Erwin A. Scharberg

Molecular basis of the JAHK (RH53) antigen

BACKGROUND: The JAHK antigen was first described in 1995 as a low‐frequency red blood cell antigen. Family studies confirmed the association of the antigen with the rare rG phenotype of the Rh blood group system, which is associated with weak expression of C and e, but normal G expression. JAHK was allocated the Rh number RH53. The serologic findings indicated the location of the antigen on the RhCE protein, although the molecular basis for JAHK has not been known.

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.

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.

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

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.

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-.

Specific amino acid substitutions cause distinct expression of JAL (RH48) and JAHK (RH53) antigens in RhCE and not in RhD

Westhoff and colleagues1 and Hustinx and coworkers2 have recently reported a total of three molecular causes for the low-prevalence Rh antigen JAL (RH48). Quantitative weakening and qualitative effects are documented for these variant RhCE proteins. Similar serological effects have been observed in the JAHK+ (RH53),3 ceSL,4 and RhCe(S122P)5 variants of the RhCE protein. We have observed 4 RhD variants harboring similar amino acid substitutions at positions 114 or 122 (Table 1). Table 1 RHD alleles encoding amino acid substitutions at positions 114 and 122 Most Rh antigens are known to be associated with molecular variants of either the RHD or RHCE gene. However, distinct Rh antigens, like c (RH4), G (RH12), Rh32 (RH32), Evans (RH37) and FPTT (RH50), can be expressed by RhD and RhCE variants (Table S1, available online). We collated all low-prevalence Rh antigens caused by single nucleotide substitutions and identified pairs of RHD and RHCE alleles that harbor identical substitutions (Table S2, available online).1–4,6 Only amino acid substitutions at positions 114 and 122 were currently found to fulfill both criteria. In RhCE these substitutions cause JAL or JAHK antigen expression. Hence, we assessed the possible expression of JAL and JAHK by RhD proteins harboring similar amino acids at positions 114 or 122. A DVII sample harboring a substitution at 110 was used as negative control. All RhD samples with amino acid substitutions at position 114, weak D type 17, type 25 and type 47, were negative for the JAL antigen (Table 2). The CeMA and Rhce(R114Q) control samples were JAL positive, as expected. Because the antigen density of weak D type 17 is very low (Table 1), we corroborated its lack of JAL expression by a negative adsorption-elution test (not shown). Table 2 Serologic results of RhD and RhCE variants with amino acid substitutions at positions 114 and 122 The weak D type 54 sample with an amino acid substitution at position 122 was negative for the JAHK antigen (Table 2). The RhCe(S122L) control sample was positive with the 3 anti-JAHK sera, as expected. ceSL has been reported to be JAHK negative.4 We confirmed this result with the original red blood cell (RBC) samples and the anti-JAHK P4.53A serum used previously. However, the two anti-JAHK sera of greater titer revealed that ceSL samples were in fact JAHK positive (Table 2). Both RhCe(S122P) samples were JAHK negative with all 3 sera. The two sera P4.53B and P4.53C were identified from a series of 138 samples collected from pregnant women with a positive antibody screen.7 Both women had neither been transfused nor were their newborns or the putative fathers positive for the JAHK antigen.7 Hence, anti-JAHK is possibly a rather frequent, naturally occurring antibody associated with pregnancy. The titers of anti-JAL (Table 2) determined with CeMA and Rhce(R114Q) differed in congruence with the known weaker expression of JAL in the Rhce variants Rhce(R114Q) and ces(340) compared to the RhCe variant CeMA.2 In contrast to Rhce(R114Q) harboring Gln114, ces(340) and CeMA are caused by mutations encoding for Trp114.1,2 The model presented by Westhoff et al.1 suggests conformational changes due to Trp114 that contribute to the expression pattern of the JAL antigen. The weak JAL expression in Rhce(R114Q) may be due to the different biochemical properties of Trp (W) and Gln (Q). However, the JAL expression in ces(340), which also harbors Trp114, is distinctly weaker than in CeMA and, hence, requires a different molecular explanation (Table S3, available online):1,2,8 The additional Leu245Val substitution, known to cause a weak expression of the e antigen,8 could also induce the weak expression of the JAL antigen. Currently observed alleles cannot exclude the possibility that the weak JAL expression in both Rhce variants is caused by the 4 amino acid residues at positions 48, 60, 68, and 103, which typically differ between the RhCe and Rhce proteins. We conclude that JAL and JAHK antigens are expressed by Ce and ce variants of the RhCE protein. We found that neither antigen was expressed by any of the 4 RhD protein variants possessing similar amino acid substitutions at positions 114 and 122.

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.

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

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 A2B. 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·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.

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.

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.