Transfusion and Apheresis Science 51 (2014) 203–208

Contents lists available at ScienceDirect

Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i

Molecular genetic analysis and structure model of a rare B(A)02 subgroup of the ABO blood group system Qing Chen a, Jiahuang Li b, Jianyu Xiao a, Leilei Du c, Min Li c, Genhong Yao d,* a

Jiangsu Province Blood Center, Nanjing 210042, China The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, China c Department of Medical Mycology, Institute of Dermatology, Chinese Academy of Medical Science, Peking Union Medical College, Nanjing 210042, China d Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China b

A R T I C L E

I N F O

Article history: Received 24 April 2014 Received in revised form 8 August 2014 Accepted 25 August 2014 Keywords: ABO blood group system B(A) ABO discrepancy ABO genotyping

A B S T R A C T

Background: Serological analysis of ABO blood group has been widely applied in transfusion medicine. However, ABO subgroups with different expression of blood group antigens sometimes cannot be determined by serological methods. Therefore, genotyping is useful to understand the variant ABO phenotypes. Material and Methods: Exon 6 to exon 7 and adjacent introns of the ABO gene from a donor with ABO typing discrepancy were amplified and sequenced. Cloning sequencing was also performed to identify the allele. To explore the effect of mutation, three dimensional model of mutant p.Pro234Ala was built and optimized. Results: The variant B (c. 700C > G) allele expressed an AweakB phenotype with anti-A in his serum with a ABO*B(A)02/O02 heterozygote genotype. Cloning sequencing confirmed that the c.700C > G single nucleotide polymorphism was associated with a B101 allele. Three dimensional molecular modeling suggested that p.Pro234Ala might affect the conformation of His233, Met266 and Ala268, which were known as critical residues for donor recognition. Conclusion: ABO genotyping is needed for correct identification subgroups to improve accuracy evaluation of blood typing and increase the safety of blood transfusion. Alteration of DNA sequence in the ABO gene resulted in amino acid substitutions and led to a weak or missing expression of ABO antigens. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Since the discovery of ABO blood group by Landsteiner, ABO remains the most important blood group system in transfusion and transplantation medicine. Correct typing of ABO blood group of donors and recipients is most important for compatible transfusion of red blood cells (RBCs) [1–4]. Up to now, serological methods are routinely used to determine the antigens of blood groups and isoaggluti-

* Corresponding author. Tel.: +86 25 80860323; fax: +86 25 80860323. E-mail address: [email protected] (G. Yao). http://dx.doi.org/10.1016/j.transci.2014.08.023 1473-0502/© 2014 Elsevier Ltd. All rights reserved.

nins. However, in some cases, the results of serological antigen (such as ABO subgroups with a weak expression of A or B antigen) and reverse typing of ABO blood group were discrepant. Therefore, some ABO subgroups were unable to be distinguished by commonly used serological methods. Many lines of evidence suggested that a clear correlation was existed between DNA sequence variation in the ABO gene and the quality (A and B antigens) and quantity (A2, A3, B3, Bel phenotype, et al.) of blood group antigens on RBCs [5–8]. The antigens of ABO blood group system are carbohydrates and are not encoded by gene directly. A specific glycosyltransferase encoded by the ABO gene plays an

204

Q. Chen et al./Transfusion and Apheresis Science 51 (2014) 203–208

important role in the modification of ABO blood group antigens. The ABO gene is located at long arm of chromosome 9 (9q34) and consists of 7 exons, among which the exon 6 and 7 harbor the majority of the exonic DNA encoding the enzyme’s entire catalytic domain of the ABO glycosyltransferases [1]. The difference between the glycosyltransferase A (GTA) and glycosyltransferase B (GTB) are Arg176Gly, Gly235Ser, Leu266Met, and Gly268Ala. The codons 266 and 268 are especially crucial in differentiating sugar specificity. Studies showed that mutations of ABO gene affected A and B glycosyltransferase activity and caused variant ABO phenotypes [1]. The decreased expression levels of A or B antigens caused the serological ABO blood grouping discrepancies and could induce transfusion reaction. To date, more than 300 ABO variant alleles have been posted in the dbRBC database. With the development of new technologies, genotyping has been shown to be a valuable tool in solving the serological grouping discrepancy in clinic samples and has been widely applied to improve patient care. In the present study, we performed a study for a B(A) phenotype blood donor previously mistyped as group B whose blood labeled B transfused to two different recipients to identify the molecular structure of his allele.

