β-Thalassaemia Major in a Spanish Patient due to a Compound Heterozygosity for CD39 C → T/−28 A → C

A Spanish male patient with β-thalassaemia major was studied. Compound heterozygosity was found for one of the most common β-globin gene mutations in the Spanish population (codon 39 C → T) and for a mutation in the TATA box element of the β-globin gene promoter (−28 A → C mutation). To our knowledge this is the first report of a CD39 C → T and −28 A → C change association and the first report of the −28 A → C substitution in a Spanish patient

Opposing effects of Protein Kinase A and C causes differential phosphatidyserine exposure in a CD47 receptor mediated erythrocyte apoptotic pathway

Erythrocyte apoptosis, like nucleated cell death, is characterised by phosphatidylserine exposure at the outer membrane leaflet. Our group has previously reported that ligation of a monoclonal antibody, BRIC 126 can mediate red cell apoptosis and subsequent PS exposure, through a CD47 receptor mediated pathway. We have also demonstrated that several membrane proteins are involved in this pathway and one such interaction is a direct protein: protein interaction between CD47 and protein 4.1R. Further studies have shown that red cells deficient in protein 4.1R undergo increased PS exposure in response to BRIC-126 than do normal cells, suggesting that protein 4.1R is critical to this pathway. In order to further elucidate the CD47 apoptotic pathway we have inhibited Protein Kinase A and Protein Kinase C whilst ligating CD47 with BRIC-126, as these kinases are known to interact with protein 4.1R at specific serine residues and are often implicated in cell signalling cascades, especially apoptosis. We have used flow cytometry in conjunction with an annexin V-FITC binding assay to compare mean percentage annexin V positive cells whilst inhibiting protein kinase A and protein kinase C using the cell permeable specific inhibitors Bisindolylmaleimide I, Go 6976 and KT 5720. Our findings suggest opposing effects of Protein kinase A and Protein Kinase C compared with control erythrocytes. We observed that PS exposure was decreased in cells with the specific cell permeable PKC inhibitors Bisindolylmaleimide I and Go 6976 present but increased in cells with the specific PKA cell permeable inhibitor KT5720 present. The increase in PS exposure when PKA inhibitor KT5720 is present is similar to the effect seen in individuals deficient in protein 4.1R. The difference in PS exposure between PKC inhibition and PKA inhibition suggests opposing effects of PKA and PKC during CD47 ligation and a possible interaction with protein 4.1R, this may also suggest that specific phosphorylation events on protein 4.1R are critical to the CD47 apoptotic pathway. Ongoing studies are investigating isoform specific inhibition of PKC and PKA along with 2D phosphoimmunoblots to look at changes in PI before and after ligation of BRIC 126

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera

Abstract JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.This research was supported by grants from the Spanish Health office FIS PI08402, FIS PI030345, PI071009 and PI12/01728 grants; RD12/10, Red de Cancer (Cancer Network of Excellence) from the Instituto de Salud Carlos III, Spain, Fundacion Mutua madrilène; Universidad Complutense groups CCG07-UCM/BIO-2555 (Santiago Barrio and Florinda Gilsanz), FMM and CRIS foundation for cancer research (Joaquin Martinez-Lopez). The Proteomics Facility of the Centro Nacional de Biotecnología is a member of ProteoRed-Spanish National Institute for Proteomics and follows the quality criteria set up by ProteoRed standards.Peer Reviewe

4.1R-deficient human red blood cells have altered phosphatidylserine exposure pathways and are deficient in CD44 and CD47 glycoproteins

Phosphatidylserine exposure on the surface of the red cell membrane initiates the process of eryptosis, the red cell death program. The 4.1R protein is a phosphatidylserine binding protein. In this article, the authors demonstrate that erythrocytes from two patients with 4.1R deficiency show alterations of other proteins of the 4.1 multicomplex such as CD44 and CD47 and significantly increased phosphatidylserine exposure, suggesting a role for 4.1 protein in a signaling pathway relevant for red cell turnover

has a key role in polycythemia Vera

JAK-STAT signaling through the JAK2 V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80 % in PV (SD 35%) vs. 23 % in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV

High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations: A Diagnostic Tool for Myeloproliferative Neoplasms

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations