Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia

C. Marconi, C.A. DI Buduo, K. LeVine, S. Barozzi, M. Faleschini, V. Bozzi, F. Palombo, S. McKinstry, G. Lassandro, P. Giordano, P. Noris, C.L. Balduini, A. Savoia, A. Balduini, T. Pippucci, M. Seri, N. Katsanis, A. Pecci

Research output: Contribution to journalArticle

Abstract

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-offunction variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis. © 2019 by The American Society of Hematology.
Original languageEnglish
Pages (from-to)1346-1357
Number of pages12
JournalBlood
Volume133
Issue number12
DOIs
Publication statusPublished - 2019

Fingerprint

Platelets
Thrombocytopenia
Blood Platelets
Mutation
Megakaryocytes
Defects
Clustered Regularly Interspaced Short Palindromic Repeats
Hemorrhage
Exome
src-Family Kinases
Hematopoiesis
Zebrafish
Ablation
Platelet Count
Siblings
Collagen
Genes
Chemical activation
Cells
Cell Line

Keywords

  • collagen
  • convulxin
  • messenger RNA
  • protein tyrosine kinase
  • adolescent
  • Article
  • autosomal recessive disorder
  • case report
  • cell migration
  • child
  • clinical article
  • CRISPR-CAS9 system
  • embryo
  • female
  • gene
  • genetic variation
  • hematopoiesis
  • human
  • human cell
  • in vivo study
  • loss of function mutation
  • male
  • megakaryocyte
  • nonhuman
  • phenotype
  • predictive value
  • priority journal
  • ptprj gene
  • school child
  • thrombocyte
  • thrombocytopenia
  • whole exome sequencing
  • zebra fish

Cite this

Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia. / Marconi, C.; DI Buduo, C.A.; LeVine, K.; Barozzi, S.; Faleschini, M.; Bozzi, V.; Palombo, F.; McKinstry, S.; Lassandro, G.; Giordano, P.; Noris, P.; Balduini, C.L.; Savoia, A.; Balduini, A.; Pippucci, T.; Seri, M.; Katsanis, N.; Pecci, A.

In: Blood, Vol. 133, No. 12, 2019, p. 1346-1357.

