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 4

In vitro–derived MK differentiation of THC2 patients.

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In vitro–derived MK differentiation of THC2 patients. MK differentiation...
MK differentiation was induced from control or patient peripheral blood CD34+ (A–D) or CD45+ progenitors (D and E) and analyzed respectively at day 10 and day 14 of culture. (A) Gates represent mature (CD41+CD42+) MKs (left panel). The ploidy level (N) was analyzed in the gate of CD41+CD42+ MKs and was based on the percentage of cells in 8N, 16N, and 32N gates. (B) Gallery of electron micrographs showing MKs from 1 control (C) and 2 patients (PD3_3, and PD1_2). (C) Expression level of CD49b (integrin α2) on the surface of CD41+CD42+ MKs of 1 control and 2 patients (PD2_1 and PD2_2). CTRneg corresponds to the CD49b staining on the CD41–CD42– cell population for each sample. The pictures show the MKs forming stress fibers after adhesion on collagen type 1. Nucleus staining (DAPI) is in blue, phalloidin (Phal) in green and vWF in red colors. (D) The percentage of PPT-forming MKs was estimated by counting MKs exhibiting 1 or more cytoplasmic processes with areas of constriction shown on the picture at the right. Data represent mean ± SD for 14 controls and for 20 THC2 patients (P). ***P < 0.001, Student’s t test. (E) Analysis of branching area in control and patients of PPT-forming MKs after adhesion on fibrinogen and staining with anti–α-tubulin Ab (green) and DAPI (blue). The number of bifurcation was counted for each PPT-forming MK in a total of 100 MKs. Data represent mean ± SD obtained for 10 controls and 17 THC2 patients. *P < 0.05, Student’s t test.