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1. The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70

Author

Marc Gastou, Sarah Rio, Michaël Dussiot, Narjesse Karboul, Hélène Moniz, Thierry Leblanc, Margaux Sevin, Patrick Gonin, Jérome Larghéro, Carmen Garrido, Anupama Narla, Narla Mohandas, William Vainchenker, Olivier Hermine, Eric Solary, and Lydie Da Costa

Subjects
Specialties of internal medicine, RC581-951
Abstract

Abstract: Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation–dependent DBA.

Published
2017
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2. Abstract 186: CD36 Antagonism Minimizes Skin Scarring By Inhibiting JUN-dependent Fibrotic Pathways Within Fibrogenic Fibroblast Subpopulations

Author

Mimi R. Borrelli, MBBS, MSc, Julia Garcia, PhD, Michelle Griffin, MD, PhD, Nestor M. Deleon Diaz, Alexandra L. Moore, MD, Shamik Mascharak, MS, Bryan Duoto, MS, Heather desJardins-Park, Sandeep Adem, MS, Tristan Lerbs, PhD, Marc Gastou, PhD, Lui Cui, PhD, Michael Januszyk, MD, PhD, Hermann P. Lorenz, MD, Derrick C. Wan, MD, Geoffrey C. Gurtner, MD, Howard Chang, MD, PhD, Gerlinde Wernig, MD, and Michael T. Longaker, MD, MA

Subjects
Surgery, RD1-811
Published
2020
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3. XPO1 regulates erythroid differentiation and is a new target for the treatment of β-thalassemia

Author

Flavia Guillem, Michaël Dussiot, Elia Colin, Thunwarat Suriyun, Jean Benoit Arlet, Nicolas Goudin, Guillaume Marcion, Renaud Seigneuric, Sebastien Causse, Patrick Gonin, Marc Gastou, Marc Deloger, Julien Rossignol, Mathilde Lamarque, Zakia Belaid Choucair, Emilie Fleur Gautier, Sarah Ducamp, Julie Vandekerckhove, Ivan C. Moura, Thiago Trovati Maciel, Carmen Garrido, Xiuli An, Patrick Mayeux, Narla Mohandas, Geneviève Courtois, and Olivier Hermine

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

β-thalassemia major (β-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human β-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of β-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of β-TM.

Published
2019
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4. Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in Diamond-Blackfan anemia

Author

Marcin W. Wlodarski, Lydie Da Costa, Marie-Françoise O’Donohue, Marc Gastou, Narjesse Karboul, Nathalie Montel-Lehry, Ina Hainmann, Dominika Danda, Amina Szvetnik, Victor Pastor, Nahuel Paolini, Franca M. di Summa, Hannah Tamary, Abed Abu Quider, Anna Aspesi, Riekelt H. Houtkooper, Thierry Leblanc, Charlotte M. Niemeyer, Pierre-Emmanuel Gleizes, and Alyson W. MacInnes

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Published
2018
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5. Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway.

Author

Harry F Heijnen, Richard van Wijk, Tamara C Pereboom, Yvonne J Goos, Cor W Seinen, Brigitte A van Oirschot, Rowie van Dooren, Marc Gastou, Rachel H Giles, Wouter van Solinge, Taco W Kuijpers, Hanna T Gazda, Marc B Bierings, Lydie Da Costa, and Alyson W MacInnes

Subjects
Genetics, QH426-470
Abstract

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.

Published
2014
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6. JUN promotes hypertrophic skin scarring via CD36 in preclinical in vitro and in vivo models

Author

Jan Sokol, Shamik Mascharak, Michelle Griffin, Heather E. desJardins-Park, Howard Y. Chang, Michael T. Longaker, Yuning Wei, Abra H. Shen, Walter L. Taylor, Bryan Duoto, Mimi R. Borrelli, Tristan Lerbs, Julia T. Garcia, Lu Cui, Alessandra L. Moore, Michael Januszyk, Gerlinde Wernig, Geoffrey C. Gurtner, Marc Gastou, Ronak A. Patel, Hermann P. Lorenz, Derrick C. Wan, Kellen Chen, Sandeep Adem, Megan King, Nestor M. Diaz Deleon, and Deshka S. Foster

Subjects
CD36 Antigens, Pathology, medicine.medical_specialty, Cicatrix, Hypertrophic, Proto-Oncogene Proteins c-jun, CD36, Skin Diseases, Article, Mice, In vivo, Hypertrophic skin, medicine, Animals, Humans, Skin pathology, Skin, integumentary system, biology, Extramural, business.industry, General Medicine, In vitro, Hypertrophic scarring, biology.protein, business, SKIN SCARRING
Abstract

Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which Jun is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. Jun overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating CD36. Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.