Research output: Contribution to journalArticle

Marconi, C, DI Buduo, CA, LeVine, K, Barozzi, S, Faleschini, M, Bozzi, V, Palombo, F, McKinstry, S, Lassandro, G, Giordano, P, Noris, P, Balduini, CL, Savoia, A, Balduini, A, Pippucci, T, Seri, M, Katsanis, N & Pecci, A 2019, 'Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia', Blood, vol. 133, no. 12, pp. 1346-1357. https://doi.org/10.1182/blood-2018-07-859496
Marconi, C. ; DI Buduo, C.A. ; LeVine, K. ; Barozzi, S. ; Faleschini, M. ; Bozzi, V. ; Palombo, F. ; McKinstry, S. ; Lassandro, G. ; Giordano, P. ; Noris, P. ; Balduini, C.L. ; Savoia, A. ; Balduini, A. ; Pippucci, T. ; Seri, M. ; Katsanis, N. ; Pecci, A. / Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia. In: Blood. 2019 ; Vol. 133, No. 12. pp. 1346-1357.
@article{432acd0ba9ff43d3b96b0a59ad9b1354,
title = "Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia",
abstract = "Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50{\%} of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-offunction variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis. {\circledC} 2019 by The American Society of Hematology.",
keywords = "collagen, convulxin, messenger RNA, protein tyrosine kinase, adolescent, Article, autosomal recessive disorder, case report, cell migration, child, clinical article, CRISPR-CAS9 system, embryo, female, gene, genetic variation, hematopoiesis, human, human cell, in vivo study, loss of function mutation, male, megakaryocyte, nonhuman, phenotype, predictive value, priority journal, ptprj gene, school child, thrombocyte, thrombocytopenia, whole exome sequencing, zebra fish",
author = "C. Marconi and {DI Buduo}, C.A. and K. LeVine and S. Barozzi and M. Faleschini and V. Bozzi and F. Palombo and S. McKinstry and G. Lassandro and P. Giordano and P. Noris and C.L. Balduini and A. Savoia and A. Balduini and T. Pippucci and M. Seri and N. Katsanis and A. Pecci",
note = "Cited By :2 Export Date: 10 October 2019 CODEN: BLOOA Correspondence Address: Seri, M.; Department of Medical and Surgical Sciences, University of Bologna, Via Massarenti 9, Italy; email: marco.seri@unibo.it Chemicals/CAS: collagen, 9007-34-5; convulxin, 37206-04-5; protein tyrosine kinase, 80449-02-1 Funding details: Duke University Funding details: Universit{\`a} di Bologna Funding details: Tufts University Funding details: Fondazione IRCCS Policlinico San Matteo Funding details: 7Department Funding details: Fondazione Cariplo, 2013-0717 Funding text 1: 1Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; 2Department of Molecular Medicine, University of Pavia, Pavia, Italy; 3Biotechnology Research Laboratories, IRCCS Policlinico San Matteo Foundation, Pavia, Italy; 4Center for Human Disease Modeling, Duke University, Durham, NC; 5Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation and University of Pavia, Pavia, Italy; 6Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 7Department of Biomedical Science and Human Oncology, Pediatric Unit, University “Aldo Moro,” Bari, Italy; 8Ferrata-Storti Foundation, Pavia, Italy; 9Department of Medical Sciences, University of Trieste, Trieste, Italy; and 10Department of Biomedical Engineering, Tufts University, Medford, MA Funding text 2: This work was supported by grants from the IRCCS Policlinico San Matteo Foundation, the IRCCS Burlo Garofolo, and the Cariplo Foundation (Italy; grant 2013-0717). References: Noris, P., Pecci, A., Hereditary thrombocytopenias: A growing list of disorders (2017) Hematology Am Soc Hematol Educ Program., 2017, pp. 385-399; Noris, P., Biino, G., Pecci, A., Platelet diameters in inherited thrombocytopenias: Analysis of 376 patients with all known disorders (2014) Blood., 124 (6), pp. e4-e10; Fixter, K., Rabbolini, D.J., Valecha, B., Mean platelet diameter measurements to classify inherited thrombocytopenias (2018) Int J Lab Hematol., 40 (2), pp. 187-195; Greinacher, A., Pecci, A., Kunishima, S., Diagnosis of inherited platelet disorders on a blood smear: A tool to facilitate worldwide diagnosis of platelet disorders (2017) J Thromb Haemost., 15 (7), pp. 1511-1521; Marconi, C., Di Buduo, C.A., Barozzi, S., SLFN14-related thrombocytopenia: Identification within a large series of patients with inherited