SCER and SCAN: two novel high-prevalence antigens in the Scianna blood group system

BACKGROUND: More than 20 years ago, two probands were described whose red blood cells (RBCs) typed Sc:1,-2,3. Their serum samples contained alloantibodies reactive with all RBCs tested except those of the Sc:-1,-2,-3 phenotype. Cloning of the Scianna gene allowed us to determine the molecular bases of these samples. STUDY DESIGN AND METHODS: In a collaborative effort, the two probands' samples and also two Sc:-1,-2,-3 samples were obtained from frozen storage. All 11 SC (ERMAP) exons and their flanking regions were sequenced. RESULTS: The two probands with antibodies to Scianna-related antigens were homozygous, respectively, for an ERMAP(R81Q) allele caused by a G to A substitution at nucleotide 242 in the ERMAP gene and for an ERMAP(H26Y,G35S) allele, in which the G35S substitution was caused by a G to A substitution at nucleotide 103. Two patients with the Sc:-1,-2,-3 phenotype both carried ERMAP(R332X) alleles caused by a C to T substitution at nucleotide 994 that differed at one nucleotide position in the noncoding region of exon 11. In eight samples carrying orphan low-prevalence antigens, no ERMAP variants were detected that could be implicated in Scianna antigen expression. CONCLUSION: SCER and SCAN expanded the Scianna blood group system to seven antigens, have been assigned the ISBT numbers 013.006 (Sc6) and 013.007 (Sc7), and were associated with ERMAP(R81Q) and ERMAP(G35S) proteins, respectively. ERMAP(R332X) is a second molecular basis for the Sc(null) phenotype. The eight low-prevalence antigens By, To(a), Pt(a), Re(a), Je(a), Li(a), SARA, and Sk(a) do not belong to the Scianna blood group system.     

Three new high-prevalence antigens in the Cromer blood group system

BACKGROUND: The Cromer blood group system consists of nine high-prevalence and three low-prevalence antigens carried on decay-accelerating factor (DAF). This report describes three new Cromer high-prevalence antigens, named ZENA, CROV, and CRAM. STUDY DESIGN AND METHODS: Sequence analyses were performed on DNA from three probands whose serum samples each contained an alloantibody to a high-prevalence antigen in the Cromer blood group system. Polymerase chain reaction-restriction fragment length polymorphism analysis to detect the mutation encoding the CROV- phenotype was performed on 100 Croatian donors. To map the respective epitopes, DAF deletion mutants were tested by immunoblotting with eluates containing the antibodies. RESULTS: In each proband, sequence analysis revealed a single-nucleotide substitution in DAF: ZENA, 726T>G mutation, predicted change His242Gln; CROV, 466G>A mutation, predicted change Glu156Lys; and CRAM, 740A>G mutation, predicted change Gln247Arg. By analysis of DAF deletion mutants, the CROV antigenic determinant mapped to the complement control protein (CCP) domain 2, which is encoded by exon 3, whereas ZENA and CRAM mapped to CCP4, which is encoded by exon 6. CONCLUSION: This study describes three novel high-prevalence antigens in the Cromer blood group system each characterized by a predicted single-amino-acid substitution. The antigens have been assigned the following International Society of Blood Transfusion (ISBT) numbers: ZENA is CROM13, CROV is CROM14, and CRAM is CROM15.     

Molecular basis of Cromer blood group antigens

BACKGROUND: The Cromer blood group system consists of 10 antigens located on decay-accelerating factor (DAF). Previous molecular genetic analysis has determined the basis for four of these antigens. The present study was undertaken to identify the mutations that determine the remaining antigens. STUDY DESIGN AND METHOD: Existing or new data were used to localize each Cromer system antigen to a specific short consensus repeat (SCR) domain of DAF. The exon encoding that SCR domain was amplified by using the polymerase chain reaction (PCR) on genomic DNA obtained from individuals of that Cromer phenotype, and the DNA product was subjected to DNA sequence analysis. RESULTS: The Tc(a)/Tc(c) polymorphism is due to an R18P amino acid substitution in SCR1 of DAF. The Es(a+)/Es(a-) polymorphism is due to an I46N mutation in SCR1 of DAF. The WES(b)/WES(a) polymorphism is due to an L48R mutation in SCR1 of DAF. The UMC+/UMC- polymorphism is due to a T216M substitution in SCR4 of DAF. CONCLUSIONS: With information from previous reports and the findings of this study, the molecular genetic basis of all known alleles of the Cromer blood group system has been elucidated. Single amino acid substitutions are responsible for 9 of the 10 antigens (all except the multiple-epitope antigen IFC).     