Published
2021
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7. Abstract 186: CD36 Antagonism Minimizes Skin Scarring By Inhibiting JUN-dependent Fibrotic Pathways Within Fibrogenic Fibroblast Subpopulations

Author

Heather E. desJardins-Park, Shamik Mascharak, Michelle Griffin, Nestor M. Deleon Diaz, Lui Cui, Michael T. Longaker, Geoffrey C. Gurtner, Bryan Duoto, Howard Y. Chang, Mimi R. Borrelli, Tristan Lerbs, Michael Januszyk, Marc Gastou, Derrick C. Wan, Sandeep Adem, Gerlinde Wernig, Hermann P. Lorenz, Julia T. Garcia, and Alexandra L. Moore

Subjects
biology, business.industry, CD36, PSRC Abstract Supplement, lcsh:Surgery, lcsh:RD1-811, medicine.anatomical_structure, medicine, Cancer research, biology.protein, Surgery, Fibroblast, business, Antagonism, SKIN SCARRING
Published
2020

8. Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism

Author

Marianna Penzo, Riekelt H. Houtkooper, Marjolijn C.J. Jongmans, Alexandra Nin-Velez, Carolina Marques dos Santos Vieira, Evelien Zonneveld-Huijssoon, Alexandra N. Olson, Roland P. Kuiper, Anne-Marie Hesse, Marie-Françoise O'Donohue, Marc Bierings, Hyung L. Elfrink, Michel van Weeghel, Marco Lezzerini, Manon Saby, Peter G. J. Nikkels, Alyson W. MacInnes, Lydie Da Costa, Illja J. Diets, Pierre-Emmanuel Gleizes, Jonathan D. Dinman, Marc Gastou, Lorenzo Montanaro, Yohann Couté, Thierry Leblanc, GuangJun Zhang, Marcin W. Wlodarski, Jutte van der Werff ten Bosch, Lezzerini, Marco, Penzo, Marianna, O'Donohue, Marie-Françoise, Marques Dos Santos Vieira, Carolina, Saby, Manon, Elfrink, Hyung L, Diets, Illja J, Hesse, Anne-Marie, Couté, Yohann, Gastou, Marc, Nin-Velez, Alexandra, Nikkels, Peter G J, Olson, Alexandra N, Zonneveld-Huijssoon, Evelien, Jongmans, Marjolijn C J, Zhang, GuangJun, van Weeghel, Michel, Houtkooper, Riekelt H, Wlodarski, Marcin W, Kuiper, Roland P, Bierings, Marc B, van der Werff Ten Bosch, Jutte, Leblanc, Thierry, Montanaro, Lorenzo, Dinman, Jonathan D, Da Costa, Lydie, Gleizes, Pierre-Emmanuel, MacInnes, Alyson W, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), The Beatson Institute for Cancer Research, University of Glasgow, Human Genetics, Radboud University Medical Center [Nijmegen], Unité d'Hémato-Immunologie pédiatrique [Hôpital Robert Debré, Paris], Service d'Immuno-hématologie pédiatrique [Hôpital Robert Debré, Paris], Hôpital Robert Debré-Hôpital Robert Debré, Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), ANR-15-CE12-0001,DBA-MULTIGENES,DBA-MULTIGENES: identification et caractérisation de gènes candidats pour la découverte de nouveaux mécanismes physiopathologiques à l'origine de l'anémie de Blackfan-Diamond.(2015), Laboratory Genetic Metabolic Diseases, AGEM - Endocrinology, metabolism and nutrition, AGEM - Inborn errors of metabolism, APH - Aging & Later Life, ACS - Diabetes & metabolism, ACS - Heart failure & arrhythmias, Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Clinical sciences, Growth and Development, and Pediatrics

