NEW ALLELES AND ANTIGENS – SHORT REPORTS Novel ABO gene variants caused by missense mutations in Exon 7 leading to discrepant ABO blood typing results Eva M. Matzhold, Andrea Wagner, Camilla Drexler, and Thomas Wagner
D
iscrepant serologic findings in ABO blood group typing often are the result of ABO gene variants, influencing the ABO transferase activity.1,2 The gene responsible for ABO blood group antigen synthesis consists of seven exons; Exons 6 and 7 encode for the catalytic domain of the protein.3 We describe two novel ABO gene variants based on missense mutations in Exon 7, identified in two Caucasians with aberrant ABO phenotypes.
MATERIALS AND METHODS ABO blood groups were determined with standard serologic and gel matrix techniques (DiaMed-ID Micro Typing System, DiaMed [GB], Midlothian, UK). Adsorption-elution studies of the patient’s red blood cells (RBCs) were carried out by use of Bioclone anti-A, consisting of two monoclonal antibodies (MH4 and 3D3, Ortho Clinical Diagnostics, Raritan, NJ) and buffering solution (Gamma ELU-Kit II, Immucor, Inc., Norcross, GA). Allele-specific amplification, based on the heterozygous single-nucleotide polymorphism 437C>T in Intron 2, was performed by long-range polymerase chain reaction (PCR; Peqlab Biotechnologie, Erlangen, Germany) to cover sequences ranging from Exon 2 to Exon 7 of the ABO gene. Regulatory regions including enhancer and promotor in 50 UTR and 15.8-kb site in Intron 1, as well as Exon 1 were amplified by genomic PCR (SuperHot Mastermix, Bioron, Ludwigshafen, Germany) and then sequenced using a cycle sequencing kit (BigDye Terminator, Version 3.1, Life Technologies, Darmstadt, Germany). In silico protein analysis was done using Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/). Prediction of possible impact of an amino acid substitution on structure and function of a protein is based on several features comprising the sequence, phylogenetic, and structural information characterizing the substitution. The calculated score represents the damage probability of a substitution. Values nearer 1 are more confidently predicted to be deleterious. Newly identified ABO alleles were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena/ data/view/LK022841-LK022844). Allele designations accord-
ing to Blood Group Antigen Gene Mutation Database4 and ISBT nomenclature are used.
RESULTS Phenotyping of both samples exhibited absence of A and B antigens, indicating blood group O (Table 1). The sera contained reactive anti-B, but discrepant findings regarding the presence of anti-A1 and antiA2 isoagglutinins were seen in reverse blood group typing. Allele-specific analysis of Sample 1 revealed an A214 (ABO*A2.14)-like allele,4 containing the A2-specific mutations c.467C>T and c.1061delC and an additional mutation c.635T>A (p.Val212Glu) in Exon 7 of the gene (LK022844). 1061delC results in the translation of 21 additional amino acids and is associated with the A2 phenotype showing weak A antigen expression.5 Whether the nucleotide substitution of thymine to adenine at Positon 635 acts alone or in combination with the typical A201 (ABO*A2.01) polymorphisms to inhibit A antigen expression remains unclear. The exchange of unpolar, hydrophobic valine to negatively charged glutamic acid in the translated protein may affect the catalytic function of the enzyme (polyphen prediction score value of 0.97). After adsorption of RBCs of Sample 1 with anti-A and subsequent elution, the eluate was tested negative for agglutination with A cells and O control cells. Allele-specific analysis of Sample 2 revealed a novel A101 (ABO*A1.01)-like allele containing a
From the Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria Address reprint requests to: Eva Maria Matzhold, Mag. Dr. scient. Med., Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Auenbruggerplatz 3, 8036 Graz, Austria; e-mail: [email protected]. Received for publication October 1, 2014; revision received December 29, 2014; and accepted December 31, 2014. doi:10.1111/trf.13025 C 2015 AABB V
TRANSFUSION 2015;55;1589–1590 Volume 55, June 2015 TRANSFUSION 1589
MATZHOLD ET AL.