STAR: a novel high-prevalence antigen in the Scianna blood group system

BACKGROUND: More than 20 years ago, a proband was described whose red blood cells (RBCs) typed Sc:1,-2,3. His serum sample contained an immunoglobulin G alloantibody that reacted with all RBCs tested except his own, his brother's, and those with the Sc:-1,-2 phenotype. Cloning of the SC gene allowed determination of the molecular basis associated with this novel high-prevalence antigen. STUDY DESIGN AND METHODS: Samples from frozen storage were obtained from the proband, his serologically matched brother, and 15 serologically mismatched family members. DNA was extracted, and amplified products from all 11 SC (ERMAP) exons and their flanking regions of the proband were sequenced. RESULTS: A single-nucleotide mutation was detected (139G>A) in Exon 3 that is predicted to encode a change of Amino Acid 47 from glutamic acid to lysine. The sequence analyses on samples from family members were as expected. CONCLUSIONS: The absence of the high-prevalence antigen STAR detected by the proband's antibody is likely associated with lysine at Position 47 of the Sc glycoprotein. This amino acid change is located on the extracellular portion of HERMAP, 10 residues upstream from the polymorphism associated with Sc1 and Sc2 (Gly57Arg). STAR expands the Sc blood group system to five antigens and has been assigned the ISBT Number 013005 (SC5).     

Prenatal typing of Rh and Kell blood group system antigens: the edge of a watershed

Knowledge of the molecular basis of the blood group systems has enabled the development of assays for blood group genotyping. At this time, polymerase chain reaction (PCR)-based assays validated on fetal material obtained by invasive means (chorionic villus sampling or amniocentesis) are available for all clinically relevant fetal blood groups, However, only Rh typing (D, C, c, E, and e) and K1 genotyping assays are discussed in this review. Importantly, one must remember that results of genotyping assays will not always be concordant with serological typing. Thus, the RhD genotyping assays have to be modified in response to increased understanding of the molecular biology of this blood group system. RhD typing assays should produce negative results when tested on the black RhD-negative RHD alleles, RHDpsi and r's. PCR-based assays can be used to determine paternal zygosity. For RhD zygosity testing, the real-time quantitative PCR approach and the direct detection of the hybrid Rhesus box, which is the result of the deletion of the RHD gene are available. Recently, methods for noninvasive prenatal genotyping have been investigated. The use of fetal cells circulating in the maternal circulation has been explored; however, the scarcity of circulating fetal cells has limited the use of this approach. More promising are the results obtained with RhD typing assays with cell-free fetal DNA, which is present in the maternal circulation in a concentration of 25 genomic equivalents per milliliter of maternal blood in early pregnancy increasing to 100 copies per milliliter in the third trimester, which is cleared from the circulation within a few hours of delivery. The positive predictive value of this approach is virtually 100%, but false-negative results are (infrequently) encountered. Therefore, this assay can at present only be used for screening of RhD-negative women to make the use of antenatal prophylaxis more targeted and hence more cost-effective. For the clinical management of the pregnancies of alloimmunized women, the development of a control for the presence and the amplification of fetal DNA is needed, which is at present only available in male pregnancies. Assays for the genotyping of the other Rh antigens or Kell antigens with cell-free fetal DNA have not yet been described.     

Molecular basis of glycophorin C variants and their associated blood group antigens

SUMMARY. The normal and variant forms of GPC and GPD molecules carry antigens of the Gerbich blood group system. This blood group system comprises three high-incidence antigens (Ge2, Ge3 and Ge4) and four low-incidence antigens (Wb, Ls^a, Dh^a and An^a). Erythrocytes of the Ge and Yus phenotypes lack normal GPC and GPD molecules but express variant molecules (denoted GPC.Ge, GPC.Yus, respectively) that functionally substitute for normal GPC and GPD in the membrane. Leach phenotype cells lack GPC and GPD molecules and are elliptocytic in shape with a membrane that is less deformable than that of normal cells. The Ls^a antigen is expressed on higher molecular-weight variants of GPC (GPC.Ls^a) and GPD (GPD.L3^a). Wb, Dh^a and An^a antigens arise from point mutations in the GYPC gene and are expressed on GPC.Wb, GPC.Dh^a and GPD.An^a, respectively. The structure of each of the variant GPC and GPD molecules and the location of the Gerbich blood group system antigens is discussed. The GYPC gene, located on chromosome 2q¹⁴-q²¹, is 13.5 kb long and comprises four exons. Exons 1,2 and most of exon 3 encode the N -terminal extracellular domain while the remainder of exon 3 and exon 4 encode transmembrane and cytoplasmic domains of GPC. Exons 2 and 3 are highly homologous, with less than 5% nucleotide divergence. The molecular basis of generation of variation GPC and GPD molecules, and the structure of the GYPC gene from different Leach phenotype individuals, is discussed.     

Functional and structural aspects of the Kell blood group system

Two covalently linked proteins, Kell and XK, constitute the Kell blood group system. Kell, a 93-Kd type II glycoprotein, is highly polymorphic and carries all but 1 of the known Kell antigens, and XK, which traverses the membrane 10 times, carries a single antigen, the ubiquitous Kx. The Kell/XK complex is not limited to erythroid tissues and may have multiple physiological roles. Absence of one of the component proteins, XK, is associated with abnormal red cell morphology and late-onset forms of nerve and muscle abnormalities, whereas the other protein component, Kell, is an enzyme whose principal known function is the production of a potent bioactive peptide, ET-3.     

