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Thromb Haemost 2021; 121(09): 1193-1205
DOI: 10.1055/s-0041-1723987
Cellular Haemostasis and Platelets

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

Ana Marín-Quílez
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Ignacio García-Tuñón 
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Cristina Fernández-Infante
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Luis Hernández-Cano
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
Verónica Palma-Barqueros
2   Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain
,
Elena Vuelta
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
3   Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain
,
Manuel Sánchez-Martín
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
3   Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain
4   Department of Medicine, University of Salamanca, Salamanca, Spain
,
José Ramón González-Porras
4   Department of Medicine, University of Salamanca, Salamanca, Spain
5   Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
,
Carmen Guerrero
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
4   Department of Medicine, University of Salamanca, Salamanca, Spain
,
Rocío Benito
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
,
José Rivera   *
2   Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain
6   On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH
,
Jesús María Hernández-Rivas   *
1   Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain
4   Department of Medicine, University of Salamanca, Salamanca, Spain
5   Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
,
José María Bastida    *
4   Department of Medicine, University of Salamanca, Salamanca, Spain
5   Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain
6   On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH
› Author Affiliations Funding This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain.
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Abstract

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Keywords

RUNX1 - inherited platelet disorders - platelet function - mouse model - CRISPR/Cas9

* These authors share senior authorship.


Supplementary Material



Publication History

Received: 29 July 2020

Accepted: 28 December 2020

Article published online:
18 February 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
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  • References