TABLE 1. Samples showing serological ABO blood grouping discrepancies and identification of novel alleles ABO phenotyping Sample ID
RBCs
Serum/plasma isoagglutinins
S1
O
S2
O
Strong reaction for anti-B Weak reaction for anti-A1 and anti-A2 Strong reaction for anti-B No reaction for anti-A1 and anti-A2
c.452T>G (p.Val151Gly) substitution in Exon 7 (LK022843). The substitution of unpolar valine with polar glycine is predicted to probably have a damaging effect on the structure and function of the translated protein (score value of 0.992). Only under high magnification were traces of RBCs, agglutinated with human polyclonal anti-AB, observed. Due to a lack of further blood samples adsorption-elution test was not performed. In both samples sequence analysis of regulatory regions and Exon 1 did not reveal any deleterious variation (data not shown). Since the new alleles were associated with common O alleles (Table 1), the expressed weak ABO subtype phenotypes may result due to the novel mutations in heterozygous state affecting the transferase activity.
ABO haplotype analysis Eluate
Identified allele 1
Identified novel allele 2
Negative for agglutination with A cells Not available
ABO*O02 (ABO*O.01.02)
ABO*A214 1 635T>A (p.Val212Glu)
ABO*O01 (ABO*O.01.01)
ABO*A101 1 452T>G (p.Val151Gly)
REFERENCES 1. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Genomic analysis of clinical samples with serologic ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles. Blood 2001;98:1585-93. 2. Seltsam A, Hallensleben M, Kollmann A, Blasczyk R. The nature of diversity and diversification at the ABO locus. Blood 2003;102:3035-42. 3. Bennett EP, Steffensen R, Clausen H, et al. Genomic cloning of the human histo-blood group ABO locus. Biochem Biophys Res Commun 1995;211:347. 4. Patnaik SK, Helmberg W, Blumenfeld OO. BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems. Nucleic Acids Res 2012;40:D1023-9. 5. Yamamoto F, McNeill PD, Hakomori S. Human histo-blood group A2 transferase coded by A2 allele, one of the A subtypes,
CONFLICT OF INTEREST
is characterized by a single base deletion in the coding sequence, which results in an additional domain at the carboxyl
The authors have disclosed no conflicts of interest.
terminal. Biochem Biophys Res Commun 1992;187:366-74.
1590 TRANSFUSION Volume 55, June 2015
D
iscrepant serologic findings in ABO blood group typing often are the result of ABO gene variants, influencing the ABO transferase activity.1,2 The gene responsible for ABO blood group antigen synthesis consists of seven exons; Exons 6 and 7 encode for the catalytic domain of the protein.3 We describe two novel ABO gene variants based on missense mutations in Exon 7, identified in two Caucasians with aberrant ABO phenotypes.
MATERIALS AND METHODS ABO blood groups were determined with standard serologic and gel matrix techniques (DiaMed-ID Micro Typing System, DiaMed [GB], Midlothian, UK). Adsorption-elution studies of the patient’s red blood cells (RBCs) were carried out by use of Bioclone anti-A, consisting of two monoclonal antibodies (MH4 and 3D3, Ortho Clinical Diagnostics, Raritan, NJ) and buffering solution (Gamma ELU-Kit II, Immucor, Inc., Norcross, GA). Allele-specific amplification, based on the heterozygous single-nucleotide polymorphism 437C>T in Intron 2, was performed by long-range polymerase chain reaction (PCR; Peqlab Biotechnologie, Erlangen, Germany) to cover sequences ranging from Exon 2 to Exon 7 of the ABO gene. Regulatory regions including enhancer and promotor in 50 UTR and 15.8-kb site in Intron 1, as well as Exon 1 were amplified by genomic PCR (SuperHot Mastermix, Bioron, Ludwigshafen, Germany) and then sequenced using a cycle sequencing kit (BigDye Terminator, Version 3.1, Life Technologies, Darmstadt, Germany). In silico protein analysis was done using Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/). Prediction of possible impact of an amino acid substitution on structure and function of a protein is based on several features comprising the sequence, phylogenetic, and structural information characterizing the substitution. The calculated score represents the damage probability of a substitution. Values nearer 1 are more confidently predicted to be deleterious. Newly identified ABO alleles were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena/ data/view/LK022841-LK022844). Allele designations accord-
ing to Blood Group Antigen Gene Mutation Database4 and ISBT nomenclature are used.