2. Material and methods 2.1. Case report An ABO typing discrepancy was observed in a 27-yearold male donor when he made third donation. He had donated 400 ml blood each in 2008 and 2009, respectively and typed B. His blood previously passed the test labeled as B and was transfused to two different patients. Ethylenediaminetetraacetate-anticoagulated blood sample from this donor was collected for the resolution of an ABO discrepancy between the forward and reverse type. Genomic DNA was extracted from a commercial kit according to the manufacturer’s instructions (TIANamp Blood DNA Kit, Tiangen Biotech Co., LTD, Beijing, China). Unfortunately, family member samples were not available. The research ethics committee of Nanjing University approved the study. Written informed consent was obtained from the donor before blood sampling.

2.3. Direct sequencing of ABO exon 6 to 7 and adjacent introns The ABO exon 6 to 7 and adjacent introns were amplified according to the method published previously [9]. After amplification, 5 μl of the PCR products were separated on 1% agarose gels and observed by gel imaging system. The PCR products were purified by a commercial kit (AxyPrep PCR clean-up kit, Axygen biosciences, Union City, CA, USA) according to the manufacturer’s instructions. The sequences of PCR products were obtained by the cycle sequencing method. Nucleotide sequencing was performed with a DNA sequencing unit (ABI PRISM Big Dye Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems, Carlsbad, CA, USA). After the sequencing reaction, the reaction mixture was purified by the BigDye Terminator Purification kit. Capillary electrophoresis and data collections were carried out on the ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems). Sequencing results were aligned to NCBI RefSeq NG_006669.1 and analyzed using Bioedit software Version 7.1.8. 2.4. Exon 6 and 7 cloning sequencing PCR product of the donor’s exon 6 and 7 was cloned into a pCRII-TOPO vector (Invitrogen, Carlsbad, CA, USA) and transformed into Escherichia coli DH5 competent cells. Plasmids were isolated using the Wizard Plus Mini Prep DNA purification system (Promega, Madison, WI, USA) for DNA sequencing. The DNA sequences of the exons and intron were analyzed using an automated DNA sequencer (ABI Prism 3730XL Genetic Analyzer, Applied Biosystems). 2.5. Three dimensional structure modeling The starting structure of the mutant was built by replacing Pro234 with the desired Ala amino acid in wild type structure (PDB ID: 2RJ8). Both wild type and mutant structure were refined by Discovery Studio 2.5 (Accelrys Software Inc., San Diego, CA, USA). Structural refinements were first accomplished by energy minimization where the initial 3000 steps of steep descent were followed by 2000 steps of conjugate gradient, and then 100 ps molecular dynamics simulation were carried out at 300K with constant temperature dynamics using Berendsen weak coupling method (NVT).

2.2. Serological test

3. Results

ABO serological test was carried out with manual tube methods according to the standard protocols. Reverse typing was also determined by tube methods using A1 cells and B cells. The results of RBCs and plasma ABO grouping by tube methods were checked by a gel column method using a DiaMed-ID microtyping system (Cressier, Switzerland). The presence of the B determinants on the RBCs was determined by adsorption-elution test with monoclonal anti-B antibody according to the standard protocol. Elution was performed using heat elution procedure. The concentration of antibody in serum or elution sample was determined by titration studies using serial doubling dilution method.

3.1. Serological phenotype The donor’s RBCs demonstrated 3 + weak agglutination with anti-A reagent and no reactivity with anti-A1 sera at room temperature. His RBCs were strongly agglutinated by anti-H sera. His serum agglutinated A standard RBCs in 2 + reaction and no anti-B was detected in his reverse typing (Table 1). The gel column test confirmed that the A antigen was weakly expressed on his RBCs while the B antigen was expressed normally, and anti-A in his serum (Fig 1). The results of the forward and reverse typing tests conducted at 4 and 37 °C were also shown in Table 1.

Q. Chen et al./Transfusion and Apheresis Science 51 (2014) 203–208

205

Table 1 Results of serological test of the donor at different temperatures. Temperature (°C)

Reactions of RBCs with

4 25 37

Reactions of serum with

Anti-A

Anti-B

Anti-A, B

Anti-A1

Anti-H

A1 cells

B cells

Self cells

4+ 3+ 2+

4+ 4+ 4+

4+ 4+ 4+

0 0 0

4+ 4+ 4+

4+ 2+ 1+

0 0 0

0 0 0

1+to 4+, agglutination of increasing strength; 0, no agglutination.

could adsorb anti-A, which was able to be eluted subsequently. Adsorption and elution tests confirmed that weak A antigen were on the RBCs of the donor. Both antibody screen and direct anti-globulin test of the donor were negative. Therefore, subgroup serological status of the donor was defined as probable AweakB or B(A).