  • 1 Kurokawa M. AML1/Runx1 as a versatile regulator of hematopoiesis: regulation of its function and a role in adult hematopoiesis. Int J Hematol 2006; 84 (02) 136-142
    CrossrefPubMedGoogle Scholar
  • 2 Ichikawa M, Asai T, Saito T. et al. AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nat Med 2004; 10 (03) 299-304
    CrossrefPubMedGoogle Scholar
  • 3 Growney JD, Shigematsu H, Li Z. et al. Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype. Blood 2005; 106 (02) 494-504
    CrossrefPubMedGoogle Scholar
  • 4 Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH, Speck NA. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci U S A 1996; 93 (08) 3444-3449
    CrossrefPubMedGoogle Scholar
  • 5 Arber DA, Orazi A, Hasserjian R. et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127 (20) 2391-2405
    CrossrefPubMedGoogle Scholar
  • 6 Song WJ, Sullivan MG, Legare RD. et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat Genet 1999; 23 (02) 166-175
    CrossrefPubMedGoogle Scholar
  • 7 Bastida JM, Benito R, Lozano ML. et al. Molecular diagnosis of inherited coagulation and bleeding disorders. Semin Thromb Hemost 2019; 45 (07) 695-707
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Stockley J, Morgan NV, Bem D. et al; UK Genotyping and Phenotyping of Platelets Study Group. Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects. Blood 2013; 122 (25) 4090-4093
    CrossrefPubMedGoogle Scholar
  • 9 Lentaigne C, Freson K, Laffan MA, Turro E, Ouwehand WH. BRIDGE-BPD Consortium and the ThromboGenomics Consortium. Inherited platelet disorders: toward DNA-based diagnosis. Blood 2016; 127 (23) 2814-2823
    CrossrefPubMedGoogle Scholar
  • 10 Bastida JM, Lozano ML, Benito R. et al. Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders. Haematologica 2018; 103 (01) 148-162
    CrossrefPubMedGoogle Scholar
  • 11 Brown AL, Arts P, Carmichael CL. et al. RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML. Blood Adv 2020; 4 (06) 1131-1144
    CrossrefPubMedGoogle Scholar
  • 12 Richards S, Aziz N, Bale S. et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17 (05) 405-424
    CrossrefPubMedGoogle Scholar
  • 13 Garcia JS, Madzo J, Cooper D. et al. Pre-Donor evaluation of an HLA matched sibling identifies a novel inherited RUNX1 mutation encoding a missense mutation found outside of the RUNT domain in familial platelet disorder. Blood 2010; 116 (21) 2709
    CrossrefPubMedGoogle Scholar
  • 14 Koh CP, Wang CQ, Ng CEL. et al. RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes. Leukemia 2013; 27 (09) 1793-1802
    CrossrefPubMedGoogle Scholar
  • 15 Prieto-Conde MI, Labrador J, Hermida G. et al. Genomic analysis of a familial myelodysplasia/acute myeloid leukemia and inherited RUNX1 mutations without a pre-existing platelet disorder. Leuk Lymphoma 2020; 61 (01) 181-184
    CrossrefPubMedGoogle Scholar
  • 16 Brown AL, Hahn C, Hiwase D, Godley LA, Scott HS. Correct application of variant classification guidelines in germline RUNX1 mutated disorders to assist clinical diagnosis. Leuk Lymphoma 2020; 61 (01) 246-247
    CrossrefPubMedGoogle Scholar
  • 17 Duployez N, Fenwarth L. Controversies about germline RUNX1 missense variants. Leuk Lymphoma 2020; 61 (02) 497-499
    CrossrefPubMedGoogle Scholar
  • 18 Sakurai M, Kunimoto H, Watanabe N. et al. Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients. Leukemia 2014; 28 (12) 2344-2354
    CrossrefPubMedGoogle Scholar
  • 19 Lamolda M, Montes R, Simón I. et al. GENYOi005-A: an induced pluripotent stem cells (iPSCs) line generated from a patient with familial platelet disorder with associated myeloid malignancy (FPDMM) carrying a p.Thr196Ala variant. Stem Cell Res (Amst) 2019; 41: 101603
    CrossrefPubMedGoogle Scholar
  • 20 Liu Y, Jennings NL, Dart AM, Du XJ. Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice. World J Exp Med 2012; 2 (02) 30-36
    Google Scholar
  • 21 Gutiérrez-Herrero S, Fernández-Infante C, Hernández-Cano L. et al. C3G contributes to platelet activation and aggregation by regulating major signaling pathways. Signal Transduct Target Ther 2020; 5 (01) 29
    CrossrefPubMedGoogle Scholar
  • 22 Lood C, Tydén H, Gullstrand B. et al. Decreased platelet size is associated with platelet activation and anti-phospholipid syndrome in systemic lupus erythematosus. Rheumatology (Oxford) 2017; 56 (03) 408-416
    PubMedGoogle Scholar
  • 23 Hardy AT, Palma-Barqueros V, Watson SK. et al. Significant hypo-responsiveness to GPVI and CLEC-2 agonists in pre-term and full-term neonatal platelets and following immune thrombocytopenia. Thromb Haemost 2018; 118 (06) 1009-1020
    Article in Thieme ConnectPubMedGoogle Scholar
  • 24 Gutiérrez-Herrero S, Maia V, Gutiérrez-Berzal J. et al. C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity. Biochim Biophys Acta 2012; 1823 (08) 1366-1377
