Inherited glycosylphosphatidylinositol defects cause the rare Emm-negative blood phenotype and developmental disorders.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors >150 proteins to the cell surface. Pathogenic variants in several genes that participate in GPI biosynthesis cause inherited GPI deficiency disorders. Here, we reported that homozygous null alleles of PIGG, a gene involved in GPI modification, are responsible for the rare Emm-negative blood phenotype. Using a panel of K562 cells defective in both the GPI-transamidase and GPI remodeling pathways, we show that the Emm antigen, whose molecular basis has remained unknown for decades, is carried only by free GPI and that its epitope is composed of the second and third ethanolamine of the GPI backbone. Importantly, we show that the decrease in Emm expression in several inherited GPI deficiency patients is indicative of GPI defects. Overall, our findings establish Emm as a novel blood group system, and they have important implications for understanding the biological function of human free GPI.

Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation.

The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.

Renal allograft DARCness in subclinical acute and chronic active ABMR.

Gene expression profiling of renal allograft biopsies revealed the Duffy antigen receptor for chemokines (DARC) as being strikingly upregulated in antibody-mediated rejection (ABMR). DARC has previously been shown to be associated with endothelial injury. This study aimed at assessing the value of DARC immunohistochemistry as diagnostic marker in ABMR. The study was performed on 82 prospectively collected biopsies of a clinically well-defined population (BORTEJECT trial, NCT01873157) of DSA-positive patients with gene expression data available for all biopsies. Diagnostic histologic assessment of biopsies was performed according to the Banff diagnostic scheme. DARC expression was focally accentuated, on peritubular capillaries (PTC) mostly in areas of interstitial fibrosis and/or inflammation. DARC positivity was associated with diagnosis of ABMR and correlated with DARC gene expression levels detected by microarray analysis. Still, as previously described, a substantial number of biopsies without signs of rejection showed DARC-positive PTC. We did not observe significantly reduced graft survival in cases showing histologic signs of ABMR and being DARC-positive, as compared to DARC-negative ABMR. In summary, the upregulation of DARC, detected by immunohistochemistry, is associated with but not specific for ABMR. We did not observe reduced graft survival in DARC-positive patients.

A new efficient tool for non-invasive diagnosis of fetomaternal platelet antigen incompatibility.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the consequence of platelet destruction by maternal alloantibodies against fetal human platelet antigens (HPA). This may result in intracranial haemorrhages (ICH) or even fetal death. Currently, fetal HPA genotyping is performed using invasive procedures. Here, we carried out a proof-of-concept study for non-invasive prenatal diagnosis of fetal platelet genotyping in four HPA systems (HPA-1, -3, -5 and-15) by droplet digital polymerase chain reaction (ddPCR) using cell-free DNA extracts from the plasma of 47 pregnant women with suspected, or history of, FNAIT. Results showed that 74% (35/47) of pregnant women presented incompatibility in at least one HPA system, and 38% (18/47) of cases presented HPA-1 incompatibility, including nine women with multiple incompatibilities. ICH occurred in one case of profound fetal thrombocytopenia with HPA-15 incompatibility, confirming the need for non-invasive prenatal genotyping in systems other than HPA-1. Fetal HPA genotypes predicted by ddPCR were confirmed in all FNAIT cases after amniocentesis or delivery. Fetal HPA genotyping on maternal plasma based on ddPCR is a fast, safe and reliable non-invasive method. This technique will be useful for the early identification of pregnancies at high risk of FNAIT requiring antenatal management to minimize the risk of fetal/neonatal haemorrhage.

Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type and ectopic mineralization.

The Augustine-negative alias At(a-) blood type, which seems to be restricted to people of African ancestry, was identified half a century ago but remains one of the last blood types with no known genetic basis. Here we report that a nonsynonymous single nucleotide polymorphism in SLC29A1 (rs45458701) is responsible for the At(a-) blood type. The resulting p.Glu391Lys variation in the last extracellular loop of the equilibrative nucleoside transporter 1 (ENT1; also called SLC29a1) is known not to alter its ability to transport nucleosides and nucleoside analog drugs. Furthermore, we identified 3 individuals of European ancestry who are homozygous for a null mutation in SLC29A1 (c.589+1G>C) and thus have the Augustine-null blood type. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis as recently suggested by the skeletal phenotype of aging Slc29a1(-/-) mice. Our results establish Augustine as a new blood group system and place SLC29A1 as a new candidate gene for idiopathic disorders characterized with ectopic calcification/mineralization.

