Occurrence of Rare Deletional Yus and Gerbich Alleles in Syria and Neighbouring Countries.

Background: Gerbich-negative phenotypes of the Gerbich Blood Group System (ISBT 020) are very rare (with the exception of Papua New Guinea). The Gerbich-negative phenotypes Yus and Gerbich are negative for the antigens Ge2, and Ge2 and Ge3, respectively. In antigen-negative individuals, anti-Ge2 and anti-Ge3 antibodies can be naturally occurring, or are triggered during pregnancies and after transfusions. Previous studies suggested an elevated frequency of Gerbich-negative phenotypes for the Middle East. In the summer of 2015, a large-scale migration of people from the Middle East to Europe occurred raising the issue of question how to guarantee blood supply for patients and manage antenatal care for pregnant women from these countries. Materials and Methods: To investigate the frequency of rare Gerbich-negative phenotypes, 1,665 immigrants to Germany originating from the Middle East were genetically tested for the presence of rare Yus, i.e., GE*01.-02, and Gerbich, i.e., GE*01-03, alleles and compared to results obtained from 507 Germans. Results: Seven Yus GE*01.-02.01 and one Gerbich GE*01.-03.02 alleles were exclusively observed among people from the Middle East, with five of them clustering among 797 Syrians. No such alleles were observed in Germans. A cumulative Yus- and GE*01.-03-type allele frequency of 0.00314 and resultant overall Gerbich-negative phenotype frequency of one among 101,633 Syrians were calculated. Conclusion: This manuscript describes for the first time an exclusively genetic screening for carriers of Gerbich-negative alleles. In conclusion, the Gerbich blood group system should be considered as one causative agent of unusual antibodies to red cell antigens, in routine patients and pregnant women, especially when originating from the Middle East.

Two novel antithetical KN blood group antigens may contribute to more than a quarter of all KN antisera in Europe.

BACKGROUND: All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown. STUDY DESIGN AND METHODS: Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed. RESULTS: Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM. CONCLUSION: A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe.

An update on the H blood group system.

CONCLUSIONS: This update of the H blood group system (Scharberg EA, Olsen C, Bugert P. The H blood group system. Immunohematology 2016;32:112-8) reports 11 new FUT1 null alleles: 8 causative for the H- phenotype (Bombay phenotype, Oh), 3 in para-Bombay cases, and 5 new FUT1 alleles causative for a weak H phenotype (para-Bombay, H+w). The H blood group system (International Society of Blood Transfusion system 18) consists of a single antigen (H) defined by a terminal fucose residue found on red blood cells (RBCs) and in secretions. The H antigen is synthesized on the RBC surface by the FUT1 gene product fucosyltransferase 1. On epithelial cells and in body fluids, the H antigen is synthesized by the FUT2 gene product fucosyltransferase 2.

Towards a Regional Registry of Extended Typed Blood Donors: Molecular Typing for Blood Group, Platelet and Granulocyte Antigens.

BACKGROUND: The provision of compatible blood products to patients is the most essential task of transfusion medicine. Besides ABO and Rh, a number of additional blood group antigens often have to be considered for the blood supply of immunized or chronically transfused patients. It also applies for platelet antigens (HPA) and neutrophil antigens (HNA) for patients receiving platelet or granulocyte concentrates. Here, we describe the molecular screening for a number of blood group, HPA, and HNA alleles. Based on the screening results we are building up a regional blood donor registry to provide extended matched blood products on demand. METHODS: We developed and validated TaqMan™ PCR and PCR-SSP methods for genetic markers defining 37 clinically relevant blood group antigens (beyond ABO and Rh), 10 HPA, and 11 HNA. Furthermore, we describe a feasible method for fast molecular screening of the HNA-2null phenotype. All data were statistically evaluated regarding genotype distribution. Allele frequencies were compared to ExAC data from non-Finnish Europeans. RESULTS: Up to now more than 2,000 non-selected regular blood donors in south-west Germany have been screened for blood group, HPA, and HNA alleles. The screening results were confirmed by serology and PCR-SSP methods for selected numbers of samples. The allele frequencies were similar to non-finnish Europeans in the ExAC database except for the alleles encoding the S, HPA-3b and HNA-4b antigens, with significantly lower prevalence in our cohort, as well as the LU14 and the HNA-3b antigens, with a higher frequency compared to the ExAC data. We identified 71 donors with rare blood groups such as Lu(a+b-), Kp(a+b-), Fy(a-b-) and Vel-, and 169 donors with less prevalent HPA or HNA types. CONCLUSION: Molecular screening for blood group alleles by using TaqMan™ PCR is an effective and reliable high-throughput method for establishing a rare donor registry.

