Discovered by Karl Landsteiner in 1900, the ABO blood type is the most important blood type for transfusions [1]. For safe blood transfusions, cell type and serum type test results of the patient should match. If the results of the tests are inconsistent, we call it ABO discrepancy. It is essential to resolve ABO discrepancies in clinical laboratories for safe blood transfusion [2]. Weak ABO subgroups are the main cause of ABO discrepancy. Various genotyping methods can be used to confirm the ABO blood group. Recently, next-generation sequencing has been used for blood grouping. However, this technique is expensive [3]. Here, we describe a weak B phenotype harboring B101/O04-variant alleles observed in a Korean family, and suggest a practical algorithm to work-up ABO subgroups without using whole-genome sequencing. The Institutional Review Board of Chonnam National University Hwasun Hospital (CNUHH), Hwasun, Korea (CNUHH-2022-096) approved this study.

A healthy 24-year-old woman visited the Chonnam National University Hospital to confirm her blood type. We carried out serological ABO typing for her and her family. ABO cell type test was performed using the tube method with anti-A, anti-B, anti-A1, and anti-H agents (Shinyang Diagnostics, Siheung, Korea). Serum type test was performed using the tube method with A1 and B cells (Shinyang Diagnostics). The proband, her father, and her half-sister presented a weak B phenotype: trace positivity (+/–) for anti-B antibodies in cell typing and strong positivity (4+) for A1 cells in serum typing (Fig. 1A). The mother of the proband presented an A phenotype. To clarify the genetic cause of the weak B phenotype, we performed a stepwise molecular work-up. For sequencing, we extracted DNA from the EDTA sample tube with QIA symphony DSP DNA mini kit (Qiagen, Hilden, Germany). PCR products were purified using QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions.

Figure 1. Summary of our work. (A) The ABO phenotypes and genotypes of the family members with the family tree and table. The ABO phenotypes are indicated below each symbol. The arrow indicates the proband. (B) A stepwise algorithm to work-up the ABO subgroups. (C) Schematic of the genetic targets used to determine ABO genotypes and the ABO phenotypes of all family members and their ABO sequencing results (exons 6 and 7).

ABO genotyping of exons 6 and 7 was performed first. Then, the other regions were genotyped sequentially (Fig. 1B). We performed PCR amplification using the primers ABOe6F and ABOe7R, and sequenced the PCR products using the primers ABOe6R, AB-Oe7F, ABOe7SF1, and ABOe7SF2. PCR amplification protocol and the cycling conditions described by Kim et al. were followed [4]. In exons 6 and 7 of the ABO gene, we found a common B101 allele in the proband, her father, and half-sister. A rare single nucleotide variant (c.579T>C) known to be associated with the O04 allele was observed in the proband and her mother (Fig. 1C).

Then, we performed PCR amplification of ABO exon 2 to coding region of exon 7 using primers ABOe27longF and ABOe27longR, and sequenced ABO exon 2 to intron 6. PCR amplification protocol and the cycling conditions described by Huh et al. [5] were followed. Further sequencing from exon 2 to intron 6 showed an insertion variant in intron 4 (c.203+1622_1623insC), which was confirmed to have originated from the O allele using allele-specific sequencing using a specific primer for ABOc.261GspR (5´-CAA TGG GAG CCA GCC AAG GGG TCA-3´). A base ‘C’, two steps away from the 3´ end, combines with a base ‘G’ at the locus c.261, which would bind specifically to the B allele. As a result, no insertion variant was found in this sequence using the specific primer. Based on these results, we made a tentative determination of the O04-variant allele (Table 1).

