Identification of a novel c.150dup variant on the ABO*B.01 allele associated with a weak B phenotype

Abbreviations

mf

mixed‐field

PTC

premature termination codon

RBC

red blood cell

SHPBC

Shanghai Hemo‐Pharmaceutical Biological Company

SMRT

single‐molecule real‐time

1. BACKGROUND

The ABO system is widely recognized as the most crucial blood group system in human blood transfusion and transplantation medicine. Most weak ABO subgroups arise from diverse genetic alterations within the coding region of the ABO gene, including nucleotide substitutions, deletions, and insertions—particularly in the last two exons (exons 6 and 7), which encode the catalytic domain of ABO glycosyltransferase. ¹ Here, we report a novel duplication variant (c.150dup) in exon 3 on an ABO*B.01 background, first identified in a Chinese family with a B_W phenotype.

2. BRIEF METHODS

With the informed consent of all participants, blood and saliva samples were collected from the proband and his family members. This study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University. ABO blood group typing was performed manually by the tube method using the following IgM monoclonal antibodies: anti‐A (clones: SRBC‐B3, 9113D10), anti‐B (clones: SRBC‐C1, 9621A8), and anti‐H (clone: 5B12) from Shanghai Hemo‐Pharmaceutical Biological Company (SHPBC); anti‐AB (clones: Birma‐1, ES4, ES15) from Immucor; and an ABO red blood cell kit (SHPBC). All procedures were carried out in strict accordance with the manufacturer's instructions. Salivary ABH antigens were detected using the saliva inhibition test. B‐galactosyltransferase activity was determined as described by Nagai et al. with modifications by Cai et al. ¹ Genomic DNA was isolated from fresh peripheral blood samples using the Column Blood Genomic DNA Purification Kit (Sangon Biotech). The seven exons and their flanking intronic regions of the ABO gene were amplified and sequenced as described previously. ¹ To confirm the novel variant and determine its haplotype, the full‐length ABO gene—including the upstream CBF/NF‐Y enhancer, proximal promoter, and the transcription factor binding site in intron 1—was amplified and subjected to single‐molecule real‐time (SMRT) sequencing on the PacBio Sequel II platform (Xi'an Haorui Genomics Technology Co., Ltd.), following the protocol described by Wang et al. ² Sequencing data were aligned and analyzed against the reference sequence (GenBank accession no. NG_006669) using Chromas (Technelysium, Australia) and SnapGene software (Dotmatics, USA).

3. RESULTS AND DISCUSSION

In forward ABO typing, red blood cells (RBCs) from the proband and his daughter exhibited 1+ to 2+ mixed‐field (mf) agglutination with anti‐B and anti‐AB, and 4+ agglutination with anti‐H. In reverse typing, their sera showed 4+ agglutination with A₁ cells, but no agglutination with B or O cells. Only H antigens were detected in their saliva, and serum B‐galactosyltransferase activity was undetectable by in vitro testing. These serological results are summarized in Table 1. Based on these characteristics, both the proband and his daughter were assigned to the B_W phenotype.

TABLE 1.

Results of serologic grouping and ABO gene analysis.

Sample	RBC grouping				Serum grouping				Serum B‐galactosyltransferase activity b	Antigens in saliva	Phenotype	ABO genotype
	Anti‐A	Anti‐B a	Anti‐AB a	Anti‐H	A1c	Bc	Oc	Selfc
Proband	0	1+~2+mf	1+~2+mf	4+	4+	0	0	0	<1	H	BW	ABO*B.01 (150dup)/ABO*O.01.01
Daughter	0	1+~2+w/mf	1+~2+mf	4+	4+	0	0	0	<1	H	BW	ABO*B.01(150dup)/ABO*O.01.02

Abbreviations: A₁c, A₁ cells; Bc, B cells; mf, mixed‐field; Oc, O cells; Selfc, self cells.

^a The proband and his daughter's RBCs showed 1+ agglutination with anti‐B and anti‐AB at the immediate spin phase, but 2+^mf agglutination following a 15‐min incubation at 4°C.

^b The unit of B‐transferase activity was the highest dilution of monoclonal anti‐B that caused detectable agglutination. Agglutination was negative using control group A₁ and O serum and 1:128 using control group B serum.

Sanger sequencing of the complete coding region of the proband's ABO gene identified heterozygous variants at nine positions: c.150G/dup, c.261del/G, c.297A/G, c.526C/G, c.657C/T, c.703G/A, c.796C/A, c.803G/C, and c.930G/A. The inferred genotype of the proband was ABO*O.01.01/ABO*B.01, with an additional novel heterozygous variant c.150dup in exon 3 (Figure S1). This variant was not found in the NCBI dbSNP database. Subsequent SMRT sequencing confirmed that c.150dup is located on the ABO*B.01 allele (Figure S2). No other variants were detected in the CBF/NF‐Y enhancer region, proximal promoter, the +5.8‐kb region of intron 1, and exon‐intron boundaries. Sanger sequencing further indicated that the proband's daughter carried the identical variant B allele, with a genotype of ABO*B.01(150dup)/ABO*O.01.02. The sequence of this novel B allele has been submitted to GenBank under Accession Number PV953302.

The c.150dup duplication causes a frameshift and introduces a premature termination codon (PTC), predicting a severely truncated protein of 56 amino acids [p.(Phe51ValfsTer6)]. Although frameshift variants generally lead to a complete loss of glycosyltransferase activity, low‐level expression of A or B antigens has been reported when such alterations occur in exons 1–4. ³ One proposed mechanism involves downstream methionine residues—such as those at codons 53 and 69 beyond the PTC—serving as alternative translation initiation sites. Previous studies have demonstrated that these downstream methionines can partially restore protein synthesis, although the resulting glycosyltransferase exhibits markedly reduced enzymatic activity. ⁴ Interestingly, prior reports on exon 3 frameshift variants indicate that c.106dup [p.(Val36GlyTer21)] on the ABO*A1.01 allele and c.106del [p.(Val36SerTer1)] on the ABO*B.01 allele are also associated with weak A and weak B phenotypes, respectively, ³ , ⁵ consistent with the present findings. In comparison, c.125_126insT [p.(Met43AsnTer12)] on the ABO*B.01 allele is linked to the B_el phenotype. ⁶ To clarify the functional impact of the c.150dup variant, further in vitro experiments are warranted, including flow cytometric verification of the phenotype and functional analysis following transient overexpression of the glycosyltransferase variant.

4. BRIEF SUMMARY

In conclusion, we identified a novel duplication variant (c.150dup) in exon 3 of the ABO*B.01 allele, which is associated with the B_W phenotype in the Chinese population.

CONFLICT OF INTEREST STATEMENT

The authors have disclosed no conflicts of interest.

Supporting information

Fig. S1. Sequence analysis of exon 3 in the ABO gene (proband). Sanger sequencing in both forward (Figure S1A) and reverse (Figure S1B) directions confirmed the presence of the novel heterozygous variant c.150dup. The duplication site of nucleotide G is indicated by a black arrow.

Fig. S2. Haplotype confirmation by SMRT sequencing. Long‐read sequencing of the proband's ABO gene confirms that the c.105dup variant resides on the ABO*B.01 allele.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov/nuccore/PV953302, reference number PV953302.