Genetic background of the rare Yus and Gerbich blood group phenotypes: homologous regions of the GYPC gene contribute to deletion alleles.

The GYPC gene encodes the glycophorins C and D. The two moieties express 12 known antigens of the Gerbich blood group system and functionally stabilize red blood cell membranes through their intracellular interaction with protein 4.1 and p55. Three GYPC exon deletions are responsible for the lack of the high-frequency antigens Ge2 (Yus type, exon 2 deletion), Ge2 and Ge3 (Gerbich type, exon 3 deletion), and Ge2 to 4 (Leach type, exons 3 and 4 deletion), but lack exact molecular description. A total of 29 rare blood samples with Yus (GE:-2,3,4) and Gerbich (GE:-2,-3,4) phenotypes, including individuals of Middle-Eastern, North-African or Balkan ancestry were examined genetically. All phenotypes could be explained by 4 different Yus alleles, characterized by deletions of exon 2 and adjacent introns, and 3 different Gerbich alleles, with deletions of exon 3 and adjacent introns. A 3600 base pair GYPC region, encompassing exon 2 and flanking region, shares a high degree of sequence homology with a region flanking exon 3, probably representing an evolutionary duplication event. Defining the expression of Gerbich variants presently relies on rare serological reagents. Our approach substitutes the serological characterization with a precise genotype approach to identify the rare Yus and Gerbich alleles.

The H blood group system.

The H blood group system, ISBT symbol H (018), consists of a single antigen (H) defined by a terminal fucose residue found on red blood cells and in secretions formed by the action of α-1,2-fucosyltransferases 1 (α2FucT1) and 2 (α2FucT2), respectively. Mutant alleles of the corresponding FUT1 and FUT2 genes result in either a H­ phenotype (Bombay phenotype, Oh) or a weak H phenotype (para-Bombay, H+w). In addition, the FUT2 gene is the molecular basis of the secretor (Se) status, and homozygosity or compound heterozygosity for null alleles is associated with the nonsecretor (se) status. H­ individuals have natural anti-H (mostly IgM), which can cause severe hemolytic transfusion reactions with intravascular hemolysis.

PCR with sequence-specific primers for typing of diallelic blood groups.

PCR with sequence-specific primers (PCR-SSP) is a cost-effective and robust method for the analysis of single nucleotide polymorphisms (SNPs). Many blood group antigens and the antithetic antigens are based on a diallelic SNP in the coding region of the corresponding blood group gene. Here, we describe PCR-SSP protocols for genotyping 24 blood group antigens based on 12 diallelic SNPs. We also provide protocols for molecular determination of the rare blood group phenotypes Yk(a-) and Vel-.

Molecular Screening for Vel- Blood Donors in Southwestern Germany.

BACKGROUND: The SMIM1 protein carries the Vel blood group antigen, and homozygosity for a 17 bp deletion in the coding region of the SMIM1 gene represents the molecular basis of the Vel- blood group phenotype. We developed PCR-based methods for typing the SMIM1 17 bp (64-80del) gene deletion and performed a molecular screening for the Vel- blood type in German blood donors. METHODS: For SMIM1 genotyping, TaqMan-PCR and PCR-SSP methods were developed and validated using reference samples. Both methods were used for screening of donors with blood group O from southwestern Germany. Heterozygotes and homozygotes for the SMIM1 64-80del allele were serologically typed for the Vel blood group antigen. In addition, the rs1175550 SNP in SMIM1 was typed and correlated to the results of the phenotyping. RESULTS: Both genotyping methods, TaqMan-PCR and PCR-SSP, represent reliable methods for the detection of the SMIM1 64-80del allele. Screening of 10,598 blood group O donors revealed 5 individuals homozygous for the deletional allele. They were confirmed Vel- by serological typing. Heterozygotes for the 64-80del allele showed different antigen expressions ranging from very weak to regular positive. CONCLUSION: Molecular screening of blood donors for the Vel- blood type is feasible and avoids the limitations of serological typing which might show false-negative results with heterozygous individuals. The identification of Vel- blood donors significantly contributes to the adequate blood supply of patients with anti-Vel.