The standard A RBCs gave a 1 reaction at a dilution of 1 in 1024 to serial dilutions of anti-A reagent before adsorption. However, the same strength of reaction was obtained at 1 in 256 after adsorption (Table 2). The RBCs of the donor

3.2. Exon 6 and exon 7 directing sequencing analysis The DNA sequences of the donor were compared with the published A consensus sequence (A101). In ABO exon 6, this donor sample was heterozygous for an A > G exchange at position 297. In exon 7, the donor sample were heterozygous for a C > G exchange at position 526, a T > A exchange at position 646, a C > T exchange at position 657, a G > A exchange at position 681, a C > G exchange at position 700, a G > A exchange at position 703, a C > T exchange at position 771, a C > A exchange at position 796, a G > C exchange at position 803, a G > A exchange at position 829, and a G > A exchange at position 930 (Table 3) (Fig 2). These nucleotide exchanges resulted in an Arg to Gly substitution at codon 176, a Pro to Ala substitution at codon 234, a Gly to Ser substitution at codon 235, a Leu to Met substitution at codon 266, and a Gly to Ala substitution at codon 268 in the ABO protein.

Fig. 1. The result of RBCs and plasma ABO grouping by gel column method.

Table 2 Semi-quantitative analysis of the monoclonal anti-A reagent. Dilution of anti-A reagent

Before adsorption After adsorption

1:1

1:2

1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

1:1024

1:2048

4+ 4+

4+ 4+

4+ 4+

4+ 4+

4+ 3+

4+ 3+

3+ 2+

3+ 2+

2+ 1+

2+ 0

1+ 0

0 0

1+to 4+, agglutination of increasing strength; 0, no agglutination.

Table 3 Sequence variations in the 6 different B(A) and cis-AB03 alleles compared with some of the ABO alleles. Alleles

A101 A102 B101 B(A)01 B(A)02 B(A)03 B(A)04 B(A)05 B(A)06 cis-AB03

Exon 6

Exon 7

297

467

526

640

641

657

700

703

796

803

930

1096

A

C T

C

A

T

C

C

G

C

G

G

G

A

A A A A A A A A

C C C C C C C C

A A A A A A A A

A

G G G G G G G G

G G G G G G G G

G

Blank boxes represent nucleotides identical to the A101 allele.

C

T T T T T T T T

G

A

T

A A A A

A A

206

Q. Chen et al./Transfusion and Apheresis Science 51 (2014) 203–208

Fig. 2. ABO gene exon 6 and 7 partial DNA cloning and sequencing results of the donor. The nucleotide sequences of the upper panel shows the heterozygous sequences (T&A), (C&T), (G&A), (C&G) and (G&A) were detected at the nucleotide position 646, 657, 681, 700, and 703. The middle panel demonstrates the cloning results of a T, G and A at nucleotide position 657, 700 and 703. The lower panel demonstrates the cloning results of A at nucleotide position 646 and 681.

3.3. Exon 6 and exon 7 cloning sequencing analysis Exon 6 and 7 cloning sequencing confirmed that c.700 C > G was associated with a B101 allele; c.646 T > A, c.681 G > A, c.771 C > T, and c.829 G > A single nucleotide polymorphisms (SNP) were associated with an O02 allele, respectively (Fig 2). In this study, the variant allele expressed an AweakB phenotype with anti-A in his serum with a ABO*B(A)02/O02 heterozygote genotype. 3.4. Homology modeling of the B(A)02 enzyme structure Three-dimensional structure modeling of B(A)02 enzyme showed that mutation of Pro234Ala might affect the conformation of His233, Met266 and Ala268, which are known as critical residues for donor recognition (Fig 3). Pro234 approach the residue involved in binding of H-antigen acceptor (HA), Asp326 and Leu330. The mutation of Pro234 to Ala could result in different conformations of side chain of two residues. 3.5. Respective analysis of recipients transfused with the B(A)02 RBC units To evaluate the risk of transfusion B(A)02 blood to B recipients, two B type recipients received the four units of B(A)02 RBCs from this donor, which had been labeled B previously, were analyzed respectively. One recipient with right pelvic osteoma had a single transfusion and was transfused total 14 RBC units during surgeon. The other recipient had multiple transfusions. Before the RBCs were issued for transfusion, routine anti-globulin cross match was performed. The results showed that RBCs samples of two B recipients were cross match-compatible with B(A)02 units.