thrombocytopenia (2016) Thromb Haemost., 115 (5), pp. 1076-1079; Johnson, B., Lowe, G.C., Futterer, J., Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects (2016) Haematologica., 101 (10), pp. 1170-1179; Pecci, A., Balduini, C.L., Lessons in platelet production from inherited thrombocytopenias (2014) Br J Haematol., 165 (2), pp. 179-192; Eto, K., Kunishima, S., Linkage between the mechanisms of thrombocytopenia and thrombopoiesis (2016) Blood., 127 (10), pp. 1234-1241; Noris, P., Guidetti, G.F., Conti, V., Autosomal dominant thrombocytopenias with reduced expression of glycoprotein Ia (2006) Thromb Haemost., 95 (3), pp. 483-489; Melazzini, F., Palombo, F., Balduini, A., Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia (2016) Haematologica., 101 (11), pp. 1333-1342; Necchi, V., Balduini, A., Noris, P., Ubiquitin/ proteasome-rich particulate cytoplasmic structures (PaCSs) in the platelets and megakaryocytes of ANKRD26-related thrombocytopenia (2013) Thromb Haemost., 109 (2), pp. 263-271; Pecci, A., Bozzi, V., Panza, E., Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells (2011) Thromb Haemost., 106 (4), pp. 693-704; Balduini, A., Malara, A., Pecci, A., Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano (2009) J Thromb Haemost., 7 (3), pp. 478-484; Bluteau, D., Balduini, A., Balayn, N., Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation (2014) J Clin Invest., 124 (2), pp. 580-591; Di Buduo, C.A., Moccia, F., Battiston, M., The importance of calcium in the regulation of megakaryocyte function (2014) Haematologica., 99 (4), pp. 769-778; Pecci, A., Malara, A., Badalucco, S., Megakaryocytes of patients with MYH9-related thrombocytopenia present an altered proplatelet formation (2009) Thromb Haemost., 102 (1), pp. 90-96; Abbonante, V., Gruppi, C., Rubel, D., Gross, O., Moratti, R., Balduini, A., Discoidin domain receptor 1 protein is a novel modulator of megakaryocyte-collagen interactions (2013) J Biol Chem., 288 (23), pp. 16738-16746; Lin, H.-F., Traver, D., Zhu, H., Analysis of thrombocyte development in CD41-GFP transgenic zebrafish (2005) Blood., 106 (12), pp. 3803-3810; Labun, K., Montague, T.G., Gagnon, J.A., Thyme, S.B., Valen, E., CHOPCHOP v2: A web tool for the next generation of CRISPR genome engineering (2016) Nucleic Acids Res., 44 (W1), pp. W272-W276; Montague, T.G., Cruz, J.M., Gagnon, J.A., Church, G.M., Valen, E., CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing (2014) Nucleic Acids Res., 42 (WEB SERVER ISSUE), pp. W401-W407; Moffat, J., Grueneberg, D.A., Yang, X., A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen (2006) Cell., 124 (6), pp. 1283-1298; Pippucci, T., Savoia, A., Perrotta, S., Mutations in the 59 UTR of ANKRD26, the ankirin repeat domain 26 gene, cause an autosomal-dominant form of inherited thrombocytopenia, THC2 (2011) Am J Hum Genet., 88 (1), pp. 115-120; Michelson, A.D., Barnard, M.R., Krueger, L.A., Frelinger, A.L., III, Furman, M.I., Evaluation of platelet function by flow cytometry (2000) Methods., 21 (3), pp. 259-270; Senis, Y.A., Tomlinson, M.G., Ellison, S., The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis (2009) Blood., 113 (20), pp. 4942-4954; Ellison, S., Mori, J., Barr, A.J., Senis, Y.A., CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases (2010) J Thromb Haemost., 8 (7), pp. 1575-1583; Pera, I.L., Iuliano, R., Florio, T., The rat tyrosine phosphatase h increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue [published correction appears in Oncogene. 2016;35(41):5456] (2005) Oncogene., 24 (19), pp. 3187-3195; Avecilla, S.T., Hattori, K., Heissig, B., Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis (2004) Nat Med., 10 (1), pp. 64-71; Lane, W.J., Dias, S., Hattori, K., Stromalderived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases (2000) Blood., 96 (13), pp. 4152-4159; Hamada, T., M{\"o}hle, R., Hesselgesser, J., Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation (1998) J Exp Med., 188 (3), pp. 539-548; Larson, M.K., Watson, S.P., Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3 (2006) Blood., 108 (5), pp. 1509-1514; Malara, A., Currao, M., Gruppi, C., Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin (2014) Stem Cells., 32 (4), pp. 