Recombinant blood group proteins facilitate the detection of alloantibodies to high-prevalence antigens and reveal underlying antibodies: results of an international study.

BACKGROUND: Alloantibodies to high-prevalence red blood cell (RBC) antigens are not easily identified by routine serologic techniques. This multicenter study was conducted to test the effectiveness of recombinant blood group proteins (rBGPs) at regional and international RBC reference laboratories. STUDY DESIGN AND METHODS: Single or mixed soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and Cr) were assessed for their ability to inhibit the reactivity of antibodies to specific antigens. Initially, the effect of rBGPs was validated by testing panels of well-characterized patient serum samples containing antibodies to high-prevalence antigens in the hemagglutination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with RBC-based diagnostics. RESULTS: Panels of predefined antibodies to high-prevalence antigens were completely and specifically neutralized by the corresponding rBGP specificities. For prospective identification, antibodies to high-prevalence antigens (n = 62) were specifically inhibited by the corresponding rBGP specificities except for some Complement Receptor 1-related antibodies, which may be directed to epitopes not expressed on the truncated recombinant Kn. In 14 cases, additional clinically relevant alloantibodies were identified. In cross-matching, the rBGPs were successfully used to inhibit the reactivity of clinically irrelevant antibodies to high-prevalence antigens to determine compatibility between donor and recipient. CONCLUSION: rBGPs enable the identification of antibodies to high-prevalence antigens without the need for rare RBC reagents, which are often unavailable. Underlying antibodies can be reliably detected and cross-matching results validated, resulting in a more efficient blood supply for immunized patients.

Development of Anti-G, Anti-C and Anti-Jk(b) in a 22-Year-Old Mother during Her Fourth Pregnancy.

BACKGROUND: Anti-G antibodies are rarely found since anti-D, in combination with anti-C, are difficult to discriminate from anti-G antibodies in routine testing. CASE REPORT: A 22-year-old, gravida-3, para-1, woman with blood group A Rh D neg ccddee and known antibody anti-Jk(b), gave birth to her second child. While anti-Jk(b) could not be detected at birth, a new anti-C was found. Antibody screening tests (IAT) were performed using gel cards and rare G positive rGr erythrocytes. Genotyping for RHD and RHCE was performed using PCR-SSP. RESULTS: The child's blood group was A Rh D neg Ccddee. Genotyping revealed Cde/cde haplotypes. The erythrocytes of the new-born showed a positive direct antiglobulin test with IgG; anti-D and anti-C could be eluted. Erythrocytes with the rare phenotype rGr were reactive with the serum of the mother. CONCLUSION: The presence of anti-D and anti-C in the eluate from then newborn's Ccddee erythrocytes proved anti-G or anti-G in combination with anti-D. When anti-C and anti-D are seen during a pregnancy, possibly anti-G is present. This observation is of relevance since women with anti-G can still develop anti-D and require rhesus prophylaxis.

RhCE protein variants in Southwestern Germany detected by serologic routine testing.

BACKGROUND: Variant RHCE alleles with diminished expression of C, c, E, and e antigens have been described and indicate the genetic diversity of this gene locus in several populations. In this study the molecular background of variant RhCE antigens identified by standard serologic routine testing in German blood donors and patients was determined. STUDY DESIGN AND METHODS: Samples from blood donors and patients were routinely analyzed for RhCE phenotype using the PK7200 analyzer with two sets of monoclonal anti-C, -c, -E, and -e reagents. Samples with confirmed variant RhCE antigens were analyzed by nucleotide sequencing of the 10 RHCE exons. A multiplex polymerase chain reaction with sequence-specific priming (PCR-SSP) method was established for rapid typing of the rare RHCE alleles. RESULTS: We identified 43 samples with serologic RhCE variants. Molecular analysis revealed variant RHCE alleles in 34 samples. Altogether 22 RHCE alleles were detected; 10 have not been published before. Twenty alleles harbored distinct single-nucleotide substitutions, 18 of which encoded amino acid changes and 2 of which occurred in noncoding regions. Two samples represented RHCE-D-CE hybrid alleles involving different segments of the RHCE Exon 5. A multiplex PCR-SSP screening for 17 RHCE alleles was negative in 1344 samples of the DNA bank GerBS. The cumulative phenotype frequency was estimated between 1 in 488 (0.20%) and 1 in 8449 (0.012%). CONCLUSION: Single-amino-acid substitutions were the molecular basis for variant RhCE antigen expression in most samples. Nucleotide substitutions in RHCE exons were excluded as possible mechanism of diminished RhCE antigen expression in one-fifth of the serologically identified samples.