RESULTS Phenotyping of both samples exhibited absence of A and B antigens, indicating blood group O (Table 1). The sera contained reactive anti-B, but discrepant findings regarding the presence of anti-A1 and antiA2 isoagglutinins were seen in reverse blood group typing. Allele-specific analysis of Sample 1 revealed an A214 (ABO*A2.14)-like allele,4 containing the A2-specific mutations c.467C>T and c.1061delC and an additional mutation c.635T>A (p.Val212Glu) in Exon 7 of the gene (LK022844). 1061delC results in the translation of 21 additional amino acids and is associated with the A2 phenotype showing weak A antigen expression.5 Whether the nucleotide substitution of thymine to adenine at Positon 635 acts alone or in combination with the typical A201 (ABO*A2.01) polymorphisms to inhibit A antigen expression remains unclear. The exchange of unpolar, hydrophobic valine to negatively charged glutamic acid in the translated protein may affect the catalytic function of the enzyme (polyphen prediction score value of 0.97). After adsorption of RBCs of Sample 1 with anti-A and subsequent elution, the eluate was tested negative for agglutination with A cells and O control cells. Allele-specific analysis of Sample 2 revealed a novel A101 (ABO*A1.01)-like allele containing a
From the Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Graz, Austria Address reprint requests to: Eva Maria Matzhold, Mag. Dr. scient. Med., Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, Auenbruggerplatz 3, 8036 Graz, Austria; e-mail: [email protected]. Received for publication October 1, 2014; revision received December 29, 2014; and accepted December 31, 2014. doi:10.1111/trf.13025 C 2015 AABB V
TRANSFUSION 2015;55;1589–1590 Volume 55, June 2015 TRANSFUSION 1589
MATZHOLD ET AL.
TABLE 1. Samples showing serological ABO blood grouping discrepancies and identification of novel alleles ABO phenotyping Sample ID
RBCs
Serum/plasma isoagglutinins
S1
O
S2
O
Strong reaction for anti-B Weak reaction for anti-A1 and anti-A2 Strong reaction for anti-B No reaction for anti-A1 and anti-A2
c.452T>G (p.Val151Gly) substitution in Exon 7 (LK022843). The substitution of unpolar valine with polar glycine is predicted to probably have a damaging effect on the structure and function of the translated protein (score value of 0.992). Only under high magnification were traces of RBCs, agglutinated with human polyclonal anti-AB, observed. Due to a lack of further blood samples adsorption-elution test was not performed. In both samples sequence analysis of regulatory regions and Exon 1 did not reveal any deleterious variation (data not shown). Since the new alleles were associated with common O alleles (Table 1), the expressed weak ABO subtype phenotypes may result due to the novel mutations in heterozygous state affecting the transferase activity.
ABO haplotype analysis Eluate
Identified allele 1
Identified novel allele 2
Negative for agglutination with A cells Not available
ABO*O02 (ABO*O.01.02)
ABO*A214 1 635T>A (p.Val212Glu)
ABO*O01 (ABO*O.01.01)
ABO*A101 1 452T>G (p.Val151Gly)
REFERENCES 1. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Genomic analysis of clinical samples with serologic ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles. Blood 2001;98:1585-93. 2. Seltsam A, Hallensleben M, Kollmann A, Blasczyk R. The nature of diversity and diversification at the ABO locus. Blood 2003;102:3035-42. 3. Bennett EP, Steffensen R, Clausen H, et al. Genomic cloning of the human histo-blood group ABO locus. Biochem Biophys Res Commun 1995;211:347. 4. Patnaik SK, Helmberg W, Blumenfeld OO. BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems. Nucleic Acids Res 2012;40:D1023-9. 5. Yamamoto F, McNeill PD, Hakomori S. Human histo-blood group A2 transferase coded by A2 allele, one of the A subtypes,
CONFLICT OF INTEREST
is characterized by a single base deletion in the coding sequence, which results in an additional domain at the carboxyl
The authors have disclosed no conflicts of interest.
terminal. Biochem Biophys Res Commun 1992;187:366-74.
1590 TRANSFUSION Volume 55, June 2015
