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Satchwell T.J.,University of Bristol | Pellegrin S.,University of Bristol | Bianchi P.,Hematology and Transplantation Unit | Hawley B.R.,University of Bristol | And 9 more authors.
Haematologica | Year: 2013

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse. © 2013 Ferrata Storti Foundation.

Lucas G.,Histocompatibility and Immunogenetics | Poles A.,Histocompatibility and Immunogenetics | Wozniak M.J.,Bristol Institute of Transfusion science | Gilmore R.,Galway University Hospital
Transfusion | Year: 2016

BACKGROUND Most recently described human platelet antigens (HPAs) have been low-frequency polymorphisms identified in cases of fetomaternal alloimmune thrombocytopenia (FMAIT). There is limited opportunity to study the clinical significance or different antenatal management strategies in cases involving low-frequency HPA antibodies because many are single pregnancies. We have previously described a low-frequency platelet (PLT) antigen, HPA-28bw, implicated in FMAIT in two of the three infants in the same family. This report describes the outcome of an additional two pregnancies in this family. STUDY DESIGN AND METHODS The fourth and fifth pregnancies in a HPA-28bw-alloimmunized mother with a heterozygous partner were investigated to determine the risk of FMAIT. The presence of anti-HPA-28bw was assessed by paternal crossmatch studies. Prenatal HPA genotyping of amniocytes was performed to inform antenatal management. RESULTS GPIIb/IIIa antibodies reactive only with paternal PLTs were detected. These antibodies had been previously identified as HPA-28bw specific using recombinant GPIIb glycoprotein mutated to contain the HPA-28bw (V740L) mutation. The fetus in the fourth pregnancy did not inherit the HPA-28bw mutation, no antenatal management was required, and the baby had a normal PLT count. The fetus in the fifth pregnancy did inherit the HPA-28bw mutation. The mother received IVIG (2 g/kg/week) and prednisolone during pregnancy, and the baby was born with a normal PLT count. CONCLUSION Study of this family has provided a unique opportunity to assess the clinical significance of antibodies against the low-frequency PLT antigen (HPA-28bw) during five pregnancies and to compare the outcomes of different antenatal treatments. © 2015 AABB.

Satchwell T.J.,University of Bristol | Satchwell T.J.,Bristol Institute of Transfusion science | Hawley B.R.,University of Bristol | Hawley B.R.,Bristol Institute of Transfusion science | And 4 more authors.
Haematologica | Year: 2015

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. © 2014 Ferrata Storti Foundation.

Bell A.J.,University of Bristol | Satchwell T.J.,University of Bristol | Heesom K.J.,University of Bristol | Hawley B.R.,University of Bristol | And 6 more authors.
PLoS ONE | Year: 2013

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes. © 2013 Bell et al.

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