    CrossrefPubMedGoogle Scholar
  • 25 De Cuyper IM, Meinders M, van de Vijver E. et al. A novel flow cytometry-based platelet aggregation assay. Blood 2013; 121 (10) e70-e80
    CrossrefPubMedGoogle Scholar
  • 26 Tucker KL, Sage T, Gibbins JM. Clot retraction. Methods Mol Biol 2012; 788: 101-107
    CrossrefPubMedGoogle Scholar
  • 27 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Δ Δ C(T)) method. Methods 2001; 25 (04) 402-408
    CrossrefPubMedGoogle Scholar
  • 28 Ortiz-Rivero S, Baquero C, Hernández-Cano L. et al. C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation. Cell Commun Signal 2018; 16 (01) 101
    CrossrefPubMedGoogle Scholar
  • 29 Trevino AE, Zhang F. Genome editing using Cas9 nickases. Methods Enzymol 2014; 546: 161-174
    CrossrefPubMedGoogle Scholar
  • 30 Cifuni SM, Wagner DD, Bergmeier W. CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets. Blood 2008; 112 (05) 1696-1703
    CrossrefPubMedGoogle Scholar
  • 31 Hayashi Y, Harada Y, Huang G, Harada H. Myeloid neoplasms with germ line RUNX1 mutation. Int J Hematol 2017; 106 (02) 183-188
    CrossrefPubMedGoogle Scholar
  • 32 Duarte BKL, Yamaguti-Hayakawa GG, Medina SS. et al. Longitudinal sequencing of RUNX1 familial platelet disorder: new insights into genetic mechanisms of transformation to myeloid malignancies. Br J Haematol 2019; 186 (05) 724-734
    CrossrefPubMedGoogle Scholar
  • 33 Owen CJ, Toze CL, Koochin A. et al. Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy. Blood 2008; 112 (12) 4639-4645
    CrossrefPubMedGoogle Scholar
  • 34 Rao AK, Poncz M. Defective acid hydrolase secretion in RUNX1 haplodeficiency: evidence for a global platelet secretory defect. Haemophilia 2017; 23 (05) 784-792
    CrossrefPubMedGoogle Scholar
  • 35 Glembotsky AC, Bluteau D, Espasandin YR. et al. Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets. J Thromb Haemost 2014; 12 (05) 761-772
    CrossrefPubMedGoogle Scholar
  • 36 Antony-Debré I, Manchev VT, Balayn N. et al. Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. Blood 2015; 125 (06) 930-940
    CrossrefPubMedGoogle Scholar
  • 37 Morowski M, Vögtle T, Kraft P, Kleinschnitz C, Stoll G, Nieswandt B. Only severe thrombocytopenia results in bleeding and defective thrombus formation in mice. Blood 2013; 121 (24) 4938-4947
    CrossrefPubMedGoogle Scholar
  • 38 Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules … or not?. J Thromb Haemost 2015; 13 (12) 2141-2151
    CrossrefPubMedGoogle Scholar
  • 39 Gabbeta J, Yang X, Sun L, McLane MA, Niewiarowski S, Rao AK. Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation. Blood 1996; 87 (04) 1368-1376
    CrossrefPubMedGoogle Scholar
  • 40 Sood R, Kamikubo Y, Liu P. Role of RUNX1 in hematological malignancies. Blood 2017; 129 (15) 2070-2082
    CrossrefPubMedGoogle Scholar
  • 41 Franke B, van Triest M, de Bruijn KMT. et al. Sequential regulation of the small GTPase Rap1 in human platelets. Mol Cell Biol 2000; 20 (03) 779-785
    CrossrefPubMedGoogle Scholar
  • 42 Harper MT, Poole AW. Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation. J Thromb Haemost 2010; 8 (03) 454-462
    CrossrefPubMedGoogle Scholar
  • 43 Jalagadugula G, Mao G, Kaur G, Dhanasekaran DN, Rao AK. Platelet protein kinase C-θ deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1. Arterioscler Thromb Vasc Biol 2011; 31 (04) 921-927
    CrossrefPubMedGoogle Scholar
  • 44 Hug BA, Ahmed N, Robbins JA, Lazar MA. A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene. J Biol Chem 2004; 279 (02) 825-830
    CrossrefPubMedGoogle Scholar
  • 45 Sun L, Mao G, Rao AK. Association of CBFA2 mutation with decreased platelet PKC-θ and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation. Blood 2004; 103 (03) 948-954
    CrossrefPubMedGoogle Scholar
  • 46 Bynagari-Settipalli YS, Cornelissen I, Palmer D. et al. Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice. Arterioscler Thromb Vasc Biol 2014; 34 (12) 2563-2569
    CrossrefPubMedGoogle Scholar
  • 47 Schlegelberger B, Heller PG. RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM). Semin Hematol 2017; 54 (02) 75-80
    CrossrefPubMedGoogle Scholar
  • 48 Jagadeeswaran P, Cooley BC, Gross PL, Mackman N. Animal models of thrombosis from zebrafish to nonhuman primates. Circ Res 2016; 118 (09) 1363-1379
    CrossrefPubMedGoogle Scholar
  • 49 Lin Q, Zhang Y, Zhou R. et al. Establishment of a congenital amegakaryocytic thrombocytopenia model and a thrombocyte-specific reporter line in zebrafish. Leukemia 2017; 31 (05) 1206-1216
    CrossrefPubMedGoogle Scholar
  • 50 Marconi C, Di Buduo CA, LeVine K. et al. Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia. Blood 2019; 133 (12) 1346-1357
    CrossrefPubMedGoogle Scholar