Joyce Poole

The Fyx phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein

The molecular basis of the three major alleles (Fya/Fyb/Fy) of the Duffy (FY) blood group system has recently been established but the Fyx phenotype associated with weak expression of the Fyb and other FY antigens is poorly understood.

Molecular basis of the globoside-deficient Pk blood group Phenotype.Identification of four inactivating mutations in the UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase gene

The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, Pk, and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood. Individuals with the rare but clinically important erythrocyte phenotypes P1 k and P2 klack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriatedEscherichia coli. As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed. To define the molecular basis of the Pk phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-β-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-β-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C202 → T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P2 k phenotype. Two individuals with P1 k and P2 k phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase. We conclude that crucial mutations in the globoside synthase gene cause the Pk phenotype.

Expression of phosphatidylserine (PS) on wild-type and Gerbich variant erythrocytes following glycophorin-C (GPC) ligation

Glycophorin‐C (GPC) is a 40 kDa glycoprotein expressed on erythrocytes and is a receptor for the malarial parasite Plasmodium falciparum to invade these cells. A link between GPC binding (ligation) and phosphatidylserine (PS) expression on erythrocytes has been suggested by its appearance on P. falciparum‐infected erythrocytes. Phosphatidylserine expression has also been shown to be a marker of cellular death in a number of biological pathways including some in erythrocytes. Using Annexin V binding, we demonstrated that ligation of GPC with mouse mAb (BRIC‐10) induced PS expression on normal erythrocytes. Phosphatidylserine exposure was prevented following tryptic digestion of intact erythrocytes. In addition, GPC variant phenotypes Yus (Δ exon 2) and Gerbich (Δ exon 3), which express a truncated extracellular domain, did not express PS following BRIC‐10 binding, whereas PS was exposed on Lsa erythrocytes (duplication of exon 3). GPC ligation was also shown to result in a concomitant loss of erythrocyte viability in wild‐type erythrocytes after 24 h in vitro. These results identify a potential pathway linking GPC to PS exposure on erythrocytes that may have a role in regulating red cell turnover. Further characterization of this pathway may also identify new targets for the treatment of P. falciparum malaria.

Novel alleles at the JK blood group locus explain the absence of the erythrocyte urea transporter in European families.

Summary.  The Kidd (JK) blood group system is of importance in transfusion medicine. The Jknull phenotype is associated with absence of the urea transporter in erythrocytes and moderately reduced ability to concentrate urine. We and others recently reported different molecular alterations in the silenced Jkb‐like alleles of Polynesians and Finns, populations with higher Jknull frequencies. Here we report novel molecular bases of this phenotype in Caucasians. Blood samples from a Swiss and an English family were investigated by serological methods, urea haemolysis test and JK genotyping. Genomic DNA and JK mRNA were sequenced. Genotyping showed homozygosity for Jka‐like alleles. The Swiss Jknull alleles deviated from wild‐type Jka sequence by a nonsense mutation in exon 7 causing an immediate stop codon (Tyr194stop). The English Jknull alleles revealed a genomic 1·6 kilobase pair deletion including exons 4 and 5, the former of which includes the translation start codon. Multiple mRNA splicing variants were detected in reticulocytes but exons 3–5 were absent in all transcripts analysed. Screening for these alleles was negative in random donors. Two novel molecular alterations at the JK locus were defined and a multiplex polymerase chain reaction method for detection of the five known silent Jk alleles was developed to complement JK genotyping in clinical transfusion medicine.

Additional molecular bases of the clinically important p blood group phenotype

BACKGROUND: The purpose of this study was to explore the molecular basis of the p phenotype by analysis of the recently cloned 4‐α‐galactosyltransferase gene responsible for synthesis of Pk (Gb3) antigen.

D variants at the RhD vestibule in the weak D type 4 and Eurasian D clusters

BACKGROUND: One branch of the RHD phylogenetic tree is represented by the weak D type 4 cluster of alleles with F223V as the primordial amino acid substitution. F223V as well as a large number of further substitutions causing D variants are located at the extracellular RhD protein vestibule, which represents the entrance to the transmembraneous channel of the RhD protein.

Transient loss of proteins carrying Kell and Lutheran red cell antigens during consecutive relapses of autoimmune thrombocytopenia

A patient is described in whom two consecutive relapses of autoimmune throbocytopenic purpura (AITP) were associated with loss of red cell antigens of the Kell and Lutheran blood group systems respectively. During the second relapse the glycoprotein CD44 and to a lesser extent the LW antigen were also depressed. Both relapses were associated with concomitant production of IgG antibody recognizing high‐frequency determinants on the corresponding antigen‐carrying protein. Blocking of antigen sites by these antibodies was not the cause of reduced antigen expression, because immunoblotting studies showed absence of Kell protein during the first relapse, and Lutheran protein during the second. On both occasions the red cell changes reverted to normal with disappearance of the antibody as the AITP entered remission. There was no evidence of clonal lymphocyte expansion as demonstrated using immunoglobulin JH and T cell receptor β chain probes.

Haemolytic Disease of the Newborn Due to Anti‐G

Mrs P. presented at 13 weeks of gestation with apparent anti‐C+D. At week 34, with antibody levels of 168 IU/ml, a D‐negative (r′r) baby was delivered with a strongly positive DAT and an Hb of 3.0 g/dl. Anti‐G in maternal serum was isolated by adsorption and elution from R2R2 cells and shown, using flow‐cytometric and chemiluminescence assays, to sensitize r′r cells at levels of cell‐bound IgG consistent with fetal haemolysis. In an analysis of 28 sera from alloimmunized women with over 5 IU/ml anti‐C+D, 2 sera were shown to contain levels of anti‐G consistent with moderate or severe haemolytic disease of the newborn (HDN). Thus HDN due to anti‐G may not be rare. An analysis of 187,037 blood donors in the south‐west of England showed the r′ gene frequency to be 0.005897 suggesting that approximately 2.9% of matings of rr women with D‐negative fathers can produce an r′r baby. These findings highlight the need for the continuous non‐invasive monitoring of D‐negative fetuses of women with apparent anti‐C+D.

A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen

BACKGROUND: The KEL2/KEL1 (k/K) blood group polymorphism represents 578C>T in the KEL gene and Thr193Met in the Kell glycoprotein. Anti‐KEL1 can cause severe hemolytic disease of the fetus and newborn. Molecular genotyping for KEL*1 is routinely used for assessing whether a fetus is at risk. Red blood cells (RBCs) from a KEL:1 blood donor (D1) were found to have abnormal KEL1 expression during evaluation of anti‐KEL1 reagents.

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.