Identification of a novel nonsense mutation (c.618C>G, p.Tyr206Ter) in the ABO gene in a Chinese individual

INTRODUCTION

The ABO system is the most important blood system in transfusion medicine^[1–2]. It contains two carbohydrate antigens, A and B. Based on the presence or absence of these two antigens on red blood cells (RBCs), four major ABO phenotypes (A, B, AB, and O) are determined. Aside from RBCs, ABO antigens are also found in platelets, endothelium, kidneys, pancreas, and lungs^[3–4]. The transfusion of ABO-incompatible blood can cause haemolytic reaction and renal failure^[5–6].

The ABO gene is located on chromosome 9q34, and consists of seven exons spanning about 20 kilobase pairs^[7]. Exons 6 and 7 are the main coding regions in the ABO gene^[8]. Based on the function, ABO alleles are mainly typed as A, B, and O. The O allele is a nonfunctional ABO allele, encoding a nonfunctional enzyme. The homozygous presence of nonfunctional O alleles causes the absence of A or B antigens and therefore is responsible for group O. Two O alleles, including ABO*O.01.01 and ABO*O.01.02, are the most common alleles in the Chinese Han population^[9]. These two O alleles are both caused by a deletion of guanine at position 261 in exon 6 (c.261delG), leading to a 117-amino-acid protein^[5]. However, other rare mechanisms for blood group O were also reported. Other than the well-known 261G deletion in exon 6, some single-nucleotide polymorphisms, splice site variations, and insertion or deletion mutations can result in unusual O alleles^[8,10–13]. Here, the study described a novel O allele with a nonsense mutation (c.618C>G) in exon 7 of the ABO gene in a Chinese individual.

MATERIALS AND METHODS

ABO blood group typing

The blood sample was collected from a 32-year-old male patient with renal failure in Sichuan province, China. Written informed consent was obtained for genetic research. Routine serologic tests were carried out first using the manual tube method. Monoclonal anti-A, anti-B, anti-A1, and anti-H (Blood Grouping Reagents, Shanghai Hemo-Pharmaceutical & Biological Inc.) were used for forward typing, while reverse typing was determined using A1, B, and O red cells (ABO Red Cell Reagent Kit, Shanghai Hemo-Pharmaceutical & Biological Inc.). Weak expression of A or B antigens on red cells was confirmed by absorption and elution test. Human anti-A and anti-B sera were used, followed by the heat elution technique.

Molecular analysis

Genomic DNA was extracted from the proband's blood sample using a blood DNA kit (TIANamp Blood DNA Kit, Tiangen Biotech Co., Ltd.). According to our previous reports^[14], the exons 6 and 7 were amplified by polymerase chain reaction (PCR) with the specific primers E67F (5′-GGCTGTTCTGAAGGTATTAG-3′) and E67R (5′-ACGGACAAAGGAAACAGAGT-3′). The amplification products were directly sequenced by an automated 3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA) with six forward or reverse primers: 1F, 5′-CCTGTCCCTTTGTTCTCCAA-3′; 2R, 5′-GCCACCCCACTCTGTCTT-3′; 3F, 5′-CCGTCCGCCTGCCTTGCAG-3′; 4R, 5′-GTAGAAATCGCCCTCGTCCT-3′; 5F, 5′-GTGGACGTGGACATGGAGTT-3′; 6R, 5′ACGGACAAAGGAAACAGA-3′. Compared with the ABO*A1.01 reference sequence (GenBank ID No. NG_006669), the ABO genotype was identified based on nucleotide polymorphism.

Haplotype analysis

The haplotypes of the ABO gene were identified by clone-sequencing. The PCR products of exon 6 and exon 7 were ligated with PMD18-T vector (Tiangen Biotech Co., Ltd. Beijing, China) and transfected into Escherichia coli via heat shock. The plasmid DNA was extracted and subsequently sequenced.

RESULTS

The forward typing results showed the proband's RBCs were not agglutinated by anti-A or anti-B. Reverse typing showed the sera contained anti-A1 and anti-B. No presence of A or B antigens on red cells were confirmed by absorption and elution test (Table 1). Thus, the proband was typed as O phenotype.

Table  1.  Results of serologic typing

Sample	RBC grouping			Serum grouping				Absorption and elution test
	Anti-A	Anti-B		A cell	B cell	O cell		A antigen	B antigen
Proband	N	N		4+	4+	N		N	N
N: negative.

 | Show Table

DownLoad: CSV

The direct sequencing results showed that the proband was heterozygous for the ABO*A1.01 allele and ABO*O.01.01 allele, except for a heterozygous c.618C>G mutation (Fig. 1A). The clone sequencing demonstrated that a nonsense mutation C>G at position 618 was identified in exon 7 of the ABO*A1.01-like allele (Fig. 1B), which caused a p.Tyr206Ter exchange in the ABO glycosyl-transferase. The truncated, 205-amino-acid ABO glycosyltransferase lost its transferase activity; thus, the novel ABO allele was classified as O allele. The nucleotide sequence was already submitted to GenBank (ON045328).

Figure  1.  Sequencing results of partial exon 7 in the ABO gene.

The C nucleotide at position 618 is replaced by the G nucleotide. A: The direct sequencing result. B: The clone sequencing result.

DownLoad: Full-Size Img PowerPoint

DISCUSSION

Group O is characterized by the absence of A or B antigens on red cells. Commonly in China, the homozygous presence of 261G-deletion O alleles results in group O, and there are few reports on other causational mechanisms for group O in Chinese individuals. Our study identified an ABO*A1.01-like allele with a nonsense mutation in exon 7, which resulted in a novel O allele.

A nonsense mutation C>G at position 618 in exon 7 caused a truncated, 205-amino-acid ABO glycosyl-transferase. The ABO gene consists of 7 exons, ranging in size from 28 to 688 base pairs (bp). Exon 6 and exon 7, which comprise 823 bp of the 1062 bp of coding region, encode for the catalytic active domain of ABO glycosyltransferases. Due to the loss of most of exon 7 coding catalytic active domain, the truncated glycosyltransferase might lead to the loss of its transferase activity. Meanwhile, decreased stability of the truncated transcripts might also impair the synthesis of the glycosyltransferase^[15–16]. Thus, the novel O allele resulted in the absence of A antigens on red cells. Lei et al. also found the c.618C>G mutation in glycosyltransferase A could lead to type O^[17].

In conclusion, our study identified a novel O allele with a nonsense mutation at position 618, which didn't rely on c.261delG. The nucleotide sequence of this novel allele was already submitted to GenBank (ON045328). The novel nonfunctional O allele is augmented in the ABO allele database.