Lethal autoimmune hemagglutination due to an immunoglobulin A autoagglutinin with Band 3 specificity.

BACKGROUND: We describe a patient with a high-titer warm immunoglobulin (Ig)A autoantibody resulting in death due to hemagglutination rather than to hemolysis. CASE REPORT: A 47-year-old male patient presented with an intriguing pronounced vascular erythema of the skin. A livedo reticularis associated with cold agglutinin of high thermal amplitude was suspected. The patient's condition unexpectedly and abruptly deteriorated resulting in death 3 days after admission. STUDY DESIGN AND METHODS: Conventional serologic procedures and immunochemical methods were used. RESULTS: Serologic and immunochemical examinations revealed a warm IgA autoantibody of high titer with anti-Band 3 specificity. Although the patient presented with severe anemia, only mild signs of hemolysis were observed, with no evidence of complement activation. The autopsy revealed an enormous accumulation of agglutinated red blood cells in liver and spleen and a B-cell lymphoma and cerebral edema. Thus, the patient's death was largely caused by hypoxia related to hemagglutination rather than to hemolysis and/or anemia per se. CONCLUSION: Strongly hemagglutinating antibodies may not only cause immune hemolysis but also hypoxia due to intravascular hemagglutination.

Molecular analysis of the rare in(Lu) blood type: toward decoding the phenotypic outcome of haploinsufficiency for the transcription factor KLF1.

KLF1 encodes an erythroid transcription factor, whose essential function in erythropoiesis has been demonstrated by extensive studies in mouse models. The first reported mutations in human KLF1 were found in individuals with a rare and asymptomatic blood type called In(Lu). Here, we show that KLF1 haploinsufficiency is responsible for the In(Lu) blood type, after redefining this peculiar blood type using flow cytometry to quantify the levels of BCAM and CD44 on red blood cells. We found 10 (seven novel) heterozygous KLF1 mutations responsible for the In(Lu) blood type. Although most were obligate loss-of-function mutations due to the truncation of the DNA-binding domain of KLF1, three were missense mutations that were located in its DNA-binding domain and impaired the transactivation capacity of KLF1 in vitro. We further showed that the levels of the hemoglobin variants HbF and HbA(2) were increased in the In(Lu) blood type, albeit differently. The levels of the membrane glycoproteins BCAM and CD44 were also differently reduced on In(Lu) red blood cells. This biochemical and genetic analysis of the In(Lu) blood type tackles the phenotypic outcome of haploinsufficiency for a transcription factor.

Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1.

We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3(-) permeability (P(HCO3(-))) sensitive to inhibition by 4,4-Diisothiocyano-2,2-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P(HCO3(-)) and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO3(-) transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid P(HCO3(-)) was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3(-) and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H(+) makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.

A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia.

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in bone marrow. We have identified a missense mutation in KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

Lack of the human choline transporter-like protein SLC44A2 causes hearing impairment and a rare red blood phenotype.

Blood phenotypes are defined by the presence or absence of specific blood group antigens at the red blood cell (RBC) surface, due to genetic polymorphisms among individuals. The recent development of genomic and proteomic approaches enabled the characterization of several enigmatic antigens. The choline transporter-like protein CTL2 encoded by the SLC44A2 gene plays an important role in platelet aggregation and neutrophil activation. By investigating alloantibodies to a high-prevalence antigen of unknown specificity, found in patients with a rare blood type, we showed that SLC44A2 is also expressed in RBCs and carries a new blood group system. Furthermore, we identified three siblings homozygous for a large deletion in SLC44A2, resulting in complete SLC44A2 deficiency. Interestingly, the first-ever reported SLC44A2-deficient individuals suffer from progressive hearing impairment, recurrent arterial aneurysms, and epilepsy. Furthermore, SLC44A2null individuals showed no significant platelet aggregation changes and do not suffer from any apparent hematological disorders. Overall, our findings confirm the function of SLC44A2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

Blood group genotyping by high-throughput DNA analysis applied to 356 reagent red blood cell samples.

