Introduction: Ael is known to be one of the weakest A subgroups and can be identified through either the adsorption-elution technique or molecular analysis. Single nucleotide variation in the ABO gene can potentially disrupt the function of ABO glycosyltransferase, resulting in decreased ABO antigen expression. Case Presentation: We reported 1 case of the missense SNV c.1024A>C in the ABO*A1.02 allele was associated with an Ael phenotype. The proband was a 19-year-old male Chinese Han blood donor who was initially stereotyped as type B. Based on the findings from absorption elution, genotype tests, and a family investigation, both the proband and his mother were classified as the AelB phenotype. An uncommon allele was detected in both the proband and his mother, differing by a single nucleotide at position 1,024 from A to C (c.1024A>C) when compared to the ABO*A1.02 allele. The c.1024A>C SNV induces an amino acid substitution, specifically p.Thr342Pro, which consequently leads to the loss of two sheets (p214–p216, p340–p344) in the wild-type A glycosyltransferase (GTA) 3D structure. The removal of these two sheets, situated at the protein’s core, implies the occurrence of an interaction within this domain that affects the stability of the protein structure. Conclusion: AelB is prone to misidentification as type B, and the accurate determination of blood type can be achieved through the integration of the adsorption-elution technique and molecular analysis.

The ABO blood group is the most important factor in blood transfusions, as it ensures a safe transfusion to the patient. ABO phenotypic expression is affected by a number of genetic events, including substitution, splice site mutations, base insertions, deletions, and hybrid alleles, which can reduce the activity of the ABO glycosyltransferase and then cause reduced or absent antigen expression in ABO blood group [1, 2]. The Ael subtype cannot be detected by anti-A or anti-A,B sera unless adsorption-elution test is performed [3]. Ael represents a rare A subtype that exhibits a low likelihood of manifestation within the Chinese Han populations. In recent years, there has been a notable surge in the study of the Ael subtype within the field of transfusion medicine, owing to the extensive utilization of molecular biology techniques [4, 5]. We report a rare A allele associated with an Ael phenotype in a Chinese Han individual.

The proband was a 19-year-old male Chinese Han blood donor. Initially, the red blood cells (RBCs) of the sample were typed as group B, but the serum sample exhibited weak anti-A activity, and the anti-A was confirmed to be anti-A1 through A2 reverse grouping cells (shown in Table 1). The presence of A antigens was confirmed through the adsorption-elution test of the RBCs using monoclonal anti-A (Shanghai Blood Bio-technology Co., Ltd., Shanghai, China). The heat elution technique (56°C, 10 min) is used to release the absorbed monoclonal anti-A. The RBCs of the proband, as detected by anti-H (Shanghai Blood Bio-technology Co., Ltd.), demonstrated a degree of agglutination similar to that seen in group B (shown in Table 1). The serologic results indicate that the proband’s phenotype is defined as AelB. The PCR-SSP analysis revealed that the ABO genotype of the proband is A/B.

Table 1.

Serological and molecular results of proband and family members

SerologyGenotypeAmino acid substitution
forward typingreverse typing cellsadsorption-elutionphenotypegenotypesequence analyze interpretation
anti-Aanti-A1anti-Banti-HA1A2BOselfA1O
Proband 4+ 1+s 1+ 3+ AelA/B ABO*A1.02+c.1024A>C/ABO*B.01 p.Thr342Pro 
Father 4+ 2+w 4+ 2+ ABO*B.01/ABO*O.01.01 
Mother 4+ 1+s 1+ 3+ AelABO*A1.02+c.1024A>C/ABO*B.01 p.Thr342Pro 
SerologyGenotypeAmino acid substitution
forward typingreverse typing cellsadsorption-elutionphenotypegenotypesequence analyze interpretation
anti-Aanti-A1anti-Banti-HA1A2BOselfA1O
Proband 4+ 1+s 1+ 3+ AelA/B ABO*A1.02+c.1024A>C/ABO*B.01 p.Thr342Pro 
Father 4+ 2+w 4+ 2+ ABO*B.01/ABO*O.01.01 
Mother 4+ 1+s 1+ 3+ AelABO*A1.02+c.1024A>C/ABO*B.01 p.Thr342Pro 

0, no agglutination; 1+∼4+, expressed in different degrees of aggregation;

w, weak reaction; s, strong reaction; /, undetected or unchanged.

There were nine sequence variants(c.297A>G; c.526C>G; c.657C>T; c.703G>A; c.796C>A; c.803G>C; c.930G>A; c.467C>T; c.1024A>C) in exons 6 and 7 according to Sanger dideoxy sequencing, which suggest the ABO genotype of the proband was ABO*A1.02/ABO*B.01 with a rare SNV (c.1024A>C). To confirm the c.1024A>C allele, PCR products of exons 6 and 7 were subcloned into the pMD18-T vector (TaKaRa, Japan) according to the manufacturer’s instructions. Plasmids were extracted from each recombinant colony and used as the template for sequencing. The primers for sequencing were I6R: 5′- GCT​GCA​TGA​ATG​ACC​TTT​CC-3′; I7F: 5′-TCT​GCT​GCT​CTA​AGC​CTT​CC-3′. The haplotype sequencing further confirmed the c.1024A>C in exon 7 based on allele ABO*A1.02 (shown in Table 1), which led to an amino acid change of p.Thr342Pro in GTA. The nucleotide sequence of this allele has been submitted to GenBank with Accession Number OR241044. Following the acquisition of informed consent, samples were collected from family members and subsequently subjected to further investigation.

