Jill R. Storry

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.

Two new molecular bases for the Dombrock null phenotype

Summary.  Red blood cells (RBCs) with the Donull phenotype lack all antigens in the Dombrock blood group system, i.e. Doa, Dob, Gya, Hy and Joa. Sequence analysis of DNA from one proband with the Donull phenotype revealed a single nucleotide mutation of t to c in the donor splice site of DO (IVS1 + 2t > c), with outsplicing of exon 2. Analysis of a second proband revealed a homozygous nonsense mutation 442 C > T in exon 2 predicting a premature stop codon (Gln148 Stop). The molecular bases described in these two probands provide an explanation for the lack of Do glycoprotein on their RBCs.

Identification of a novel hybrid glycophorin gene encoding GP.Hop

BACKGROUND: The GP.Hop (Mi.IV) phenotype expresses the MNS low‐incidence antigens Mur, Hop, TSEN, MINY, and MUT. Because serologically similar MNS phenotypes expressing some or all of these antigens were shown to be carried by hybrid GP(B‐A‐B) proteins, it was proposed that a similar protein would be found for GP.Hop. The identification of a second GP.Hop propositus (ES) initiated a study to determine the molecular basis of this phenotype.

SERF: a new antigen in the Cromer blood group system

Summary.  The Cromer blood group system consists of eight high incidence and three low incidence antigens carried on decay‐accelerating factor (DAF). This report describes the identification and characterization of a new Cromer high incidence antigen, named SERF.

The MNS Blood Group Antigens, Vr (MNS12) and Mta (MNS14), Each Arise from an Amino Acid Substitution on Glycophorin A

Background and Objectives: The antigens, Vr (MNS12) and Mta (MNS14), are low–incidence antigens of the MNS blood group system. The Vr antigen has been found only on the red blood cells (RBCs) of persons of Dutch ancestry whereas the Mta antigen has been found on the RBCs of persons from a wide geographic distribution. The objective of this study was to determine the molecular basis of Vr and Mta. Materials and Methods: Following RT–PCR amplification of total RNA isolated from one Vr+ person (G488) and one Mt(a+) person (GH), the genes encoding glycophorin A (GYPA) and glycophorin B (GYPB) were cloned and sequenced. To confirm the point mutation observed in the cDNA from G488 (Vr+), GYPA exon 3 was cloned and sequenced from the genomic DNA of G488 and a second unrelated Vr+ person (MU). A restriction fragment length polymorphism (RFLP) assay was used to analyze genomic DNA from 11 Mt(a+) persons (10 unrelated) following PCR amplification of GYPA exon 3. Results: The coding sequence of GYPB was normal in both G488 (Vr+) and GH (Mt(a+)). Sequencing data from GYPA clones derived from G488 showed to full length GYPA sequences: A normal GYPA M allele and a GYPA M allele with a point mutation 197C→A. Sequencing of GYPA exon 3 from G488 and MU confirmed the point mutation. Sequencing data drom GYPA clones derived from GH showed two full length GYPA sequences: a normal GYPA M allele and a GYPA N allele with a point mutation 230C→T. RFLP analysis based on the point mutation showed that DNA from 11 Mt(a+) samples were heterozygous for the point mutation. Conclusion: The Vr antigen arises from a point mutation 197C→A on GYPA which is predicted to change serine at position 47 to tyrosine. This change introduces a new α–chymotrypsin cleavage site. The Mta antigen arises from a point mutation 230C→T which is predicted to change threonine at position 58 to isoleucine.

Rh haplotypes that make e but not hrB usually make VS.

Background and objectives: The Rh phenotypes hrB– and VS+ are both rare in Whites but more common in Blacks. The high‐incidence antigen hrB is present on most red cells that are e+. The presence of VS on red cells is associated with an aberrant expression of e, often called eS. Materials and methods: Using conventional serologic methods, including a monoclonal anti‐hrB‐like antibody, we studied 65 e+ samples that were apparently hrB–. Results: Of the 65, we found that 59 (91%) were VS+. Recent findings have indicated that in VS+ persons a change from leucine to valine occurs at amino acid 245 of the RHCE‐encoded polypeptide. While this residue is predicted to lie within the red cell membrane bilayer, the change presumably affects alanine 226 (that is present when e is expressed) in such a way that eS is seen. Conclusions: Our findings suggest that the change from e to eS may result in nonexpression or marked depression of expression of hrB that is, perhaps, an epitope of e. While the molecular basis of the hrB– phenotype is not known, it is unlikely that the leucine‐to‐valine change at residue 245, resulting in the aberrant form of e, explains all hrB– samples. First, hrB– VS+ and hrB– VS– samples must differ. Second, some hrB– VS+ samples are C+, some are C–. Presumably diverse molecular bases are involved in hrB– phenotypes.

