A novel B allele with c.28 + 5885C>T substitution in the erythroid cell–specific regulatory element identified in an individual with phenotype B₃

Abstract

The ABO blood group system is the most clinically important in blood transfusion. The decreased expression levels of A or B antigens of the ABO subtype often present difficulties in blood typing and cross-matching for clinical transfusion. B 3 is the most common B subtype, which has a frequency of approximately 1 in 900 in the Chinese population. The underlying mechanisms of the B 3 phenotype were diversified. Several mutations in the exons, splicing site, and mutations in the promoter have been reported for B 3.1,2 Recently, a regulatory element was identified in the first intron of the ABO gene with a functional GATA-1 erythroid transcription factor binding site. 3 Studies have shown that mutations in the erythroid cell-specific regulatory element (15.8 kb) can cause variant ABO phenotypes. 2,4 Here, we report a novel single-nucleotide substitution within the GATA motif (C>T substitution at Position c.28 1 5885) in the 15.8-kb site on the B allele in one individual with the B 3 phenotype. METHODS AND RESULTSA blood sample from a 44-year-old Chinese Han male patient who was admitted to our hospital for treatment of arrhythmia was referred to our laboratory because the B antigen of red blood cells (RBCs) demonstrated mixedfield agglutination when tested with routine ABO blood group typing. He had no history of blood transfusion. The blood sample was collected after informed consent was obtained, and genomic DNA was extracted by a commercial kit according to the manufacturer's instructions (TIANamp blood DNA kit, Tiangen Biotech Co.). ABO blood group typing was routinely determined by a gel test kit (Ortho ABO-Rh/reverse grouping cassette, Ortho-Clinical Diagnostics). The forward typing was further confirmed by a manual tube test using monoclonal anti-A, anti-B, anti-AB, and anti-H (Blood Grouping Reagent, Shanghai Hemo-Pharmaceutical & Biological, Co., Ltd) according to the standard protocols. Reverse typing was also performed with a tube test using A 1 , B, and O RBCs.The upstream CBF/NF-Y enhancer region, promoter, all seven exons and adjacent introns of the ABO gene were amplified and sequenced according to previously published methods. 5 The method of amplification for the ABO 15.8-kb site is shown in Appendix S1 (available as supporting information in the online version of this paper).On forward typing by the gel column method, the RBCs of the patient had no reaction with anti-A and mixed-field agglutination (21) with anti-B and anti-AB reagents, but there was a strong reaction with anti-H sera (41). On reverse typing, the serum of the patient was agglutinated by standard A 1 RBCs (21), and it was not agglutinated by standard B and O RBCs.Direct sequencing demonstrated the B101/O02 genotype in this proband. However, one point mutation in the element was identified with the presence of a novel single-nucleotide substitution within the GATA motif From the

Human ABO gene transcriptional regulation

“…) . Similarly, single‐nucleotide substitution of c.28 + 5885C > T was reported around the RUNX1 motif in an individual with B 3 . These genetic studies confirmed the regulatory significance of the +5.8‐kb site for erythroid cell–specific expression of ABO in vivo.…”

Sequence variants in the proximal promoter and +5.8‐kb site of ABO in Koreans with weak B phenotypes

“…The erythroid cell‐specific regulatory activity of the +5.8‐kb site was found to be dependent on the binding of the haematopoietic transcription factors GATA and runt‐related transcription factor 1 (RUNX1), and variants in the binding motifs of these factors were shown to reduce the transcriptional activity of the +5.8‐kb site [4, 7]. To date, seven variants in the +5.8‐kb site have been reported to cause weak B phenotypes, most of which have been identified in Chinese and Japanese individuals [4, 5, 8, 10, 11, 13]. In the Japanese population, B m is the most common B subgroup, the main mechanism of which is the 5.8‐kb deletion (g.10157_15927del) [4, 8, 19].…”

“…In the Japanese population, B m is the most common B subgroup, the main mechanism of which is the 5.8‐kb deletion (g.10157_15927del) [4, 8, 19]. In contrast, in the Chinese and Korean populations, B 3 is the most common B subgroup [10, 14], and the 5.8‐kb deletion has not been reported. In this study, which included 16 Koreans with weak B phenotypes, the most frequent variant was g.10925C>T located in the RUNX1 motif of the +5.8‐kb site (Figure 1b).…”

“…The majority of ABO subgroups are caused by variants in the coding exons and flanking introns of ABO , that is, missense, nonsense, frameshift and splice site variants [1, 2]. However, some weak ABO subgroups result from variants in the ABO regulatory regions, including the proximal promoter, +5.8‐kb site and CCAAT‐binding factor (CBF)/NF‐Y binding site [1, 3–13]. These subgroups are often misclassified as having common ABO genotypes such as ABO * B.01 / O.01.01 because ABO genotyping in many blood banks examines only the coding exons and flanking introns of the ABO gene.…”

ABO*B.01+c.464A>C represents a missense variation in the ABO gene and encodes a weak B phenotype

“…The full coding regions of the ABO gene, the upstream CBF/NF-Y enhancer region, promoter, and the erythroid cell-specific regulatory elements in intron 1 were nucleotide sequenced as previously published. 5 Exons 6 and 7 of the ABO gene and the intervening intron were cloned and the two haplotypes sequenced (TOPO TA cloning kit; Life Technologies, Carlsbad, CA).…”