Kit (TIANGEN Biotech, China). Sanger sequencing was performed in accordance with a previously described method.2 Furthermore, nanopore sequencing was employed to obtain a full-length haplotype of the ABO gene, based on our previous report.3 The vibrational entropy difference between the wild-type and variant A glycosyltransferase was constructed via the Dynamut server (https://biosig.lab.uq.edu.au/dynamut/). The functional impact of the variant was evaluated using PROVEAN v1.1.3 (http://provean.jcvi.org/index.php) and PolyPhen-2 v2.2.3 (http://genetics.bwh.harvard.edu/pph2/). Default parameters were employed for both tools: a cutoff score of −2.5 for PROVEAN and the humDiv model (score ≥0.85) for PolyPhen-2. One Chinese individual was serologically phenotyped as blood type O by forward typing, while reverse typing demonstrated markedly weaker agglutination with A1 red blood cells than with B red blood cells (Table 1). This serological profile was consistent with the Ael subgroup, which is characterized by extremely weak expression of A antigens. Subsequently, adsorption and elution experiments were performed. The eluent obtained from the proband's red blood cells after incubation with human polyclonal anti-A serum agglutinated reagent A red blood cells, confirming that the sample belongs to the Ael subgroup (Table 1). Sanger sequencing of the entire ABO gene coding region demonstrated that the individual had an ABO*A1.01/ABO*O.01.01 genotype and a heterozygous c.674T>C variant. To determine which haplotype carried the c.674T>C variant, the entire ABO gene, covering the full coding region, promoter region, and intron 1 enhancer region, was sequenced using nanopore sequencing. The results confirmed that the individual has an ABO*O.01.01 haplotype and an ABO*A1.01 haplotype with the c.674T>C variant, which aligns with the Sanger sequencing result. The haplotype sequence of this novel Ael allele has been submitted to GenBank with an accession number PV804810. The c.674T>C variant in ABO*A1.01 is predicted to introduce a leucine to proline substitution at amino acid position 225, which is located within the catalytic domain of the A glycosyltransferase.4 To assess the potential impact of this change on the enzyme, we performed computational analyses. Using the Dynamut server, we found a vibrational entropy difference (ΔΔSvib) of 0.292 kcal·mol−1·K−1 between the wild-type and variant enzymes, indicating an alteration in the enzyme's structural integrity. Furthermore, we employed the PROVEAN and PolyPhen-2 algorithms to evaluate the variant's functional impact. The variant was categorized as deleterious by PROVEAN, with a score of −6.961 (deleterious range: <−2.5; neutral: >−2.5); this finding was supported by PolyPhen-2, which classified it as probably damaging with a score of 1.000 (damaging range: >0.5; benign: <0.5). Collectively, these results suggest that this novel A allele generates a compromised A glycosyltransferase, leading to impaired A antigen expression. In summary, we report a novel A allele (c.674T>C) in a Chinese blood donor that causes significantly weakened A antigen expression on red blood cells, resulting in the Ael phenotype. This work was supported by the Chengdu Medical Research Fund (2025014). The authors have disclosed no conflicts of interest. Research data are not shared.