Aberrant ABO B Phenotype with Irregular Anti-B Caused by a Para-Bombay FUT1 Mutation

Abstract

Background

Routine ABO blood group typing for pre-transfusion testing of a male Austrian patient of Far Eastern origin showed discrepant results with an apparently weak blood group B phenotype and irregular anti-B.

Materials and Methods

ABH phenotyping and cross-matching was done by standard serologic techniques and levels of H expression were determined by flow cytometry. ABO gene sequencing including regulatory regions as well as analysis of FUT1 (H), FUT2 (Secretor), and FUT3 (Lewis) were carried out.

Results

While monoclonal ABO antigen typing indicated blood group O, weak agglutination reactions using polyclonal human anti-B and anti-AB were seen. In reverse typing at room temperature, the plasma was reactive with A1 and A2 RBCs and negative with B and O cells, whereas at 4°C, anti-B reactivity was found. The indirect anti-globulin cross-match of the patient's plasma was positive with group B RBCs and negative with group O RBCs. Sequencing analysis showed the presence of ABO*B.01 (B114) allele and homozygosity for the FUT1 mutation c.551_552delAG. Flow cytometry demonstrated trace amounts of H antigen on the patient's RBCs.

Conclusion

While a functional B allele was found, analysis of FUT1 and FUT2 genes revealed the presence of a rare para-Bombay genotype O_h^B. Interestingly, no anti-H but irregular anti-B was found in the patient's plasma, responsible for the positive cross-match with group B RBCs. Even though very rare and not reported for the European population, the presence of an H-deficient phenotype should be considered when investigating individuals with an unusual ABO blood group type.

Introduction

Blood group A and B antigen synthesis is dependent on the presence of precursor H carbohydrate structures. H antigen expression on red blood cells (RBCs) is regulated by the fucosyltransferase 1, product of the FUT1 gene, while fucosyltransferase 2, encoded by the FUT2 gene, regulates the expression of soluble ABH antigens in secretions. Mutations in the FUT1 gene may result in reduced or absent H antigen production on RBCs, while nonfunctional FUT2 alleles when homozygous result in the non-secretor phenotype. Inactivity of both, fucosyltransferase 1 and 2, characterizes the Bombay phenotype (O_h). In the para-Bombay phenotypes, ABH antigens are deficient in secretors or partially deficient in both secretors and non-secretors [1, 2].

Genetic polymorphisms of FUT1 and FUT2 are ethnically and geographically specific. Alleles of FUT1 are rare and occur sporadically, whereas the FUT2 gene is highly polymorphic and is distributed in a population-specific manner [3, 4, 5, 6, 7]. Variants of fucosyltransferases 1 and 2 may be the reason for discrepant serologic ABO blood group typing results. Here we report an aberrant B phenotype with irregular anti-B caused by a disruptive mutation in FUT1of a male Austrian patient of Far Eastern origin.

Materials and Methods

Serologic ABO and Lewis blood typing was performed using gel matrix techniques (MicroTyping system, Bio-Rad, Vienna, Austria). The presence of H antigen was tested using two monoclonal anti-H (MIMA134, provided by G. Halverson, New York Blood Center, New York, NY; 10934C11, BAG, Lich, Germany) in gel matrix (Bio-Rad). Additionally, H antigen expression was analyzed by flow cytometry using fluorescein isothiocyanate-conjugated anti-H (BRIC198, International Blood Group Reference Laboratory, Bristol, UK; FACSCanto II, Becton Dickinson, Heidelberg, Germany). Polyclonal anti-A or anti-B (Biotest, Dreieich, Germany) was adsorbed onto the patient's RBCs, followed by acid elution (DiaCidel, DiaMed). Eluates were tested against A1, A2, B, and O RBCs. Cross-matching of the patient's plasma with group O and B RBCs was performed in anti-human globulin and neutral gel cards (Bio-Rad) at 37°C androom temperature, respectively. Anti-erythrocyte antibody screening and the direct anti-human globulin test (DAT) was done as described [8]. Allele-specific sequencing analyses of the ABO gene and its regulatory regions, as well as of FUT1, FUT2, and FUT3 genes were performed as previously described [6, 9].

Results

Testing the patient's RBCs with monoclonal ABO typing reagents indicated blood group O. In contrast, when using polyclonal reagents after RBC pretreatment with bromelin, weak agglutination reactions were seen with anti-B and anti-AB (−/+ and +++, respectively; Fig. 1A and B). In reverse blood group typing at room temperature, the serum was strongly reactive with A1 RBCs, ++ reactive with A2, and negative with B and O cells. However, reverse typing at 4°C showed the presence of anti-B (++) (Fig. 1C and D). The patient's plasma showed a negative antibody screening, and his RBCs were negative in the DAT.

Fig. 1.