Subjects
Male, INTELLECTUAL DISABILITY, Ribosomopathy, [SDV]Life Sciences [q-bio], Ribosome biogenesis, medicine.disease_cause, Ribosome, PATHWAY, 0302 clinical medicine, RPL9, diamond blackfan anemia, DBA, ribosomes, ribosomopathy, Tumours of the digestive tract Radboud Institute for Molecular Life Sciences [Radboudumc 14], RNA Precursors, Missense mutation, FAILURE, ASSAY, Variants in ribosomal protein, RNA Processing, Post-Transcriptional, Child, Cancer, Anemia, Diamond-Blackfan, Medicine(all), Genetics, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 0303 health sciences, Mutation, ABNORMALITIES, [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, 3. Good health, 030220 oncology & carcinogenesis, Carcinoma, Squamous Cell, Female, Eukaryotic Ribosome, Adult, Ribosomal Proteins, Adolescent, DATABASE, DIAMOND-BLACKFAN ANEMIA, Biology, bone marrow failure syndrome, 03 medical and health sciences, Erythroid Cells, Ribosomal protein, Exome Sequencing, medicine, Humans, RNA, Messenger, Gene, Molecular Biology, 030304 developmental biology, PREDISPOSITION, MODEL, Genes, Cellular Metabolism, 5' Untranslated Regions, Ribosomes, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Contains fulltext : 218593.pdf (Publisher’s version ) (Open Access) Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

Published
2020
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9. Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in diamond-blackfan anemia

Author

Dominika Danda, Marcin W. Wlodarski, Riekelt H. Houtkooper, Ina Hainmann, Marie-Françoise O'Donohue, Victor B Pastor, Charlotte M. Niemeyer, Amina Szvetnik, Thierry Leblanc, Narjesse Karboul, Anna Aspesi, Marc Gastou, Lydie Da Costa, Nahuel A. Paolini, Abed Abu Quider, Alyson W. MacInnes, Pierre-Emmanuel Gleizes, Hannah Tamary, Franca di Summa, Nathalie Montel-Lehry, Landsteiner Laboratory, Laboratory Genetic Metabolic Diseases, AGEM - Endocrinology, metabolism and nutrition, AGEM - Inborn errors of metabolism, APH - Aging & Later Life, ARD - Amsterdam Reproduction and Development, AP-HP, Service d'Hématologie Biologique, Hôpital Robert-Debré, Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de recherche translationnelle, Institut Curie [Paris], Pediatric Hematology Unit (Pediatric Hematology Unit), Schneider Children's Medical Center of Israel, Dept. Medical Sciences, Università degli Studi del Piemonte Orientale - Amedeo Avogadro (UPO), The Beatson Institute for Cancer Research, University of Glasgow, Jet Propulsion Laboratory (JPL), NASA-California Institute of Technology (CALTECH), Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Mutation, business.industry, [SDV]Life Sciences [q-bio], Point mutation, Bone marrow failure, Hematology, Gene mutation, medicine.disease_cause, medicine.disease, 3. Good health, 03 medical and health sciences, 030104 developmental biology, Hydrops fetalis, Immunology, Genotype, medicine, Missense mutation, Diamond–Blackfan anemia, business, ComputingMilieux_MISCELLANEOUS, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Published
2018
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10. Recurring mutations in

Author

Marcin W, Wlodarski, Lydie, Da Costa, Marie-Françoise, O'Donohue, Marc, Gastou, Narjesse, Karboul, Nathalie, Montel-Lehry, Ina, Hainmann, Dominika, Danda, Amina, Szvetnik, Victor, Pastor, Nahuel, Paolini, Franca M, di Summa, Hannah, Tamary, Abed Abu, Quider, Anna, Aspesi, Riekelt H, Houtkooper, Thierry, Leblanc, Charlotte M, Niemeyer, Pierre-Emmanuel, Gleizes, and Alyson W, MacInnes

Subjects
Erythrocyte Indices, Male, Ribosomal Proteins, Genotype, Hydrops Fetalis, DNA Mutational Analysis, Pregnancy Complications, Hematologic, Apoptosis, Cell Differentiation, Genes, p53, Article, Cell Line, Pedigree, Phenotype, Pregnancy, Protein Biosynthesis, Mutation, Humans, Female, Genetic Predisposition to Disease, Red Cell Biology & its Disorders, Biomarkers, Genetic Association Studies, Anemia, Diamond-Blackfan, Cell Proliferation
Abstract