Table 1 . Several substitutions of the ABO gene found in the proband indicating the B101/O04-variant allele

Exon/Intron	Location	Site number in the Reference*	Nucleotide in the Reference allele (A101)	Nucleotide substitution in the template allele	Tentative origin
Intron 2	c.98+362	18478	C	T	O04-variant allele
	c.98+369	18485	C	G	O04-variant allele
	c.98+396	18512	T	C	O04-variant allele
Intron 3	c.155+377	19274	C	T	O04-variant allele
	c.155+969	19866	T	C	O04-variant allele
	c.155+1357	20254	C	T	O04-variant allele
Intron 4	c.203+28	20424	G	C	O04-variant allele
	c.203+74	20470	A	T	O04-variant allele
	c.203+215	20611	A	G	O04-variant allele
	c.203+216	20612	A	C	O04-variant allele
	c.203+673	21069	A	T	O04-variant allele
	c.203+738	21134	T	G	O04-variant allele
	c.203+1467	21863	G	A	O04-variant allele
	c.203+1496	21892	T	C	O04-variant allele
	c.203+1511	21907	T	G	O04-variant allele
	c.203+1622_1623	22020	C	insC	O04-variant allele
Intron 5	c.239+530	22648	A	G	B101 allele
Exon 6	c.261	22694	G	delG	O04-variant allele
	c.297	22730	A	G	B101 allele
Intron 6	c.374+42	22849	G	T	B101 allele
	c.374+163	22970	C	T	B101 allele
	c.374+179	22986	T	C	B101 allele
	c.374+271	23078	A	G	B101 allele
	c.374+280	23087	C	T	B101 allele
	c.374+446	23253	A	G	B101 allele
	c.374+628	23435	A	G	B101 allele
	c.374+686	23493	C	A	B101 allele
	c.374+786	23593	A	G	B101 allele
	c.374+891	23698	A	G	B101 allele
	c.374+901	23708	G	A	B101 allele
	c.374+950	23757	A	G	B101 allele
Exon 7	c.526	24011	C	G	B101 allele
	c.579	24064	T	C	O04-variant allele
	c.657	24142	C	T	B101 allele
	c.703	24188	G	A	B101 allele
	c.796	24281	C	A	B101 allele
	c.803	24288	G	C	B101 allele
	c.930	24415	G	A	B101 allele
	c.1096	24581	G	A	B101 allele

*The nucleotide sequence of GenBank accession no. NG_006669.2 was used as a refeFtrence.

Then, we performed PCR amplification, including exon 1, adjacent ABO promoter site, and CBF/NF-Y enhancer site using primers ABOenhe1longF and ABOenhe1longR and sequenced each region. We did not find any remarkable variants for these regions [5, 6]. We followed the PCR amplification protocol and the cycling conditions described by Huh et al. [5].

Next, we focused on intron 1. Long PCR using the primers ABO+4419S and ABO+11078AS was performed as described by Sano et al. [7]. PCR amplification was performed in a final reaction volume of 50 °L, containing 25 °L of KOD One™ PCR Master Mix (TOYO BO, Osaka, Japan), 1.0 °M of each primer, and 3 °L of genomic DNA. The PCR amplification was carried out in Veriti 96-well Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The cycling conditions were: initial denaturation at 98°C for 180 seconds, 35 cycles of 98°C for 10 seconds, and 68°C for 60 seconds, followed by a final elongation at 68°C for 180 seconds. The PCR product was electrophoresed on 0.7% agarose gel. It revealed no deletion of the +5.8-kb site, including the erythroid cell-specific regulatory element in intron 1. Sequencing using the primers ABO+5.8kbseqF and ABO+5.8kbseqR showed no remarkable variants on this site. We also amplified the 9.9 kb region that has CBF/NF-Y enhancer site, the binding site of the ABO+4419S, and the GATA motif in the +5.8-kb site. The heterozygosity of the 41st nucleotide in ABO minisatellite in the PCR product was maintained, confirming that the binding site of the PCR primer and the GATA motif was not deleted.

Lastly, we focused on the +22.6-kb enhancer region within the 3´ UTR region. PCR amplification was performed using the primers ABO+22.6kbpcrF and ABO+22.6kbpcrR, and the PCR product was sequenced using the primers ABO+22.6kbseqF and ABO+22.6kbpcrR as described by Sano et al. [8]. PCR amplification was performed in a final reaction volume of 50 °L, containing 25 °L of 2x Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific), 1.0 °M of each primer, and 3 °L of genomic DNA. The PCR amplification was carried out in Veriti 96-well Thermal Cycler (Thermo Fisher Scientific). The cycling conditions were: initial denaturation at 98°C for 180 seconds, 35 cycles of 98°C for 15 seconds, and 72°C for 60 seconds, followed by a final elongation at 72°C for 180 seconds. The PCR product was electrophoresed on 1.0% agarose gel. We did not find any deletion or nucleotide variants, or large deletion in the amplified sequences. During sequencing, the nucleotide sequence of GenBank accession no. NG_006669.2 was used as a reference. Sequencher software (Gene Codes, Ann Arbor, MI, USA) was used for sequence analysis. We described primer sequences and their targets in the ABO gene used in this study (Table 2).