926-937; Davis, E.E., Frangakis, S., Katsanis, N., Interpreting human genetic variation with in vivo zebrafish assays (2014) Biochim Biophys Acta., 1842 (10), pp. 1960-1970; Rost, M.S., Grzegorski, S.J., Shavit, J.A., Quantitative methods for studying hemostasis in zebrafish larvae (2016) Methods Cell Biol., 134, pp. 377-389; Balduini, C.L., Savoia, A., Genetics of familial forms of thrombocytopenia (2012) Hum Genet., 131 (12), pp. 1821-1832; Sanna-Cherchi, S., Khan, K., Westland, R., Exome-wide association study identifies GREB1L mutations in congenital kidney malformations [published correction appears in Am J Hum. 2017;101(6):1034] (2017) Am J Hum Genet., 101 (6), p. 1034; Greenberg, S.M., Rosenthal, D.S., Greeley, T.A., Tantravahi, R., Handin, R.I., Characterization of a new megakaryocytic cell line: The Dami cell (1988) Blood., 72 (6), pp. 1968-1977; Zhang, N., Zhi, H., Curtis, B.R., CRISPR/ Cas9-mediated conversion of human platelet alloantigen allotypes (2016) Blood., 127 (6), pp. 675-680; Burkhart, J.M., Vaudel, M., Gambaryan, S., The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways (2012) Blood., 120 (15), pp. e73-e82; Zhu, J.W., Brdicka, T., Katsumoto, T.R., Lin, J., Weiss, A., Structurally distinct phosphatases CD45 and CD148 both regulate B cell and macrophage immunoreceptor signaling (2008) Immunity., 28 (2), pp. 183-196; Hermiston, M.L., Zikherman, J., Zhu, J.W., CD45, CD148, and Lyp/Pep: Critical phosphatases regulating Src family kinase signaling networks in immune cells [published correction appears in Immunol Rev. 2009;229(1):387] (2009) Immunol Rev., 228 (1), pp. 288-311; Senis, Y.A., Barr, A.J., Targeting receptor-type protein tyrosine phosphatases with biotherapeutics: Is outside-in better than inside-out? (2018) Molecules., 23 (3), p. E569; Senis, Y.A., Protein-tyrosine phosphatases: A new frontier in platelet signal transduction (2013) J Thromb Haemost., 11 (10), pp. 1800-1813; Mori, J., Wang, Y.J., Ellison, S., Dominant role of the protein-tyrosine phosphatase CD148 in regulating platelet activation relative to protein-tyrosine phosphatase-1B (2012) Arterioscler Thromb Vasc Biol., 32 (12), pp. 2956-2965; Mori, J., Nagy, Z., Di Nunzio, G., Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148 (2018) Blood., 131 (10), pp. 1122-1144; Senis, Y.A., Mazharian, A., Mori, J., Src family kinases: At the forefront of platelet activation (2014) Blood., 124 (13), pp. 2013-2024; Ballmaier, M., Germeshausen, M., Congenital amegakaryocytic thrombocytopenia: Clinical presentation, diagnosis, and treatment (2011) Semin Thromb Hemost., 37 (6), pp. 673-681; Pecci, A., Ragab, I., Bozzi, V., Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim (2018) EMBO Mol Med., 10 (1), pp. 63-75; Albert, M.H., Bittner, T.C., Nonoyama, S., X-linked thrombocytopenia (XLT) due to WAS mutations: Clinical characteristics, long-term outcome, and treatment options (2010) Blood., 115 (16), pp. 3231-3238; Larson, M.K., Watson, S.P., A product of their environment: Do megakaryocytes rely on extracellular cues for proplatelet formation? (2006) Platelets., 17 (7), pp. 435-440; Sabri, S., Foudi, A., Boukour, S., Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment (2006) Blood., 108 (1), pp. 134-140; Mazharian, A., Thomas, S.G., Dhanjal, T.S., Buckley, C.D., Watson, S.P., Critical role of Src-Syk-PLCgamma2 signaling in megakaryocyte migration and thrombopoiesis (2010) Blood., 116 (5), pp. 793-800; Kunishima, S., Okuno, Y., Yoshida, K., ACTN1 mutations cause congenital macrothrombocytopenia (2013) Am J Hum Genet., 92 (3), pp. 431-438; Palazzo, A., Bluteau, O., Messaoudi, K., The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation (2016) J Thromb Haemost., 14 (12), pp. 2524-2535; Elbatarny, M., Mollah, S., Grabell, J., Normal range of bleeding scores for the ISTH-BAT: Adult and pediatric data from the merging project (2014) Haemophilia., 20 (6), pp. 831-835; Lowe, G.C., Lordkipanidz{\'e}, M., Watson, S.P., Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders (2013) J Thromb Haemost., 11 (9), pp. 1663-1668",
year = "2019",
doi = "10.1182/blood-2018-07-859496",
language = "English",
volume = "133",
pages = "1346--1357",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "12",

