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Qadah T.,Head of Experimental Haematology Research Unit | Qadah T.,University of Western Australia | Qadah T.,King Abdulaziz University | Finlayson J.,Head of Experimental Haematology Research Unit | And 5 more authors.
Hemoglobin | Year: 2014

In this study, we describe the clinical features and provide experimental analyses of a novel point mutation affecting the penultimate nucleotide of the first exon of the HBA2 (HBA2: c.94A>G) gene identified in a 26-year-old female who also carries a heterozygous Hb E (HBB: c.79G>A) variant. The aim of the study was to investigate the impact of this point mutation on the transcriptional activity of the HBA2 gene using a combination of an initial in silico prediction followed by in vitro mutagenesis and transcriptional activity assessment. The analyses revealed that the HBA2: c.94A>G point mutation causes the activation of a cryptic splice site located 49bp upstream of the exon1-intron1 boundary in both HBA2 long and short isoforms, thus generating a frameshift and a premature termination codon between codons 48 and 49 in the second exon. A rapid degradation of the aberrantly spliced transcripts by the nonsense mediated decay (NMD) surveillance system is highly indicative of an α-thalassemia (α-thal) phenotype. However, the abnormal mRNA may not be entirely degraded since the proband presents a slight splenomegaly that could be the sign of extra vascular hemolysis. © 2014 Informa Healthcare USA, Inc. Source


Joly P.,Unite de Pathologie Moleculaire du Globule Rouge | Wajcman H.,French Institute of Health and Medical Research | Francina A.,Unite de Pathologie Moleculaire du Globule Rouge
Annales de Biologie Clinique | Year: 2010

The Library of variants (LOV) v. 1.0 CD-Rom is a digital library of more than 200 typical cation-exchange HPLC graphs, for the phenotype determination of a haemoglobin disorder. These graphs, presented on a case-report form, have been obtained with the 4 Bio-Rad liquid chromatography devices available for haemoglobin analysis: D-10™, Variant™, Variant II™ and Variant nbs™. In case of an atypical HPLC pattern obtained in clinical practice, this library will be of a potential useful help for the biologist to make a presumptive diagnosis of the type of haemoglobinopathy. Nevertheless, the definitive characterization will have to be made by molecular biology in a reference laboratory. Source


Lapillonne H.,French Institute of Health and Medical Research | Lapillonne H.,University Pierre and Marie Curie | Kobari L.,French Institute of Health and Medical Research | Kobari L.,University Pierre and Marie Curie | And 16 more authors.
Haematologica | Year: 2010

Background: Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine. Design and Methods: We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin. Results: We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form. Conclusions: Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients. © 2010 Ferrata Storti Foundation. Source


Kobar L.,University Pierre and Marie Curie | Kobar L.,French Institute of Health and Medical Research | Yates F.,University Paris - Sud | Oudrhiri N.,University Paris - Sud | And 24 more authors.
Haematologica | Year: 2012

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. © 2012 Ferrata Storti Foundation. Source


Joly P.,Unite de Pathologie Moleculaire du Globule Rouge | Lacan P.,Unite de Pathologie Moleculaire du Globule Rouge | Garcia C.,Unite de Pathologie Moleculaire du Globule Rouge | Desbree A.,Jean Monnet University | And 2 more authors.
Hemoglobin | Year: 2013

We report two new variants of the δ-globin gene: Hb A2-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A2-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon. © 2013 Informa Healthcare USA, Inc. Source

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