FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia
Hana Raslova, … , William Vainchenker, Rémi Favier
Hana Raslova, … , William Vainchenker, Rémi Favier
Published July 1, 2004
Citation Information: J Clin Invest. 2004;114(1):77-84. https://doi.org/10.1172/JCI21197.
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Article Hematology

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

Abstract

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34+ cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41+CD42– progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41+CD42– cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41+CD42– cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1+ or FLI1– CD41+CD42– cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.

Authors

Hana Raslova, Emiko Komura, Jean Pierre Le Couédic, Frederic Larbret, Najet Debili, Jean Feunteun, Olivier Danos, Olivier Albagli, William Vainchenker, Rémi Favier

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Construction and validation of a lentiviral vector encoding FLI1 cDNA. (...
Construction and validation of a lentiviral vector encoding FLI1 cDNA. (A) Construction of a lentiviral expression vector coding for FLI1. FLI1 expression was driven by the PGK promoter. (B) Immunoblot analysis of FLI1 protein in HEL cells expressing endogenous Fli1 (lane 1), in 293T cell line transiently transfected with either the control lentiviral vector (lane 2) or with the lentiviral vector encoding FLI1 cDNA (lane 3). Five hundred × 103 cells were loaded in lane 1 and 50 × 103 in lanes 2 and 3. (C) Single-cell RT-PCR detection of eGFP in transduced CD34+ cells. Peripheral blood CD34+ cells obtained from healthy individuals were investigated for the presence of eGFP 6 days after infection with PGK-Fli1-IRES-eGFP virus. β2-M was used as an internal control of mRNA integrity and cell sorting. Safety control for the experiment was performed in the absence of sorted cells (lane 2: Control). (D) Immunolabeling and flow-cytometry analysis of CD34+ cells transduced with the lentiviral vector. CD34+ cells were stained with anti–CD42-PE and anti–CD41-APC Ab’s 9 days after transduction with the control lentivirus vector (dotted line) or with the FLI1 encoding lentivirus vector (thin line). Analysis of CD42 expression was performed in the cell population expressing high level of CD41 (MKs).