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

Published
2017

11. The severe phenotype of Diamond-Blackfan anemia is modulated by heat shock protein 70

Author

Narla Mohandas, Hélène Moniz, Michael Dussiot, Carmen Garrido, Sarah Rio, Narjesse Karboul, Olivier Hermine, Marc Gastou, Jérôme Larghero, Lydie Da Costa, Margaux Sevin, Thierry Leblanc, Eric Solary, Patrick Gonin, William Vainchenker, Anupama Narla, Hématopoïèse normale et pathologique ( U1170 Inserm ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge ( Labex Gr-Ex ), Université Paris Diderot - Paris 7 ( UPD7 ) -Université Sorbonne Paris Cité ( USPC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Centre de recherche sur l'inflamation - UMR 1149, Université Paris Diderot - Paris 7 ( UPD7 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Imagine - Institut des maladies génétiques ( IMAGINE - U1163 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Unité d'Hémato-Immunologie pédiatrique [Hôpital Robert Debré, Paris], Service d'Immuno-hématologie pédiatrique [Hôpital Robert Debré, Paris], Hôpital Robert Debré-Hôpital Robert Debré, Lipides - Nutrition - Cancer [Dijon - U1231] ( LNC ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Plateforme d’évaluation préclinique ( PFEP ), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse ( AMMICa ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Hôpital Saint-Louis, Unité de Thérapie Cellulaire, Institut Universitaire d’Hématologie, Stanford University School of Medicine [Stanford], Stanford University [Stanford], Red Cell Physiology Laboratory [New York, USA], New York Blood Center - NYBC, Centre de recherche sur l'Inflammation ( CRI ), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Service d'Hématologie Biologique [Hôpital Robert Debré, Paris], AP-HP Hôpital universitaire Robert-Debré [Paris], Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Paris Diderot - Paris 7 (UPD7)-Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche sur l'Inflammation (CRI (UMR_S_1149 / ERL_8252 / U1149)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lipides - Nutrition - Cancer [Dijon - U1231] (LNC), Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Plateforme d’évaluation préclinique (PFEP), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Alloimmunité-Autoimmunité-Transplantation (A2T), Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Stanford School of Medicine [Stanford], Stanford Medicine, Stanford University-Stanford University, New York Blood Center, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, congenital, hereditary, and neonatal diseases and abnormalities, Identification, Apoptosis-Inducing Factor, Gata1 Mutations, Inhibits Apoptosis, Biology, Hsp70, 03 medical and health sciences, Germline mutation, Red Cells, Iron, and Erythropoiesis, hemic and lymphatic diseases, medicine, [ SDV.MHEP.HEM ] Life Sciences [q-bio]/Human health and pathology/Hematology, Nuclear Import, Erythropoiesis, Diamond–Blackfan anemia, Human Erythroblasts, Bone marrow failure, [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, GATA1, Hematology, medicine.disease, Phenotype, Molecular biology, 3. Good health, 030104 developmental biology, Ribosomal-Proteins, Protein Gene Deletions, Haploinsufficiency
Abstract

International audience; Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation–dependent DBA.

Published
2017
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12. HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

Author

Narjesse Karboul, Olivier Bluteau, Narla Mohandas, Marc Gastou, Margaux Sevin, Sarah Rio, Mickael Dussiot, Thierry Leblanc, William Vainchenker, Eric Solary, Lydie Da Costa, Carmen Garrido, and Olivier Hermine

Subjects
Programmed cell death, Gene knockdown, Ribosomopathy, Immunology, GATA1, Cell Biology, Hematology, Gene mutation, Biology, medicine.disease, Biochemistry, hemic and lymphatic diseases, medicine, Cancer research, Erythropoiesis, Diamond–Blackfan anemia, Haploinsufficiency
Abstract

Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

Published
2015
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13. Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Author

Narla Mohandas, Aurore Cretien, Olivier Bluteau, G. Tchernia, L. Da Costa, L. Croisille, M. Faubladier, Jérôme Larghero, H Moniz, Marc Gastou, Yann Lécluse, Thierry Leblanc, William Vainchenker, Larissa Lordier, Hana Raslova, and Corinne Hurtaud

Subjects
p53, Male, Ribosomal Proteins, Cancer Research, RPS19, Cellular differentiation, Immunology, CD34, Antigens, CD34, Apoptosis, Gene mutation, Biology, Small hairpin RNA, Cellular and Molecular Neuroscience, RPL11, Erythroid Cells, Humans, Progenitor cell, RNA, Small Interfering, Cells, Cultured, Anemia, Diamond-Blackfan, Cell Proliferation, Cell growth, Infant, Newborn, Infant, Cell Differentiation, Cell Biology, Molecular biology, Phenotype, G1 Phase Cell Cycle Checkpoints, Haematopoiesis, Child, Preschool, Original Article, cell cycle, Female, RNA Interference, Tumor Suppressor Protein p53, DBA
Abstract

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34+ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G0/G1 cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G0/G1 cell cycle arrest with activation of p53. Infection of cord blood CD34+ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.

Published
2012

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