Following serological testing, it was determined that the ABO blood type of the proband’s father was B, while the proband’s mother exhibited a group B with weak anti-A1 activity (shown in Table 1). The results of adsorption-elution experiment provided evidence indicating that the blood type of the proband’s father was type B, while the mother possessed the AelB blood type. Subsequent gene sequencing analysis revealed that the paternal genotype of the proband was ABO*B.01/ABO*O.01.01, while the maternal genotype was identified as ABO*A1.02+c.1024A>C/ABO*B.01 (shown in Fig. 1).

Fig. 1.

Genealogy of the ABO blood group of the proband’s family member. Conventional pedigree symbols are used to represent the family members and their interconnections. The proband is denoted by an arrow.

Fig. 1.

Genealogy of the ABO blood group of the proband’s family member. Conventional pedigree symbols are used to represent the family members and their interconnections. The proband is denoted by an arrow.

Close modal

An in vitro study was conducted to investigate the conversion of group O RBCs by glycosyltransferase in plasma, with the aim of comparing the GTA activity of group A with that of the p.Thr342Pro variant. A 10% suspension of washed group O RBCs in plasma from donors of group A and the proband were incubated at 37°C with a final concentration of 0.35 mmol/L UDP-GalNAc donor substrate (MCE, NJ, USA) and 10 mmol/L MnCl2 (MCE) for 24 h in a total volume of 250 μL [6]. Following incubation, the treated cells underwent washing and were subsequently analyzed using the monoclonal anti-A and anti-B in DiaClon ABD-Confirmation (DiaMed GmbH, Cressier, Switzerland) by column agglutination method. The wild-type GTA (the plasma from donors of group A) can convert the O type RBCs into the A type with a score of 4+ agglutination (shown in Fig. 2a), while the p.Thr342Pro variant GTA cannot convert the O type into the A type with a score of 0 agglutination (shown in Fig. 2b). The experiment was repeated three times with consistent results. The results of in vitro study are consistent with the proband’s serological results, suggesting that the p.Thr342Pro variant may play a role in the reduced A antigen expression on RBCs.

Fig. 2.

The in vitro study suggests that the p.Thr342Pro variant may impact GTA function. a The results of the DiClon ABD-Confirmation shows that the wild-type GTA can change the O type RBCs into the A type. b The p.Thr342Pro variant GTA cannot change O type to A type. A(ABO1), monoclonal anti-A; B(ABO2), monoclonal anti-B; DVI-(RH1), monoclonal anti-D; ctl, blank control.

Fig. 2.

The in vitro study suggests that the p.Thr342Pro variant may impact GTA function. a The results of the DiClon ABD-Confirmation shows that the wild-type GTA can change the O type RBCs into the A type. b The p.Thr342Pro variant GTA cannot change O type to A type. A(ABO1), monoclonal anti-A; B(ABO2), monoclonal anti-B; DVI-(RH1), monoclonal anti-D; ctl, blank control.

Close modal

To understand the effect of variants on the structure or activity of GTs produced from the allele, 3D molecular models of the single nucleotide variation (SNV) c.1024A>C were generated from the template structure of wild-type GTA (PDB code: 1LZ0) using the PyMOL software and DynaMut (https://biosig.lab.uq.edu.au/dynamut/) platform. The SNV c.1024A>C results in an amino acid substitution, specifically p.Thr342Pro. The DVD motif, a distinctive structure found in GTA, is known to coordinate with a Mn2+ ion, which is crucial for donor binding and catalysis [7]. The amino acid change (p.Thr342Pro) leads to two sheets [p214–p216, p340–p344] loss on wild-type GTA 3D structure, one of which is located downstream [p214–p216] of the DVD motif [p211–p213] (shown in Fig. 3b,c). The absence of two β-sheets in the variant GTA p.Thr342Pro may impacts the functionality of the DVD motif, preventing it from effectively interacting with Mn2+ ions to fulfill its role in donor binding and catalysis. Furthermore, we compared interatomic interactions such as hydrophobic bonds and ionic interactions between the wild-type and the p.Thr342Pro variant (shown in Fig. 3d, e). Pro342 substitution can alter interatomic contacts, including loss of ionic interactions (yellow dotted line), changes in hydrophobic contacts (green dotted lines), loss of hydrogen bonds (red dotted line), and addition of weak hydrogen bonds with (orange dotted lines) 215Glu. The 215Glu in the β-sheet ([p214–p216]) center and the changes of interatomic contacts in two β-sheets [p214–p216, p340–p344] may affect DVD motif ability and protein stability. The stability changes of the GTA variant were forecasted by utilizing DynaMut, which calculated the difference in Gibbs free energy between the ΔG variant and the ΔG wild-type protein as ΔΔG = ΔGm - ΔGw. This calculation indicates the effect of the substituted variant on protein stability. The predicted DynaMut ΔΔG value of −0.607 kcal/mol suggests that the variant destabilized the protein in comparison to the wild-type. The findings from the protein model analysis indicated that the Pro342 substitution can alter the structure and interatomic interactions of GTA, potentially impacting its stability. This impairment is likely to contribute to the significantly reduced expression of the A antigen.