A novel KEL*1,3 allele with weak Kell antigen expression confirming the cis-modifier effect of KEL3.

BACKGROUND: KEL1 (K) is the most immunogenic red blood cell antigen of the Kell blood group system. The frequently occurring anti-KEL1 alloantibodies may cause hemolytic transfusion reactions as well as severe hemolytic disease of the fetus and newborn. So far, reports on weak KEL phenotypes are scarce. STUDY DESIGN AND METHODS: Blood samples of two unrelated Central European propositi with faint reactions in routine KEL1 typing were analyzed by extended serologic phenotyping, flow cytometry, genotyping by polymerase chain reaction with sequence-specific priming, and genomic DNA sequencing of separated parental KEL alleles. RESULTS: Both propositi exhibited an unusual KEL:1,2,3,4 phenotype: markedly weakened and negative reactions were observed in serologic KEL1 typing in gel and tube technique, respectively. No KEL1 epitope loss was detected using five different monoclonal anti-KEL1 reagents. KEL genotyping confirmed KEL*1/2 and KEL*3/4 (Kpa/Kpb) heterozygosity of both individuals. Importantly, DNA sequencing of the two separated parental alleles of both propositi revealed a KEL*1-specific coding nucleotide T578 and a KEL*3-specific T841 on the same allele. This novel KEL*1,3 (KKpa)allele was associated with an approximately 80 percent reduction in KEL1 expression, compared to KEL:1,2,-3,4 controls. The low KEL1 density was attributed to acis-modifier effect of KEL3, so far only reported in association with weakened expression of KEL2 (k). CONCLUSION: This is the first description of the KEL*1,3 allele encoding KEL1 and KEL3 on the same molecule. In individuals with weakened KEL1 because of KEL3 in cis, very sensitive serologic or molecular genetic techniques may be required for reliable Kell typing.

A Novel Variant B Allele of the ABO Blood Group Gene Associated with Lack of B Antigen Expression.

SUMMARY: BACKGROUND: The gene locus for the ABO blood group system encodes a glycosyltransferase. Alterations in the DNA sequence are associated with the blood groups and the expression levels of antigens on red blood cells. A number of ABO alleles have been described as the molecular basis of weak A or B antigens. PATIENTS AND METHODS: Here, we describe a novel variant B allele in a blood donor with discrepant results in routine forward (group A) and reverse (very weak anti-B isoagglutinins) ABO blood grouping. RESULTS: Determination of the ABO genotype using polymerase chain reaction-sequence-specific primers (PCR-SSP) indicated blood group A(2)B. Sequencing of the ABO gene exons 6 and 7 showed for 1 allele a G insertion into the GGGGGG sequence at position 811-816 of exon 7. The 816insG mutation (designated ABO*Bw20) led to a frame shift of the coding sequence and subsequent alteration of the protein sequence. The location of the mutation on a B allele was proven by PCR-SSP. Screening for the novel mutation in 211 blood donors with regular ABO phenotypes indicated that *Bw20 is a rare variant. CONCLUSIONS: The low levels of anti-B isoagglutinins associated with this novel variant indicate that residual undetectable amounts of B antigen may be present on red blood cells. The serological and molecular analysis of members of the blood donor's family further proved the phenotype-genotype correlation of the *Bw20 allele with antigen O and individually variable levels of anti-B isoagglutinins. The characterization of novel alleles associated with ABO subgroups may ensure the correct determination of blood groups in which serological methods are combined with molecular genetic approaches.