Identification of a novel <scp>c.506A&gt;C</scp> variant on the <i>ABO</i>*<i>A1.02</i> allele in a <scp>Chinese</scp> individual

Identification of the ABO blood group is crucial for ensuring the safety of blood transfusion and transplantation. Discrepancies in serologic forward and reverse typing are often indicative of ABO subtypes. In 1990, Yamamoto et al. made a significant contribution by cloning and elucidating the ABO gene,1 enabling genotype analysis of the ABO blood group. Combined serologic and genotypic testing can lead to a deeper understanding of how ABO subtypes are formed at the molecular level. This report presents a case of Aw subtype, in which the genotype was identified using third-generation long-read sequencing. This subtype is characterized by a new allele with c.506A>C (rs number not available in dbSNP) variant on the ABO*A1.02 for one haplotype and a hybrid allele of ABO*O.01.02-ABO*A1.01 for the other haplotype. The proband in this study was a 29-year-old female who visited a general outpatient clinic. Her informed consent was obtained to collect 3–5 mL of EDTA-anticoagulated whole blood. The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (2023-KY-0870-003). Serologic tests were performed using the tube method, and the following reagents were used: anti-A, anti-B (Beijing Jinhao), anti-A1, anti-H (Sanquin), A1c, Bc, and Oc (Changchun Bioxun), and A2c (Immucor). DNA extraction was carried out using the Blood Genome Kit (Beijing TIANGEN) to obtain the genomic DNA of the proband. The extracted sample was then sent to Xi'an Haorui Gene Technologies Ltd for third-generation long-read sequencing, and the protocol used for sequencing was based on the method established by Wang et al.2 The International Society of Blood Transfusion (ISBT)-recommended NG_006669 was used as the reference genomic sequence for comparison. To understand the effect of amino acid substitutions on glycosyltransferase A (GTA) structure and function, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) was also queried. Serological analysis (Table 1) revealed weak expression of A antigen (1+) in forward typing, with weak anti-A1 antibody (1+) detected in reverse typing, suggesting that the proband has an Aw subtype. Third-generation long-read sequencing results demonstrated that one haplotype of the proband exhibited c.467C>T, c.506A>C compared to A1.01. These variants formed a new ABO allele, which has been assigned GenBank accession number OR538568. The other haplotype of the proband displayed c.106G>T, c.188G>A, c.189C>T, c.220C>T, c.261delG, and c.297A>G. Based on the presence of the c.261delG variant, it is inferred that the haplotype belongs to the O.01.XX group. The allelic variations observed in exons 1–6 corresponded to the O.01.02 allele, whereas exon 7 matched either the A1.01 or O.01.01 allele, indicating a recombination event between exons 6 and 7. Third-generation long-read sequencing pinpointed the potential recombination breakpoint to a region extending from nucleotide 1014 in intron 6 to nucleotide 645 in exon 7, spanning a sequence length of 310 base pairs. This hybrid allele was initially reported by Suzuki and colleagues,3 who provided sequencing details for exons 6 and 7, as well as intron 6, however, sequence data for exons 1–5 were not included in their report. Additionally, this allele was not found in Genbank, Erythrogene, and ISBT ABO allele database. The c.467C>T variant (p.Pro156Leu) is a common polymorphic site in the Chinese population, and it does not significantly affect the structure and activity of the GTA enzyme.4 However, the p.Gln169Pro caused by c.506A>C is located in the Rossmann fold region, which is close to the N-terminal domain and primarily associated with sugar donor binding.5 As predicted by PolyPhen-2 (“probably damaging” with a score of 0.997 by HumDiv and 0.984 by HumVar), the substitution is probably to impair protein function due to its impact on the local structure of the Rossmann fold region, which likely reduces the recognition of the UDP-GalNAc donor substrate by the GTA enzyme. As a result, it is hypothesized that p.Gln169Pro ultimately leads to a decrease in GTA enzyme activity. Our results suggest that c.467C>T and c.506A>C form a novel ABO allele, and that the c.506A>C variant results in an alteration of p.Gln169Pro in the Rossmann fold region, which may be responsible for the reduced activity of GTA synthesized by the AW.new allele. The authors declare no conflicts of interest.