Alloanti-c in a c-positive, JAL-positive patient.

Background and Objectives  In the Rh blood group system, partial D, C, and e antigens are well‐known, but a partial c antigen resulting in the production of alloanti‐c in a c+ individual is rare. One example of an alloanti‐c in a c+ person was an anti‐Rh26, which can appear as anti‐c, and another was an alloanti‐c in a c+ person with a presumed R1r phenotype. The finding of an apparent alloanti‐c in a transfused c+ patient initiated this investigation.

First Example of Hemolytic Disease of the Newborn Caused by Anti-Or and Confirmation of the Molecular Basis of Or

Background and Objectives: The rare MNS antigen Or (MNS31) is sensitive to ficin, papain and sialidase, but partially resistant to trypsin (0.05%); the effect of α-chymotrypsin is not known. A point mutation, 204C → T in exon 3 of GYPA, is associated with the Or+ phenotype. We report here the first case of hemolytic disease of the newborn (HDN) caused by anti-Or, and expand the information on the nature of the Or determinant. Materials and Methods: A woman, gravida 4, para 0, delivered a baby whose red blood cells (RBCs) were positive (2+) on the direct antiglobulin test (DAT). The mother’s serum, an eluate made from the baby’s RBCs and the RBCs of the baby’s father were investigated. Exon 3 of GYPA, extracted from the father’s genomic DNA, was amplified and sequenced. Results: The mother’s serum reacted at room temperature, 37°C and on the indirect antiglobulin test with RBCs from the baby’s father. The father’s RBCs were M+N+S–s+Or+. The antibody in the mother’s serum and in the baby’s eluate was identified as anti-Or. The serum did not react with the father’s RBCs treated with trypsin (180,000 U/ml), but did react with his α-chymotrypsin-treated RBCs. Amplification and sequencing of DNA from the father revealed a single point mutation, 204C → T, in GYPA exon 3. At birth, the baby had clinical symptoms of HDN and was transfused with 36 ml of packed RBCs and received phototherapy for eight days. At week 11, the baby’s M+N+S+s+Or+ RBCs were negative on the DAT. Conclusion: This is the first case of HDN caused by anti-Or. The observed point mutation, 204C → T, confirms that of a previous report and predicts a change of Arg (Or–) to Trp (Or+) at amino acid 31.

Four Examples of Anti-TSEN and Three of TSEN-Positive Erythrocytes

Abstract Background: TSEN (MNS33) is a low‐incidence antigen in the MNS blood group system encoded by hybrid glycophorin genes. TSEN is expressed by a unique amino acid sequence that results from the junction of GPA58 to GPB27, if the GPB carries S antigen. Until this study, only one example of anti‐TSEN had been found. Antibody screening red blood cells (RBCs) positive for both S and s (ref. No. C873) reacted with four patient sera. Initially, the RBCs had been typed as S+s+, but later were typed as S‐s+ in another laboratory. Two other RBC samples, one from a volunteer blood donor (D.L.), the other from a patient whose serum contained anti‐EnaFR (J.S.), also gave anomalous results when tested with anti‐S. We sus pected the presence of TSEN‐positive hybrids on all three RBC samples. Materials and Methods: Reactive sera (O.B., E.C., S.K., R.F.) were tested against RBCs with normal MNS phenotypes and with TSEN‐positive RBCs. The RBCs of D.L., J.S. and C873 were tested with anti‐S whose reactivity with S+s+ TSEN+RBCs had been established previously, and with the original example of anti‐TSEN. Immunoblotting was performed on the C873, D.L. and J.S. RBC membranes using a monoclonal antibody to an epitope common to both glycophorin A and glycophorin B. Results: The sera from O.B., E.C., S.K. and R.F. were strongly reactive on the indirect antiglobulin test with TSEN+ RBCs. The RBCs of C873, D.L. and J.S. were typed as TSEN+. Immunoblotting pattern of D.L. and C873 were consistent with TSEN heterozygotes, while that of J.S. was consistent with a TSEN homozygote. Conclusions: Based on the estimated number of screening events with C873 RBCs, the incidence of anti‐TSEN is approximately 1 in 20,000 sera. The antibody is found in patients with and without documented exposure to allogeneic RBCs. All known examples of anti‐TSEN are lgG, but their clinical significance is not known.