INTRODUCTION
The ABO blood group system represents the most significant blood group system in humans, and plays a pivotal role in numerous fields, including transfusion medicine and organ transplantation. ABO subtypes represent further classifications within the four major blood groups (A, B, O, and AB), demonstrating notable specific frequencies within human populations. ABO subgroups are prone to being missed or misidentified during routine serologic testing, posing a significant risk to transfusion safety1. In this study, we used third-generation single-molecule sequencing technology to identify a B subgroup and discovered a novel ABO allele harboring the c.298T>A variant.
MATERIALS AND METHODS
The proband was a 26-year-old female voluntary blood donor from Yinchuan, Ningxia, China, who met all eligibility criteria for blood donation.
ABO forward grouping
Red blood cells (RBCs) were tested using anti-A, anti-B, anti-A,B (Diagast, Loos France), and anti-H reagents (Shanghai Blood Biopharmaceutical Co. Ltd., Shanghai, China) to detect the A, B, and H antigens on the RBC surface, respectively.
ABO reverse grouping and adsorption - elution
Serum was tested against A1, B, and O standard RBCs (Shanghai Blood Biopharmaceutical Co. Ltd.).
Genetic sequencing analysis
Full-length sequencing of the ABO gene was performed using third-generation single-molecule sequencing (PacBio sequencing; Xi’an Haorui Genome Technologies Co. Ltd., Fengdong New City, Xi’an, Shaanxi, China). The resulting DNA sequences were then aligned against the reference allele ABO*B.01 using SnapGene software to identify sequence variations (GraphPad, Boston, MA, USA).
PolyPhen-2 was used to predict the impact of the amino acid substitution on the structure and function of glycosyltransferase A3, yielding a score ranging from 0.0 (tolerated) to 1.0 (deleterious).
RESULTS AND DISCUSSION
The proband’s RBCs showed no agglutination with anti-A, and a weak agglutination (1+) with anti-B at 4°C. Reverse typing indicated blood type B, and the adsorption-elution test detected the presence of B antigen at all temperatures (room temperature, 4°C, 37°C). The proband is not a typical type B but exhibits a weak B phenotype. Third-generation single-molecule sequencing analysis revealed that one haplotype carries a novel variant c.298T>A (p.Phe100Ile) in exon 6, while the other haplotype is an O allele. No abnormal variants were detected in other exons, introns, or regulatory regions. The ABO genotype of the proband was determined to be ABO*Bvar/ABO*O.01.01 ( Table I). The c.298T>A variant was not detected in a randomly selected cohort of 60 blood donors. Pedigree analysis indicated that the novel c.298T>A (p.Phe100Ile) variant in the proband was inherited from the father.
Table I.
Serologic and genetic results of the proband
| Forward typing | Reverse typing | Adsorption-elution | Genetic results | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Anti-A | Anti-B | Anti-AB | Anti-H | A1 cell | B cell | O cell | A1 cell | B cell | O cell | Phenotype | Haplotype1/Variant | Haplotype2/Variant | Genotype | |
| RT | 0 | ± | 1+ | 4+ | 4+ | 0 | 0 | 0 | 1+ | 0 | Bweak | c.297A>G;c.298T>A;c.526C>G;c.657C>T;c.703G>A;c.796C>A;c.803G>C;c.930G>A | c.261delG | Bvar/ABO*O.01.01 |
| 4°C | 0 | 1+ | 2+ | 4+ | 4+ | 0 | 0 | 0 | 2+ | 0 | ||||
| 37°C | 0 | 0 | 1+ | 4+ | 4+ | 0 | 0 | 0 | 1+ | 0 | ||||
1–4+ represented different agglutination strengths; 0 represented no agglutination. Bvar : a novel ABO*B allele. RT: room temperature.
Variants in the ABO gene determine the type and quantity of antigens present on the surface of RBCs, thereby giving rise to different blood group phenotypes2,3. The c.298T>A variant in the proband results in an amino acid substitution at position 100 of the glycosyltransferase encoded by the B allele, changing phenylalanine to isoleucine. Analysis using Polyphen-2 software yielded a score of 0.998, indicating that the c.298T>A (p.Phe100Ile) variant is highly likely to disrupt the structure of the glycosyltransferase, thereby impairing its catalytic activity. This leads to weak expression of the B antigen on the proband’s RBCs, resulting in a B subgroup phenotype. The c.298T>A variant identified in this study has not been previously reported in the literature. The nucleotide sequence of this novel allele has been deposited in GenBank (NCBI accession number: PV232733). The discovery of this new allele is of significant importance for deepening our understanding of the genetic diversity within the ABO blood group system4.
CONCLUSIONS
In conclusion, we have identified a novel B allele with c.298T>A (p.Phe100Ile) in a Chinese blood donor population, and this variant is associated with a weak B subtype phenotype. This hypothesis, however, requires further validation through functional studies in vitro5.
Acknowledgments
This work was funded by The Scientific Research Project of Ningxia Health System (Grant number: 2022-NWKY-040) and the Ningxia Nature Science Foundation (Grant Number: 2025AAC030472).
Footnotes
Authors’ contributions: J-CL is responsible for experimental design, third-generation single-molecule sequencing, data interpretation, and manuscript drafting. WZ and FS conducted a critical review and gave final approval of the manuscript. X-YM, X-YB, JY and JH is responsible for phenotypic analysis.
The Authors declare no conflicts of interest.
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