}

TY - JOUR

T1 - Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia

AU - Marconi, C.

AU - DI Buduo, C.A.

AU - LeVine, K.

AU - Barozzi, S.

AU - Faleschini, M.

AU - Bozzi, V.

AU - Palombo, F.

AU - McKinstry, S.

AU - Lassandro, G.

AU - Giordano, P.

AU - Noris, P.

AU - Balduini, C.L.

AU - Savoia, A.

AU - Balduini, A.

AU - Pippucci, T.

AU - Seri, M.

AU - Katsanis, N.

AU - Pecci, A.

N1 - Cited By :2 Export Date: 10 October 2019 CODEN: BLOOA Correspondence Address: Seri, M.; Department of Medical and Surgical Sciences, University of Bologna, Via Massarenti 9, Italy; email: marco.seri@unibo.it Chemicals/CAS: collagen, 9007-34-5; convulxin, 37206-04-5; protein tyrosine kinase, 80449-02-1 Funding details: Duke University Funding details: Università di Bologna Funding details: Tufts University Funding details: Fondazione IRCCS Policlinico San Matteo Funding details: 7Department Funding details: Fondazione Cariplo, 2013-0717 Funding text 1: 1Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy; 2Department of Molecular Medicine, University of Pavia, Pavia, Italy; 3Biotechnology Research Laboratories, IRCCS Policlinico San Matteo Foundation, Pavia, Italy; 4Center for Human Disease Modeling, Duke University, Durham, NC; 5Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation and University of Pavia, Pavia, Italy; 6Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy; 7Department of Biomedical Science and Human Oncology, Pediatric Unit, University “Aldo Moro,” Bari, Italy; 8Ferrata-Storti Foundation, Pavia, Italy; 9Department of Medical Sciences, University of Trieste, Trieste, Italy; and 10Department of Biomedical Engineering, Tufts University, Medford, MA Funding text 2: This work was supported by grants from the IRCCS Policlinico San Matteo Foundation, the IRCCS Burlo Garofolo, and the Cariplo Foundation (Italy; grant 2013-0717). References: Noris, P., Pecci, A., Hereditary thrombocytopenias: A growing list of disorders (2017) Hematology Am Soc Hematol Educ Program., 2017, pp. 385-399; Noris, P., Biino, G., Pecci, A., Platelet diameters in inherited thrombocytopenias: Analysis of 376 patients with all known disorders (2014) Blood., 124 (6), pp. e4-e10; Fixter, K., Rabbolini, D.J., Valecha, B., Mean platelet diameter measurements to classify inherited thrombocytopenias (2018) Int J Lab Hematol., 40 (2), pp. 187-195; Greinacher, A., Pecci, A., Kunishima, S., Diagnosis of inherited platelet disorders on a blood smear: A tool to facilitate worldwide diagnosis of platelet disorders (2017) J Thromb Haemost., 15 (7), pp. 1511-1521; Marconi, C., Di Buduo, C.A., Barozzi, S., SLFN14-related thrombocytopenia: Identification within a large series of patients with inherited thrombocytopenia (2016) Thromb Haemost., 115 (5), pp. 1076-1079; Johnson, B., Lowe, G.C., Futterer, J., Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects (2016) Haematologica., 101 (10), pp. 1170-1179; Pecci, A., Balduini, C.L., Lessons in platelet production from inherited thrombocytopenias (2014) Br J Haematol., 165 (2), pp. 179-192; Eto, K., Kunishima, S., Linkage between the mechanisms of thrombocytopenia and thrombopoiesis (2016) Blood., 127 (10), pp. 1234-1241; Noris, P., Guidetti, G.F., Conti, V., Autosomal dominant thrombocytopenias with reduced expression of glycoprotein Ia (2006) Thromb Haemost., 95 (3), pp. 483-489; Melazzini, F., Palombo, F., Balduini, A., Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia (2016) Haematologica., 101 (11), pp. 1333-1342; Necchi, V., Balduini, A., Noris, P., Ubiquitin/ proteasome-rich particulate cytoplasmic structures (PaCSs) in the platelets and megakaryocytes of ANKRD26-related thrombocytopenia (2013) Thromb Haemost., 109 (2), pp. 263-271; Pecci, A., Bozzi, V., Panza, E., Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells (2011) Thromb Haemost., 106 (4), pp. 693-704; Balduini, A., Malara, A., Pecci, A., Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano (2009) J Thromb Haemost., 7 (3), pp. 478-484; Bluteau, D., Balduini, A., Balayn, N., Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation (2014) J Clin Invest., 124 (2), pp. 580-591; Di Buduo, C.A., Moccia, F., Battiston, M., The importance of calcium in the regulation of megakaryocyte function (2014) Haematologica., 99 (4), pp. 769-778; Pecci, A., Malara, A., Badalucco, S., Megakaryocytes of patients with MYH9-related thrombocytopenia present an altered proplatelet formation (2009) Thromb Haemost., 102 (1), pp. 90-96; Abbonante, V., Gruppi, C., Rubel, D., Gross, O., Moratti, R., Balduini, A., Discoidin domain receptor 1 protein is a novel modulator of megakaryocyte-collagen interactions (2013) J Biol Chem., 288 (23), pp. 16738-16746; Lin, H.