Molecular basis of the K:6,-7 [Js(a+b−)] phenotype in the Kell blood group system

BACKGROUND: The Kell blood group system consists of at least 21 antigens. KEL6(Jsa) is a low-incidence antigen that has an antithetical relationship with the high-incidence KEL7(Jsb) antigen. The molecular basis of KEL6 that appears in less than 1.0 percent of the general population, but in up to 19.5 percent of African Americans, was unknown. STUDY DESIGN AND METHODS: Nineteen exons of the Kell gene (KEL) were amplified by polymerase chain reaction (PCR) assays of genomic DNA obtained from individuals with K:6,-7 [Js(a+b-)] phenotype. The PCR products were sequenced. A comparison was made of the sequence of the PCR products and the sequence of K:-6,7, the common phenotype. RESULTS: KEL from individuals with the K:6,-7 phenotype had two base substitutions in exon 17. One was a missense mutation (T-to-C base substitution) at nucleotide (nt) 1910, which predicts an amino acid change from leucine to proline; the other was a silent substitution (A- to-C) at nt 2019. The T-to-C substitution eliminated a restriction site for Mnl I, whereas the A-to-G substitution eliminated a Dde I site. Analyses of exon 17 in seven unrelated persons with K:6,-7 phenotype by Mnl I and Dde I enzymes showed the expected presence of restriction fragment length polymorphisms. CONCLUSION: The base substitutions T-to- C at nt 1910 and A-to-G at nt 2019 are unique to KEL6. The predicted Leu–>Pro change may disrupt the alpha-helical structure and thus form the epitope for KEL6.     

Molecular bases of the antigens of the Lutheran blood group system

BACKGROUND: Lutheran is a complex blood group system consisting of 18 identified antigens. There are four pairs of allelic antigens, whereas others are independently expressed antigens of a high frequency. Lutheran antigens are carried by the Lutheran glycoproteins, which are a product of a single gene LU. STUDY DESIGN AND METHODS: Genomic DNA from 21 individuals of 12 Lutheran phenotypes was used for PCR amplification of selected LU exons that were directly sequenced and compared to control DNA of a common Lutheran phenotype. RESULTS: Lutheran phenotypes were mostly caused by single-nucleotide polymorphisms within LU, resulting in single amino acid changes. The following mutations were observed: in LU:-4, G524A, Arg175Gln; in LU:-5, G326A, Arg109His; in LU:-6,9, C824T, Ser275Phe; in LU:-8,14, T611A, Met204Lys; in LU:-13, three point mutations (C1340T, Ser447Leu, C1671T silent mutation for Ser557 and A1742T, Gln581Leu); in LU:-16, C679T, Arg227Cys; in LU:-17, G340A, Glu114Lys; and in LU:-20, C905T, Thr302Met. Two LU:-12 samples had differing results: one individual had a deletion 99GCGCTT, Arg34 and Leu35, whereas the second LU:-12 sample had a point mutation G419A, Arg140Gln. CONCLUSION: The results revealed the genetic background of 11 Lutheran antigens and suggested their placement on the Lutheran glycoprotein.     

Molecular genotyping of Indian blood group system antigens in Indian blood donors

Background

Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, In^a and In^b. The In^a /In^b polymorphism arises from 252C?>?G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles.

Unusual suppression of Kell system antigens in a healthy blood donor

Red cells of a Kp(a+) donor, ascertained as a result of screening donor red cell units with anti-Kpa, reacted only very weakly with anti-Kpb and demonstrated weakened expression of various other Kell and para-Kell antigens. Family studies revealed a Kp(a+) sister with similarly weakened expression of Kpb and other Kell and para-Kell antigens. Red cell morphology was normal in both of these individuals. This phenotype is different from any previously reported and is named the Allen phenotype.     

The KEL24 and KEL14 alleles of the Kell blood group system

BACKGROUND: The Kell blood group system consists of at least 21 antigens, which may be classified into five sets of alleles and at least 10 independently expressed antigens. The molecular basis of four of the five sets of alleles has been described; point mutations in KEL leading to amino acid substitutions characterize the alleles. In this study, the point mutation associated with the remaining allele, KEL14/KEL24, was determined. STUDY DESIGN AND METHODS: The 19 exons of KEL were amplified from genomic DNA by a polymerase chain reaction (PCR) procedure. The PCR products were sequenced. DNA sequences from unrelated KEL:14,24 and KEL:-14,24 individuals were compared to the DNA sequence of the common KEL:14,-24 phenotype. RESULTS: DNA from the KEL:14,24 person yielded both G and C at nt 659, indicating an Arg and Pro polymorphism in amino acid residue 180 of Kell protein. DNA from the KEL:-14,24 person had a G659C mutation in exon 6, indicating an Arg180Pro substitution. The G659C change introduces an Hae III restriction enzyme site, which was used to confirm the base mutations by restriction fragment length polymorphism analysis of the PCR products. CONCLUSION: A G659C mutation, predicting an Arg180Pro change in Kell protein, is associated with the KEL14/KEL24 allele.