ABO forward and reverse typing. ABO antigen typing using monoclonal reagents (A) showed generally negative results, as opposed to positive reactions with polyclonal reagents (B) enhanced by bromelin pretreatment of red cells (slightly positive and +++ with anti-B and anti-AB, respectively). ABO reverse typing of the patient's plasma in neutral gel showed reactivity with A1 and A2 cells at room temperature (RT; C), and additionally with B cells at 4°C (D).

Adsorption-elution studies with the patient's RBCs revealed negative results with anti-A, but positive reactions with anti-B, confirming a weak B phenotype. Cross-match of the patient's plasma was positive with two of three group B RBC samples in both, the anti-human-globulin phase at 37°C and saline phase at room temperature. Cross-match was generally negative with group O RBCs (data not shown). H antigen was not detected in RBC hemagglutination typing (Fig. 2A). Flow cytometry indicated trace amounts of H antigen on the patient's RBCs, even less than on A1 control cells (Fig. 2B).

Fig. 2.

H antigen expression. Serologic H antigen typing of the patient's RBCs (Pat.) using monoclonal anti-H (HIMA134) in neutral gel cards was negative (A). Identical results were obtained with another anti-H (10934C11; not shown). Flow cytometric analysis using fluorochrome-conjugated anti-H (BRIC198) detected minimal amounts of H antigens on the patient's RBCs (B). For comparison, A1, B, and O control RBCs are shown.

By sequencing analysis, the alleles ABO*O.01.01 and a variant of ABO*B.01 (B114)were identified. The FUT1 genotype showed homozygosity for FUT1*01N.06allele, containinga nucleotide deletion of one of three AG repeats (c.551_552delAG, p.Glu184Valfs*85) in the coding region of the gene [10]. FUT2 was homozygous for the functional Se357 allele (c.357C>T, p.Asn119Asn119) [11], indicating secretor status. Consistent with the patient's Le(a-b-) phenotype, FUT3 sequencing detected homozygosity for inactive Lewis alleles based on the presence of c.59T>G (p.Leu20Arg) and c.1067T>A (p.I356K) [12, 13].

Discussion

The presence of a functional B allele in the genotype does not reflect the aberrant ABO blood group phenotype of the investigated patient. However, the detected sequence variant in FUT1 gene encodesa mutation previously found in a few individuals in Taiwan. Since the deletion results in a reading frame shift creating a premature stop codon in the coding region, nonfunctional α(1,2)fucosyltransferase enzymes may be expressed [10]. Functional FUT2 alleles are present, indicating an H-deficient secretor type O_h^B-secretor.

The minimal presence of H antigens on the patient's RBCs was only recognized applying flow cytometry analysis, known to be more sensitive than serological typing by direct agglutination. Due to the secretor status of the patient, soluble H antigen synthesized by FUT2, as well as the B antigens detected may be adsorbed on the RBCs described as O_h^B phenotype [14, 15].

Like those with the Bombay phenotype, para-Bombay individuals may develop antibodies against the H antigens [16, 17]. However, no anti-H was observed in the serum of our patient.

Interestingly, anti-B was found in the patient's plasma at 4°C, suggesting the presence of irregular anti-B being responsible for a positive crossmatch with group B RBCs.

No previous blood transfusion is known and unfortunately, family history data were not available.

When transfusion is indicated, group O RBCs appear to be compatible for this patient. Donor blood of the same phenotype is - aside from family donors - exceedingly rare in Europe and was no option in this case. In case of allo-anti H development, the transfusion strategy has to be revised.

The presence of anti-H in H-deficient secretors was reported [16, 17]; however, as para-Bombay individuals usually retain some H antigens on their RBCs, only weak or no anti-H activity may be observed in their plasma.

The para-Bombay phenotype is slightly more common in Taiwan with a frequency of about 1 in 8,000 people [18], but not reported in the Austrian population. Since anti-H antiserum is not routinely applied for ABO typing, cases of para-Bombay phenotype would remain unidentified. Even though very rare, especially when investigating individuals of Far Eastern origin showing an unusual ABO blood group type, the presence of a H-deficient phenotype should be considered. In such phenotypes, particular attention should be paid to potential aberrant reverse typing results to ensure transfusion safety.

Statement of Ethics

The patient has given informed consent. No ethical approval was required.

Disclosure Statement

The authors have no conflicts of interest to declare.

Funding Sources

The study did not receive any funding.

Author Contributions

Eva Maria Matzhold wrote the manuscript, which was conceptually designed by Günther F. Körmöczi and Thomas Wagner. Marlies Schönbacher, Eva Maria Matzhold, and Günther F. Körmöczi performed the laboratory tests and compiled the data. Camilla Drexler revised the work critically and prepared the figures. All authors approved the manuscript for submission.

Web-Based Resources

http://www.isbtweb.org/fileadmin/user_upload/Working_parties/WP_on_Red_Cell_Immunogenetics_and/018_H_Alleles_v5.1_170221.pdf. The URL was accessed on April 5, 2018.