Novel KEL allele associated with loss of Kpb identified in a white blood donor.

The importance of identifying variant alleles among blood donors is significant to the safety of transfusion for recipients. Molecular methods have become more prominent in the routine process of antigen typing donor units. Some variant antigens cannot be detected using only serologic methods. Molecular testing allows the determination of nucleotide sequences that are used to predict a phenotype. Antigens of the Kell blood group system are known for being highly immunogenic and causing adverse reactions upon antibody formation. A female white blood donor who typed Kp(b-) using serologic methods on multiple donations since 2005 was the subject of a typing discrepancy investigation. Routine genotyping using a commercial genotyping kit (HemoID DQS Panel; Agena Bioscience, San Diego, CA) predicted the donor to type Kp(a+b+). Investigation of the discrepancy between these two results identified a rare single nucleotide variant in the KEL gene at nucleotide position c.948G>T that alters amino acid residue 316 from tryptophan (Trp) to cysteine (Cys). After discovery of the novel allele, adsorption and elution studies were performed to see if there was weakened Kpb expression. The elution studies yielded negative results, which indicated that Kpb is not expressed. The KEL transcripts expressed by the donor were determined using cDNA analysis, and the predicted amino acid sequence of the novel allele was modeled to investigate the impact of the amino acid sequence on the structure of the KEL polypeptide. Both SWISS-MODEL and Robetta software were used to evaluate the impact of the p.Trp316Cys on the three-dimensional protein structure. There was no conformational change noted with SWISS-MODEL, whereas the Robetta software showed a significant conformational change compared with the normal Kp(b+) reference sequence. Because the donor is homozygous for variants associated with k and Jsb expression, it was not possible to determine whether the novel allele is associated with loss of Kpb only or loss of all Kell antigens.

Possible suppression of fetal erythropoiesis by the Kell blood group antibody anti-Kp(a).

Antibodies to antigens in the Kell blood group system are usually immunoglobulin G, and, notoriously, anti-K, anti-k, and anti-Kp(a) can cause severe hemolytic transfusion reactions, as well as severe hemolytic disease of the fetus and newborn (HDFN). It has been shown that the titer of anti-K does not correlate with the severity of HDFN because, in addition to immune destruction of red blood cells (RBCs), anti-K causes suppression of erythropoiesis in the fetus, which can result in severe anemia. We report a case involving anti-Kp(a) in which one twin was anemic and the other was not. Standard hemagglutination and polymerase chain reaction (PCR)-based tests were used. At delivery, anti-Kp(a) was identified in serum from the mother and twin A, and in the eluate prepared from the baby's RBCs. PCR-based assays showed twin A (boy) was KEL*841T/C (KEL*03/KEL*04), which is predicted to encode Kp(a+b+). Twin B (girl) was KEL*841C/C (KEL*04/KEL*04), which is predicted to encode Kp(a­b+). We describe the first reported case of probable suppression of erythropoiesis attributable to anti-Kp(a). One twin born to a woman whose serum contained anti-Kp(a) experienced HDFN while the other did not. Based on DNA analysis, the predicted blood type of the affected twin was Kp(a+b+) and that of the unaffected twin was Kp(a­b+). The laboratory findings and clinical course of the affected twin were consistent with suppression of erythropoiesis in addition to immune RBC destruction.

Changes in red cell ion transport, reduced intratumoral neovascularization, and some mild motor function abnormalities accompany targeted disruption of the Mouse Kell gene (Kel).

Kell (ECE-3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo-antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin-3-converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(-/-) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(-/-) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin-3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild-type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(-/-) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell glycoprotein.

Onset of expression of the components of the Kell blood group complex.

BACKGROUND: Kell and XK, two distinct red blood cell membrane proteins, are linked by a disulfide bond and form the Kell blood group complex. Kell surface antigens are expressed early during erythropoiesis but the onset of expression of XK which carries the Kx antigen is unknown. STUDY DESIGN AND METHODS: To determine whether Kell and XK are synchronously expressed, sorted human hematopoietic progenitor cells and mouse progenitor cells of defined lineage were studied. To determine the onset of expression, human marrow and cord blood cells were sorted into three subpopulations, representing stem, multipotent, and erythroid progenitor cells, and the expression of Kell and XK was determined by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS) analysis. Mouse Kell and XK transcripts were determined by cDNA blotting of progenitor cells of defined lineage. RESULTS: By RT-PCR, human peripheral blood progenitor cells had weak expression of Kell and XK transcripts but FACS analysis did not detect surface antigens. Kell and XK transcripts are expressed in multipotent progenitor cells and these cells express Kell surface antigens. The expression of Kx antigen in progenitor cells was not determined owing to nonspecific reactions with the antibody. By cDNA blotting, mouse Kell expression was detected in bipotential megakaryocytes-erythroid cells and in colony-forming units-erythroid (CFU-E) and burst-forming units-erythroid (BFU-E), whereas XK was only detected in CFU-E and BFU-E. CONCLUSION: Both Kell and XK transcripts occur early during erythropoiesis; however, expression may not be coincident because, in mice, Kell transcripts, but not XK, occur in bipotential megakaryocytes-erythroid progenitor cells.

Kell expression on myeloid progenitor cells.

Kell is one of the major human red blood cell groups and comprises 22 antigens. These antigens are produced by alleles located on chromosome 7, including sets of antithetical antigens such as Kell (K, K1) and cellano (k, K2), which differ in a single amino acid change (T193M). It consists of a 93-Kd transmembrane glycoprotein that is surface-exposed and shares sequence and structural homology with zinc endopeptidases, which are involved in regulating bioactive peptides. Anti-Kell antibodies have been shown to suppress fetal erythropoiesis. Recently published data indicate a similar effect on myeolopoiesis and megakaryopoiesis. Substantial thrombocytopenia in fetuses affected with HDN due to anti-K antibodies led to the discovery of the inhibitory effect of Kell-related antibodies on CFU-MK growth. In addition to its inhibitory effect on BFU-E growth, anti-Kell antibodies significantly reduced CFU-GM colony formation from haematologically normal individuals. Moreover, anti-cellano and anti-Kp(b) antibodies also inhibited the growth of CFU-GM from antigen positive MNC. These data indicate that Kell is not restricted to erythroid blood cells, but is expressed on a broader spectrum of haematopoietic cells than previously believed.

Kx, a quantitatively minor protein from human erythrocytes, is palmitoylated in vivo.

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.