Table 2 . Primer sequences and their targets in the ABO gene used in this study

Name	Primer sequence	Primer binding sites*	Purpose	Reference
ABOe6F	5´-GCTGAGTGGAGTTTCCAGGT-3´	Intron 5, 22573–22592	PCR amplification	[4]
ABOe7R	5´-AACAGGACGGACAAAGGAAA-3´	Exon 7, 24633–24652	PCR amplification	[4]
ABOe27longF	5´-TACTCACCTATTATTGGCCTTTGGTT-3´	Intron 1, 17421–17446	PCR amplification	[5]
ABOe27longR	5´-TAGGCTTCAGTTACTCACAACAGGAC-3´	Exon 7, 24645–24670	PCR amplification	[5]
ABOenhe1longF	5´-CTTACCAAAGGAGTCACACCCTCAAA-3´	Pre-enhancer region, 42–67	PCR amplification	[5]
ABOenhe1longR	5´-GAACTCAGCGATACTGAACACAGTGC-3´	Intron 1, 6119–6144	PCR amplification	[5]
ABO+4419S	5´-TGGAATTGCTGTCTCCTCTTTTAGTCC-3´	Intron 1, 9458–9484	PCR amplification	[7]
ABO+11078AS	5´-GGTCCCTCCTGACCCTGACAA-3´	Intron 1, 16070–16090	PCR amplification	[7]
ABO+22.6kbpcrF	5´-CAAGGACGAGGGCGATTTCTACTAC-3´	Exon 7, 24256–24280	PCR amplification	[8]
ABO+22.6kbpcrR	5´-CTCTGACACCCGATTGCTGCT-3´	Exon 7, 28089–28109	PCR amplification	[8]
ABOenhsF	5´-GCTCTTGCTCCTAGATGAT-3´	Enhancer, 1065–1083	CBF/NF-Y enhancer	[5]
ABOenhsR	5´-CAGGGAAGGACTTGGTTCAG-3´	Enhancer, 1591–1610	CBF/NF-Y enhancer	[5]
ABOe1sF	5´-GGCGCCGTCCCTTCCTAG-3´	Promoter, 4838–4855	Promoter, Exon 1	[5]
ABOe1sR	5´-CGAGGAGAGGCTGGAGAC-3´	Intron 1, 5350–5367	Promoter, Exon 1	[5]
ABO+5.8kbseqF	5´-TCATGTATTGCTGCGGGATAAT-3´	Intron 1, 10558–10579	+5.8-kb site	[7]
ABO+5.8kbseqR	5´-ACCATGTTCGCCAGGCTAGT-3´	Intron 1, 11257–11276	+5.8-kb site	[7]
ABOe2sF	5´-ACCATCTTGGCAGATGAAGG-3´	Intron 1, 17923–17942	Exon 2	[5]
ABOi2sR	5´-CCCCAGACTCCACACTTAG-3´	Intron 2, 18341–18359	Intron 2	[5]
ABOi2sF	5´-TTAGTCGCTTCCAGACACAG-3´	Intron 2, 18431–18450	Intron 2	[5]
ABOe3sF	5´-ACCAACAGGCAGTCTTCGTT-3´	Intron 2, 18785–18804	Exon 3	[5]
ABOi3sF1	5´-TCTTTCCAGAACATAAGGTAGG-3´	Intron 3, 19388–19409	Intron 3	[5]
ABOi3sF2	5´-GCTGGGTGGTTCACTTTGGG-3´	Intron 3, 19718–19737	Intron 3	[5]
ABOe4sF	5´-TGCCCTAAATCCTGCTCCTA-3´	Intron 3, 20294–20313	Exon 4	[5]
ABOi4sF1	5´-CCTGGGCTCAAGTGATTCTC-3´	Intron 4, 20717–20736	Intron 4	[5]
ABOi4sF2	5´-CTGTTGTTATGAGTCTGCTAC-3´	Intron 4, 21269–21289	Intron 4	[5]
ABOe5sF	5´-GCTGAATCAGAGACTCTGAG-3´	Intron 4, 21869–21888	Exon 5	[5]
ABOe5sR	5´-AAGAGACGCAAGTCAGAGAAAG-3´	Intron 5, 22290–22311	Exon 5	[5]
ABOi5sF1	5´-GAAGGTATTAGAGGGCGGTT-3´	Intron 5, 22170–22189	Intron 5	[5]
ABOi5sF2	5´-GGGTTTGTTCCTATCTCTTTG-3´	Intron 5, 22403–22423	Intron 5	[5]
ABOc.261GspR	5´-CAATGGGAGCCAGCCAAGGGGTCA-3´	Exon 6, 22693–22716	c.203+1622_1623insC	This study
ABOe6R	5´-CCACCCCACTCTGTCTTGAA-3´	Intron 6, 22884–22903	Exon 6	[4]
ABOi6sF1	5´-CGAGTGACTGTGGACATTGAG-3´	Intron 6, 22850–22870	Intron 6	[5]
ABOi6sR1	5´-CTGCCGAGAAGTCAAGTATGTGT-3´	Intron 6, 23347–23369	Intron 6	[5]
ABOi6sF2	5´-GAATGACTTACTCTTAGGAATAG-3´	Intron 6, 23408–23430	Intron 6	[5]
ABOi6sR2	5´-GGTGAAGACATAGTAGTGGAC-3´	Exon 7, 23924–23944	Intron 6	[5]
ABOe7F	5´-TCTGCTGCTCTAAGCCTTCC-3´	Exon 7, 23746–23765	Exon 7	[4]
ABOe7SF1	5´-TCCTCAGCGAGGTGGATTAC-3´	Exon 7, 24084–24103	Exon 7	[4]
ABOe7SF2	5´-ACGAAGAGAGCCACCTGAA-3´	Exon 7, 24387–24405	Exon 7	[4]
ABO+22.6kbseqF	5´-ATGGCTATTCCTGACCGTTG-3´	Exon 7, 27547–27566	+22.6-kb enhancer	[8]
ABO+22.6kbpcrR	5´-CTCTGACACCCGATTGCTGCT-3´	Exon 7, 28089–28109	+22.6-kb enhancer	[8]

