Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation
Dominique Bluteau, … , Remi Favier, Hana Raslova
Dominique Bluteau, … , Remi Favier, Hana Raslova
Published January 16, 2014
Citation Information: J Clin Invest. 2014;124(2):580-591. https://doi.org/10.1172/JCI71861.
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Research Article Hematology

Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation

Abstract

Point mutations in the 5′ UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5′ UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5′ UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.

Authors

Dominique Bluteau, Alessandra Balduini, Nathalie Balayn, Manuela Currao, Paquita Nurden, Caroline Deswarte, Guy Leverger, Patrizia Noris, Silverio Perrotta, Eric Solary, William Vainchenker, Najet Debili, Remi Favier, Hana Raslova

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Figure 2

ANKRD26 expression is maintained during megakaryopoiesis of THC2 patients.

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ANKRD26 expression is maintained during megakaryopoiesis of THC2 patient...
In vitro MK differentiation was induced from control or patient peripheral blood CD34+ progenitors in the presence of TPO. RT-PCR analysis of ANKRD26 mRNA was performed at different stages of MK differentiation. (A) CD34+ cells were recovered before culture (D0) and at different days (D1, D4, D7, D10) of culture. (B) CD41+ cells were sorted at day 6, put in culture, and then studied on 3 different days of culture (D6 to D12, corresponding to cells with increasing maturity). (C) CD41+CD42– (immature MKs) and CD41+CD42+ (mature MKs) were sorted at day 6 of culture. (A–C) Data are normalized to PPIA transcript level and represent mean ± SD of triplicate. (D) CD41+CD42+ mature MKs were sorted at day 10 of culture. MKs of 5 THC2 patients (PD1_1, PD3_1, PD3_2, PD3_3, PD3_4) and of 3 control individuals were studied. (E) Platelets were purified from platelet-rich plasma. Platelets from 9 THC2 patients (PD1_1, PD1_2, PD2_1, PD2_2, PD3_1, PD3_2, PD3_3, PD4_1, PD4_2) and from 5 control individuals were studied. Data are normalized to PPIA transcript level, and expression is compared with control C1. Data represent mean ± SEM. *P < 0.05; **P < 0.001, Mann-Whitney test.