Fig. 3.

3D modeling of the GTA was performed with the PyMOL software. a The variant residues is illustrated in sticks, whereas a purple sphere indicates the site of the p.Thr342Pro variant. b Structure of the wild-type GTA which the amino acid residue position with the rare SNV. c Structure of the GTA with p.Thr342Pro. The amino acid change (p.Thr342Pro) leads to two sheets [p214–p216, p340–p344] loss on wild-type GTA 3D structure, one of which is located downstream [p214–p216] of the DVD motif [p211–p213]. The interatomic interaction between wild-type (d) and variant structures (e) on the GTA structure is depicted through the visualization of wild-type and variant residues as light green sticks, along with the surrounding residues involved in interactions. The substitution of Pro342 may result in alterations in interatomic contacts.

Fig. 3.

3D modeling of the GTA was performed with the PyMOL software. a The variant residues is illustrated in sticks, whereas a purple sphere indicates the site of the p.Thr342Pro variant. b Structure of the wild-type GTA which the amino acid residue position with the rare SNV. c Structure of the GTA with p.Thr342Pro. The amino acid change (p.Thr342Pro) leads to two sheets [p214–p216, p340–p344] loss on wild-type GTA 3D structure, one of which is located downstream [p214–p216] of the DVD motif [p211–p213]. The interatomic interaction between wild-type (d) and variant structures (e) on the GTA structure is depicted through the visualization of wild-type and variant residues as light green sticks, along with the surrounding residues involved in interactions. The substitution of Pro342 may result in alterations in interatomic contacts.

Close modal

In this study, we identified an ABO variant in the Ael phenotype that may impair the function and stability of the GTA, resulting in significantly reduced synthesis of A antigens on the RBCs. The Ael subgroup can produce unexpected anti-A1 antibodies that exhibit reduced activity compared to anti-A antibodies. The ABO blood type of the proband is easily misidentified as type B, necessitating additional measures such as absorption-elution or genetic testing to determine the cause for weak anti-A1 activity of reverse typing to ensure the accuracy of results. We have detected an inherited A allele in exon 7 of the ABO gene exhibiting a variant c.1024A>C (p.Thr342Pro) in a healthy male Chinese Han with an Ael phenotype. An article reported a variant p.L319V in GTA that leads to an Ael phenotype. Although the expression of GTA on HeLa cells transfected with the c.955C>G (p. L319V) variant remains unaffected, the reduced expression of A antigens on RBCs may be attributed to the dysfunction of the variant GTA [8]. In our study, the variant p.Thr342Pro results in the loss of two sheets [p214–p216, p340–p344] surrounding the DVD motif, in contrast to the wild-type GTA. The DVD motif is a characteristic structure of GTA that coordinates to a Mn2+ ion, shown to be critical in donor binding and catalysis [9]. The β-sheet is a recurring structural motif that can be broadly categorized into parallel and antiparallel orientations, both of which are upheld by hydrogen bonding interactions between peptide chains or peptides [10]. Moreover, a hydrophilic and polar molecule threonine in residue 342 in the wild-type GTA was replaced by a hydrophobic and nonpolar molecule proline and the proline-introducing variant can lead to some changes in interatomic contacts. It was hypothesized that there may be an interaction within the DVD motif and the two β-sheets [p214–p216, p340–p344], which can influence the stability or catalysis ability of the variant GTA p.Thr342Pro. The removal of these two β-sheets [p214–p216, p340–p344] implies the disappearance of an interaction within the DVD motif that impacts the stability or catalytic activity of variant GTA, resulting in the loss of its ability to convert O type into A type. This is likely to ultimately result in the manifestation of an Ael phenotype.

The proband and his parents have provided their written informed consent to gain access to and use of confidential and personal information used to write the manuscript and publish the manuscript in a scientific journal. This study protocol was reviewed and approved by the Medical Ethics Review Committee of Xuzhou Red Cross Blood Center, approval number xzbcec-2023-2.

The authors have no conflicts of interest to declare.

This work was supported by Xuzhou Pharmaceutical and Health Project (KC22230), Jiangsu Blood Transfusion Association InTec Fund (JS2022013), in vitro study of the effect of apheresis platelets transfusion on immune function in blood recipients (GSWS2022097).

Zhaoze Ma and Mengyuan Ding contributed to the serological testing of the samples and family investigation. Yan Li performed the molecular assays and wrote the manuscript. Chenlong Wang and Liling Zhou designed the experiments and critically examined the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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