BACKGROUND: DNA analysis for the prediction of blood group antigen expression has broad implications in transfusion medicine. It may be of particular interest especially to detect variants, when antigen expression is weak or altered. The use of high-throughput DNA analysis has never been applied to donors whose red blood cells (RBCs) are selected for reagent RBCs. The aim of this study was to analyze the concordance between the serologic phenotype and that predicted from DNA analysis in panel donors, to determine the benefit of the use of DNA analysis in reagent RBC selection strategy. STUDY DESIGN AND METHODS: The "Panel National de Référence du Centre National de Référence sur les Groupes Sanguins" is a reference reagent mainly used for antibody identification. DNA genotyping of 356 panel donors was performed with BeadChips (human erythrocyte antigen v1.2 BeadChips, BioArray Solutions). The comparison between serologic phenotype and that predicted from DNA analysis held on 8876 paired results obtained from 10 blood group systems and 25 antigens. RESULTS: A 99.95% concordance was observed. Discrepancies in four cases (RH, KEL, LU, and DO systems) were analyzed. Genotyping precisions on the Duffy system were of particular interest. No new rare blood group was observed. CONCLUSION: Systematic DNA analysis of panel donors should unquestionably change the management of reagent RBC selection. The notion of "antigens in double dose" should evolve regarding data obtained from DNA analysis, allowing an improved quality of reagent RBCs for antibody screening and identification.

Generation and characterisation of Rhd and Rhag null mice.

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

Intercellular adhesion molecule-4 and CD36 are implicated in the abnormal adhesiveness of sickle cell SAD mouse erythrocytes to endothelium.

BACKGROUND: Abnormal adhesiveness of red blood cells to endothelium has been implicated in vaso-occlusive crisis of sickle cell disease. The present study examined whether the SAD mouse model exhibits the same abnormalities of red blood cell adhesion as those found in human sickle cell disease. DESIGN AND METHODS: The repertoire of adhesive molecules on murine erythrocytes and bEnd.3 microvascular endothelial cells was determined by flow cytometry using monoclonal antibodies or by western blotting. Adhesion was investigated in dynamic conditions and measured at different shear stresses. RESULTS: CD36, CD47 and intercellular adhesion molecular-4, but not Lutheran blood group antigen/basal cell adhesion molecule, are present on mouse mature erythrocytes. alpha(4)beta(1) are not expressed on SAD and wild type reticulocytes. Endothelial bEnd.3 cells express alpha(V)beta(3), alpha(4)beta(1), CD47, vascular cell adhesion molecule-1, and Lutheran blood group antigen/basal cell adhesion molecule, but not CD36. Adhesion of SAD red cells is: (i) 2- to 3-fold higher than that of wild type red cells; (ii) further increased on platelet activating factor-activated endothelium; (iii) not stimulated by epinephrine; (iv) inhibited after treating the endothelium with a peptide reproducing one of the binding sequences of mouse intercellular adhesion molecular-4, or with mon-oclonal antibody against murine alpha(v) integrin; and (v) inhibited after pretreatment of red blood cells with anti-mouse CD36 monoclonal antibodies. The combination of treatments with intercellular adhesion molecular-4 peptide and anti-CD36 monoclonal antibodies eliminates excess adhesion of SAD red cells. The phosphorylation state of intercellular adhesion molecular-4 and CD36 is probably not involved in the over-adhesiveness of SAD erythrocytes. CONCLUSIONS: Intercellular adhesion molecular-4/alpha(v)beta(3) and CD36/thrombospondin interactions might contribute to the abnormally high adhesiveness of SAD red cells. The SAD mouse is a valuable animal model for investigating adhesion processes of sickle cell disease.

A functional AQP1 allele producing a Co(a-b-) phenotype revises and extends the Colton blood group system.

