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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.


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.


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.


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.


We present here seven new hemoglobin (Hb) variants identified during routine Hb analysis. All of them are caused by a missense mutation except Hb Saint Chamond, which results from an in-frame deletion of the asparagine residue at β80. All these variants are clinically silent in the heterozygous state but two of them (Hb Cap d'Agde and Hb Dompierre) may be unstable, whereas Hb Nîmes could present a very slightly elevated oxygen affinity. These data are to be confirmed by appropriate biochemical tests. © 2015 © 2015 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted.


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 | Francina A.,Unite de Pathologie Moleculaire du Globule Rouge
Hemoglobin | Year: 2013

We present two case reports in which an HBD mutation is present with a rare α hemoglobinopathy that substantially complicates the associated phenotype. In the first case, a new δ-globin variant, Hb A2-Pierre-Bénite [δ83(EF7)Gly→Arg; HBD: c.250G>C] is associated with Hb Groene Hart [α119(H2)Pro→Ser (α1); HBA1: c.358C>T], an α-thalassemic variant. In the second case, a δ+-thalassemic variant, δ4(A1)Thr→Ile; HBD: c.14C>T, is associated with a newly described deletion of the hypersensitive site 40 (HS-40) region on the α-globin gene cluster. In both patients, a δ-globin mutation was suspected because of an abnormally low Hb A2 level, whereas the α hemoglobinopathy was sought to explain the slight microcytosis and hypochromia presented by the probands. Copyright © Informa Healthcare USA, Inc.


Joly P.,Unite de Pathologie Moleculaire du Globule Rouge | Joly P.,University of Lyon | Lacan P.,Unite de Pathologie Moleculaire du Globule Rouge | Garcia C.,Unite de Pathologie Moleculaire du Globule Rouge | And 2 more authors.
Clinical Biochemistry | Year: 2010

Objectives: The Mediterranean and A(-) G6PD variants are particularly prevalent in Africa and Southern Europe. Our study was aimed to develop an assay for the rapid genotyping of these two variants by HRM. Methods: After PCR reactions corresponding to the G6PD Mediterranean (exon 6), G6PD (A-) (exon 4) and G6PD (A-) (exon 5) mutations, amplicons were submitted to HRM. This protocol was applied to a cohort of 132 patients suffering from sickle cell disease. Results: Wild, homozygous or hemizygous and heterozygous states were fully discriminated by HRM for all three mutations. HRM results were in total accordance with DNA sequencing for 22 patients of our cohort with a 'A' genotype: presence of the (A-) (exon 5) mutation but absence of the (A-) (exon 4) mutation. Conclusions: Our HRM protocols allow a rapid, simple and cost-effective screening of G6PD deficiency in patients originating from the Mediterranean and the African areas. © 2009 The Canadian Society of Clinical Chemists.


PubMed | Unite de Pathologie Moleculaire du Globule Rouge
Type: Case Reports | Journal: Hemoglobin | Year: 2011

Deletions in the -globin locus control region (-LCR) lead to ()(0)-thalassemia [()(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect -LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp -LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the -LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an -thal trait. Consequently, a complete - and -globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling.


PubMed | Unite de Pathologie Moleculaire du Globule Rouge
Type: Case Reports | Journal: Hemoglobin | Year: 2012

We report a new mutation on the 2-globin gene causing (+)-thalassemia ((+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47.


We present here seven new hemoglobin (Hb) variants identified during routine Hb analysis. All of them are caused by a missense mutation except Hb Saint Chamond, which results from an in-frame deletion of the asparagine residue at 80. All these variants are clinically silent in the heterozygous state but two of them (Hb Cap dAgde and Hb Dompierre) may be unstable, whereas Hb Nmes could present a very slightly elevated oxygen affinity. These data are to be confirmed by appropriate biochemical tests.

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