*The nucleotide sequence of GenBank accession no. NG_006669.2 was used as a reference.

Whole-genome sequencing may be ideal for determining ABO genotypes [3], but it is not readily available in routine practice yet. Here, we used a stepwise procedure to identify genetic variations in the ABO gene based on previously reported causes of a weak phenotype [6-9]. We made a tentative determination of the B101/O04-variant, which harbored c.203+1622_1623insC in addition to c.579T>C in this case. The O04 allele is rarely observed, alth-ough a case of Ael02/O04 presenting as a typical O phenotype and a chimera with A102/B101/O04 have been reported in Korea [10]. A recent Chinese report suggested the potential effect of the O04 allele associated with the expression of weak A antigen in a case of B101/O04 with a weak AB phenotype [11]. However, the O04-variant allele was unlikely to be the main cause of the weak B phenotype in our case, since the father and half-sister had the common O allele, rather than the O04-variant allele, but still expressed a weak B phenotype. Regrettably, we could not clarify the cause of the weak B phenotype as it is difficult to find the exact cause of the ABO mismatch based on general PCR techniques. However, we found a new O04-variant allele that has both c.579T>C and c.203+1622_1623insC. In conclusion, we described a stepwise algorithm to work-up ABO subgroups, which can be used instead of whole-genome sequencing in a real-world laboratory.

None declared.

This study was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (grant no. NRF-2019M3E5D1A02067953) and by the Chonnam National University Hospital Biomedical Research Institute (HCRI22005).