BACKGROUND: The Colton blood group system currently comprises three antigens, Co(a) , Co(b) , and Co3. The latter is only absent in the extremely rare individuals of the Colton "null" phenotype, usually referred to as Co(a-b-), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a-b-) individual with no AQP1 deficiency suggested another molecular basis for the Co(a-b-) phenotype. STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped-flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction-restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells. RESULTS: Although Co(a-b-), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single-amino-acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K-562 cells, the Q47R substitution was shown to inhibit the expression of both Co(a) and Co(b) antigens. Other naturally occurring single-amino-acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system. CONCLUSIONS: The Co(a-b-) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Co(b) antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.

Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule.

Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH(3) across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, "twin-Phe." In this study, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH(3) permeability of RhCG was determined and was found to be close to that of AmtB. With a site-directed mutagenesis approach, critical residues involved in Rh NH(3) channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid was shown. In contrast to AmtB, individual mutations of each phenylalanine of the twin-Phe impaired the function while the removal of both resulted in recovery of the transport activity. The impact of the mutations suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the NH(3) transport mechanisms.

RhAG protein of the Rhesus complex is a CO2 channel in the human red cell membrane.

We have determined CO2 permeabilities, P(CO2), of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While P(CO2) of normal red cells is approximately 0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rh(null)) a significantly reduced P(CO2) of 0.07 cm/s +/-0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1(null)), suggesting that each of the Rh and AQP-1 proteins is responsible for approximately 1/2 of the normal CO2 permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO2 permeability. The CO2 pathway constituted by Rh proteins was inhibitable at pH(e)= 7.4 by NH4Cl with an I50 of approximately 10 mM corresponding to an I50 for NH3 of approximately 0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH4Cl/NH3. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal P(CO2), while in contrast another AE1 inhibitor, DiBAC, does not inhibit P(CO2), although it markedly reduces P(HCO3-). We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO2 in addition to NH3.

Identification and characterization of a novel XK splice site mutation in a patient with McLeod syndrome.

BACKGROUND: McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype-genotype correlation has been found, especially for the point mutations affecting splicing sites. STUDY DESIGN AND METHODS: A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription-polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele-specific PCR. RESULTS: A novel XK splice site mutation (IVS1-1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3' acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane. CONCLUSION: The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype-genotype correlation in the McLeod syndrome.

Heterogeneous molecular background of the weak C, VS+, hr B-, Hr B- phenotype in black persons.

BACKGROUND: The rare Hr(B)- phenotype is encoded by the (C)ce(s) haplotype when present at the homozygous state. This haplotype contains two altered genes: a hybrid RHD-CE-D(s) gene segregated with a ce(s) allele of RHCE (733C>G and 1006G>T substitutions in Exon 5 and Exon 7 respectively). The aim of this study was to further investigate the molecular background of the (C)ce(s) haplotype. STUDY DESIGN AND METHODS: Twelve individuals with depressed C and/or depressed e phenotype were selected from their genomic DNA analysis showing both 733C>G and 1006G>T substitutions. Phenotypic expression of low- and high-prevalence Rh antigens was studied. Complete sequences of RHD and RHCE transcripts were analyzed when obtained. RESULTS: A new hybrid RHD-CE-D(s) gene (Exons 1 and 2; complete Exon 3; Exons 8, 9, and 10 from RHD; and Exons 4 through 7 from RHCE) segregated with a ce(s) allele, which genomic organization was almost identical to that of the classical (C)ce(s) haplotype, is described. The two different (C)ce(s) haplotypes encoded two different patterns of Rh antigen expression. Although both encoded weak e, VS, and did not produce D, V, hr(B), or Hr(B) antigens, the new haplotype encoded a much weaker C antigen and red blood cells lacked expression of Rh42, in contrast to the classic (C)ce(s) haplotype. CONCLUSION: The study showed the heterogeneity of the molecular background of the weak C, VS+, hr(B)-, Hr(B)- phenotype in the black population. The screening of blood donors in this population for hr(B)- or Hr(B)- phenotype should implement the molecular characterization of Rh genes.