-F., Traver, D., Zhu, H., Analysis of thrombocyte development in CD41-GFP transgenic zebrafish (2005) Blood., 106 (12), pp. 3803-3810; Labun, K., Montague, T.G., Gagnon, J.A., Thyme, S.B., Valen, E., CHOPCHOP v2: A web tool for the next generation of CRISPR genome engineering (2016) Nucleic Acids Res., 44 (W1), pp. W272-W276; Montague, T.G., Cruz, J.M., Gagnon, J.A., Church, G.M., Valen, E., CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing (2014) Nucleic Acids Res., 42 (WEB SERVER ISSUE), pp. W401-W407; Moffat, J., Grueneberg, D.A., Yang, X., A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen (2006) Cell., 124 (6), pp. 1283-1298; Pippucci, T., Savoia, A., Perrotta, S., Mutations in the 59 UTR of ANKRD26, the ankirin repeat domain 26 gene, cause an autosomal-dominant form of inherited thrombocytopenia, THC2 (2011) Am J Hum Genet., 88 (1), pp. 115-120; Michelson, A.D., Barnard, M.R., Krueger, L.A., Frelinger, A.L., III, Furman, M.I., Evaluation of platelet function by flow cytometry (2000) Methods., 21 (3), pp. 259-270; Senis, Y.A., Tomlinson, M.G., Ellison, S., The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis (2009) Blood., 113 (20), pp. 4942-4954; Ellison, S., Mori, J., Barr, A.J., Senis, Y.A., CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases (2010) J Thromb Haemost., 8 (7), pp. 1575-1583; Pera, I.L., Iuliano, R., Florio, T., The rat tyrosine phosphatase h increases cell adhesion by activating c-Src through dephosphorylation of its inhibitory phosphotyrosine residue [published correction appears in Oncogene. 2016;35(41):5456] (2005) Oncogene., 24 (19), pp. 3187-3195; Avecilla, S.T., Hattori, K., Heissig, B., Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis (2004) Nat Med., 10 (1), pp. 64-71; Lane, W.J., Dias, S., Hattori, K., Stromalderived factor 1-induced megakaryocyte migration and platelet production is dependent on matrix metalloproteinases (2000) Blood., 96 (13), pp. 4152-4159; Hamada, T., Möhle, R., Hesselgesser, J., Transendothelial migration of megakaryocytes in response to stromal cell-derived factor 1 (SDF-1) enhances platelet formation (1998) J Exp Med., 188 (3), pp. 539-548; Larson, M.K., Watson, S.P., Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3 (2006) Blood., 108 (5), pp. 1509-1514; Malara, A., Currao, M., Gruppi, C., Megakaryocytes contribute to the bone marrow-matrix environment by expressing fibronectin, type IV collagen, and laminin (2014) Stem Cells., 32 (4), pp. 926-937; Davis, E.E., Frangakis, S., Katsanis, N., Interpreting human genetic variation with in vivo zebrafish assays (2014) Biochim Biophys Acta., 1842 (10), pp. 1960-1970; Rost, M.S., Grzegorski, S.J., Shavit, J.A., Quantitative methods for studying hemostasis in zebrafish larvae (2016) Methods Cell Biol., 134, pp. 377-389; Balduini, C.L., Savoia, A., Genetics of familial forms of thrombocytopenia (2012) Hum Genet., 131 (12), pp. 1821-1832; Sanna-Cherchi, S., Khan, K., Westland, R., Exome-wide association study identifies GREB1L mutations in congenital kidney malformations [published correction appears in Am J Hum. 2017;101(6):1034] (2017) Am J Hum Genet., 101 (6), p. 1034; Greenberg, S.M., Rosenthal, D.S., Greeley, T.A., Tantravahi, R., Handin, R.I., Characterization of a new megakaryocytic cell line: The Dami cell (1988) Blood., 72 (6), pp. 1968-1977; Zhang, N., Zhi, H., Curtis, B.R., CRISPR/ Cas9-mediated conversion of human platelet alloantigen allotypes (2016) Blood., 127 (6), pp. 675-680; Burkhart, J.M., Vaudel, M., Gambaryan, S., The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways (2012) Blood., 120 (15), pp. e73-e82; Zhu, J.W., Brdicka, T., Katsumoto, T.R., Lin, J., Weiss, A., Structurally