The clinic records showed that the hemoglobin, hematocrit and indirect bilirubin level remained in normal range in these two B recipients after transfusion. No significant evidence of acute or delayed hemolytic transfusion reactions were observed after transfusion of B(A)02 RBCs. 4. Discussion In the present study, this 27-year-old male donor, who had made two donations previously and typed B, was observed an ABO forward and reverse typing discrepancy during his third blood donation. Since molecular testing provided a more precise means to predict the phenotype from the genotype, genetic analysis was performed in the donor with ABO serological discrepancy. This donor harbored the B(A)02 allele, which was differed from the normal B101 allele only by one nucleotide substitution. The single nucleotide substitution caused amino acid change at position Pro234Ala. The allele was responsible for ABO discrepancy in this donor with a weak expression of A antigen and anti-A in his serum. Similar phenomena were also observed in some donors with B(A)02 phenotypes occasionally tested in some labs in China [10,11] and a Bw03 subtype donor by Xia et al. [12]. The reason for typing discrepancies in the same donor during different donations may be due to monoclonal typing reagents from different manufactures (e.g., for the reagents used in this donor, previously two donations reagents from one manufacturer, and this time from another manufacturer). B(A) red cells have a variable reactivity with monoclonal anti-A reagents. B(A) phenotypes could not be detected if the monoclonal typing reagents do not harbor the MHO4 clone [13]. To date, six different B(A) subtypes have been reported. The molecular structure of B(A) phenotype was first

Q. Chen et al./Transfusion and Apheresis Science 51 (2014) 203–208

207

Fig. 3. (A) structure of wild type GTB in complex with UDP, Mn ion (ball) and HA (PDB ID: 2RJ8). Pro234 is labeled in stick. (B) superposition of the structures of native GTB and A234.

determined in 1993 and named ABO*B(A)01 [14]. ABO*B(A)02 was first reported in 1999 by Yu et al. in Taiwan [15]. B(A)03 was first observed in donors from the Bavarian region of Germany characterized as AweakB [16]. B(A)04 to B(A)06 were first identified in mainland China [17,18]. B(A) phenotypes were sporadic observed in China. The B(A)02 allele is identical to B101 allele except for the SNP at c.700C > G, which is associated with AweakB phenotype. It is interesting to note that c.700C > T in the B101 allele backbone caused the amino acid change Pro234Ser, which was identified as cis-AB03 [19]. Studies have shown that amino acid position 176 and 235 of ABO protein were associated with

affecting the enzyme turnover rate and did not confer the ability to differentiate UDP-GalNac from UDP-Gal, while amino acid positions 266 and 268 were associated with enzyme’s specificity [20]. Pro234Ala was just adjacent to the second of the four specific amino acids. The 3D modeling revealed that the Pro234Ala might change the conformation of Met266 and Ala268, thus compromised the B transferase specificity and increased capacity to use UDPN-acetylgalactosamine, in addition to UDP-galactose, resulting in detectable A antigen synthesis. Previous study also reported the same position Pro234Ser mutation completely reversed the specificity of the GTB [21].

208

Q. Chen et al./Transfusion and Apheresis Science 51 (2014) 203–208

Anti-A and anti-B are the most clinically important antibodies of all antibodies to RBC blood group antigens due to natural occurring antibodies to the A or B antigens lacking on their RBCs. The clinical significance of anti-A and anti-B is also important in hematopoietic and solid organ transplantation. In this study, the B(A)02 donor was previously mistyped as B in 2008 and 2009. His donated blood was labeled B and transfused to two B type recipients respectively. Therefore, retrospective studies of his two donations were performed to evaluate the safety of B(A)02 blood transfusion. Fortunately, the clinic records showed that no hemolytic transfusion reaction was observed in the patients transfused with the donated RBCs and plasma. Our results suggested that the quantity of A antigen on the B(A) cells of the proband with AweakB phenotype was so much lower than that on normal group A cells. The reduced A antigen might not result in significant hemolytic transfusion reactions. No hemolytic transfusion reaction and other side effects were also observed by Zheng et al. in a B type patient transfused with B(A) RBCs mistyped as B type [22]. However, another study showed that a slight transfusion reaction was detected in a B type patient transfused with B(A) RBCs. And no transfusion reaction was shown in the B(A) type patients transfused with O type washed red blood cells [11]. More clinic transfusion data needed to be collected to get more information about ABO subtype blood transfusion. To exclude an even slight chance of mild hemolytic transfusion, B(A) phenotype individuals should be transfused with B or O blood group RBCs. However, B(A) phenotype RBCs should only be transfused to B(A) or AB patients, or excluded as donor. By correct identifying the B(A) phenotype, genotyping can be an assistance to serological test and is very useful for clinical transfusion practice. In summary, in the present study, the difficult-todefine B(A) phenotype was identified by ABO genotyping. The data of ABO sequencing indicated that the variant allele that expressed an AweakB phenotype with anti-A was ABO*B(A)02/O02 heterozygote genotype. The mutation of ABO gene led to the change of amino acid and structure and thus altered the specificity of ABO enzymes. Contribution statement Genhong Yao and Qing Chen conceived and designed the study, and drafted the manuscript. Jiahuang Li, Jianyu Xiao, Leilei Du and Min Li participated in the conception of the study, the analysis and revising of the manuscript. Acknowledgements This work was supported by “Jiangsu Province Medical Elite Program (No. RC2011088)”, “333” Projects of Jiangsu