distinct phosphatases CD45 and CD148 both regulate B cell and macrophage immunoreceptor signaling (2008) Immunity., 28 (2), pp. 183-196; Hermiston, M.L., Zikherman, J., Zhu, J.W., CD45, CD148, and Lyp/Pep: Critical phosphatases regulating Src family kinase signaling networks in immune cells [published correction appears in Immunol Rev. 2009;229(1):387] (2009) Immunol Rev., 228 (1), pp. 288-311; Senis, Y.A., Barr, A.J., Targeting receptor-type protein tyrosine phosphatases with biotherapeutics: Is outside-in better than inside-out? (2018) Molecules., 23 (3), p. E569; Senis, Y.A., Protein-tyrosine phosphatases: A new frontier in platelet signal transduction (2013) J Thromb Haemost., 11 (10), pp. 1800-1813; Mori, J., Wang, Y.J., Ellison, S., Dominant role of the protein-tyrosine phosphatase CD148 in regulating platelet activation relative to protein-tyrosine phosphatase-1B (2012) Arterioscler Thromb Vasc Biol., 32 (12), pp. 2956-2965; Mori, J., Nagy, Z., Di Nunzio, G., Maintenance of murine platelet homeostasis by the kinase Csk and phosphatase CD148 (2018) Blood., 131 (10), pp. 1122-1144; Senis, Y.A., Mazharian, A., Mori, J., Src family kinases: At the forefront of platelet activation (2014) Blood., 124 (13), pp. 2013-2024; Ballmaier, M., Germeshausen, M., Congenital amegakaryocytic thrombocytopenia: Clinical presentation, diagnosis, and treatment (2011) Semin Thromb Hemost., 37 (6), pp. 673-681; Pecci, A., Ragab, I., Bozzi, V., Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim (2018) EMBO Mol Med., 10 (1), pp. 63-75; Albert, M.H., Bittner, T.C., Nonoyama, S., X-linked thrombocytopenia (XLT) due to WAS mutations: Clinical characteristics, long-term outcome, and treatment options (2010) Blood., 115 (16), pp. 3231-3238; Larson, M.K., Watson, S.P., A product of their environment: Do megakaryocytes rely on extracellular cues for proplatelet formation? (2006) Platelets., 17 (7), pp. 435-440; Sabri, S., Foudi, A., Boukour, S., Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment (2006) Blood., 108 (1), pp. 134-140; Mazharian, A., Thomas, S.G., Dhanjal, T.S., Buckley, C.D., Watson, S.P., Critical role of Src-Syk-PLCgamma2 signaling in megakaryocyte migration and thrombopoiesis (2010) Blood., 116 (5), pp. 793-800; Kunishima, S., Okuno, Y., Yoshida, K., ACTN1 mutations cause congenital macrothrombocytopenia (2013) Am J Hum Genet., 92 (3), pp. 431-438; Palazzo, A., Bluteau, O., Messaoudi, K., The cell division control protein 42-Src family kinase-neural Wiskott-Aldrich syndrome protein pathway regulates human proplatelet formation (2016) J Thromb Haemost., 14 (12), pp. 2524-2535; Elbatarny, M., Mollah, S., Grabell, J., Normal range of bleeding scores for the ISTH-BAT: Adult and pediatric data from the merging project (2014) Haemophilia., 20 (6), pp. 831-835; Lowe, G.C., Lordkipanidzé, M., Watson, S.P., Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders (2013) J Thromb Haemost., 11 (9), pp. 1663-1668

PY - 2019

Y1 - 2019

N2 - Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-offunction variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis. © 2019 by The American Society of Hematology.

AB - Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-offunction variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients' megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis. © 2019 by The American Society of Hematology.

KW - collagen

KW - convulxin

KW - messenger RNA

KW - protein tyrosine kinase

KW - adolescent

KW - Article

KW - autosomal recessive disorder

KW - case report

KW - cell migration

KW - child

KW - clinical article

KW - CRISPR-CAS9 system

KW - embryo

KW - female

KW - gene

KW - genetic variation

KW - hematopoiesis

KW - human

KW - human cell

KW - in vivo study

KW - loss of function mutation

KW - male

KW - megakaryocyte

KW - nonhuman

KW - phenotype

KW - predictive value

KW - priority journal

KW - ptprj gene

KW - school child

KW - thrombocyte

KW - thrombocytopenia

KW - whole exome sequencing

KW - zebra fish

U2 - 10.1182/blood-2018-07-859496

DO - 10.1182/blood-2018-07-859496

M3 - Article

VL - 133

SP - 1346

EP - 1357

JO - Blood

JF - Blood

SN - 0006-4971

IS - 12

ER -