Province, “Jiangsu Provincial Nature Science Foundation (No. BK2011573)”, and “Jiangsu Health International Exchange Program”.

References [1] Daniels G. Human blood groups. 3nd. Oxford, UK: Wiley-Blackwell; 2013. p. 11–95. [2] Franchini M, Liumbruno GM. ABO blood group: old dogma, new perspectives. Clin Chem Lab Med 2013;51:1545–53. [3] Daniels G, Reid ME. Blood groups: the past 50 years. Transfusion 2010;50:281–9. [4] Hosoi E. Biological and clinical aspects of ABO blood group system. J Med Invest 2008;55:174–82. [5] Bugert P, Scharberg EA, Janetzko K, et al. A novel variant B allele of the ABO blood group gene associated with lack of B antigen expression. Transfus Med Hemother 2008;35:319–23. [6] Blumenfeld OO. The ABO gene-more variation. Blood 2003;102:2715. [7] Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Genomic analysis of clinical samples with serological ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles. Blood 2001;98:1585–93. [8] Seltsam A, Hallensleben M, Kollmann A, et al. The nature of diversity and diversification at the ABO locus. Blood 2003;102:3035–42. [9] Chen Q, Xiao J, Wang S, et al. ABO sequence analysis in an AB type with anti-B patient. Chin Med J (Engl) 2014;127:971–2. [10] Duan FC, Song N, Tian L, et al. Serologic and molecular biological detection of rare B(A) blood group. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2013;21:478–80. [11] Huang XY, Duan FC, Li DY, et al. Rare blood group B (A) detection and safe transfusion. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2013;21:1280– 4. [12] Li GJ, Zhang X. Molecular genetic analysis of two individuals with Bw subtypes of ABO variant. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2013;30:733–5. [13] Roback JD, Grossman BJ, Harris T, Hillyer CD, editors. Technical manual. 17th ed. Bethesda, MD: AABB; 2011. p. 368. [14] Yamamoto F, McNeill PD, Yamamoto M, et al. Molecular genetic analysis of the ABO blood group system: 3. A(X) and B(A) alleles. Vox Sang 1993;64:171–4. [15] Yu LC, Lee HL, Chan YS, et al. The molecular basis for the B(A) allele: an amino acid alteration in the human histoblood group B alpha(1,3)-galactosyltransferase increases its intrinsic alpha-(1,3)-Nacetylgalactosaminyltransferase activity. Biochem Biophys Res Commun 1999;262:487–93. [16] Seltsam A, Hallensleben M, Kollmann A, et al. Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles. Transfusion 2003;43:428–39. [17] Deng ZH, Yu Q, Wu GG, et al. Molecular genetic analysis for Ax phenotype of the ABO blood group system in Chinese. Vox Sang 2005;89:251–6. [18] Deng ZH, Yu Q, Liang YL, et al. Characterization of a novel B(A) allele with BBBA type at the ABO blood group. J Hum Genet 2006;51:732–6. [19] Roubinet F, Janvier D, Blancher A. A novel cis AB allele derived from a B allele through a single point mutation. Transfusion 2002;42:239– 46. [20] Yamamoto F, McNeill PD. Amino acid residue at codon 268 determines both activity and nucleotide-sugar donor substrate specificity of human histo-blood group A and B transferases. In vitro mutagenesis study. J Biol Chem 1996;271:10515–20. [21] Marcus SL, Polakowski R, Seto NO, et al. A single point mutation reverse the donor specificity of hauman blood group B-synthesizing galactosyltransferasel. J Biol Chem 2003;278:12403–5. [22] Zheng W, Luo H, Ye Y, et al. Identification and clinical transfusion of B(A) blood group. J Clin Transfus Lab Med 2013;15:135–8.