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. 2004 Jul;114(1):77-84.
doi: 10.1172/JCI21197.

FLI1 Monoallelic Expression Combined With Its Hemizygous Loss Underlies Paris-Trousseau/Jacobsen Thrombopenia

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Free PMC article

FLI1 Monoallelic Expression Combined With Its Hemizygous Loss Underlies Paris-Trousseau/Jacobsen Thrombopenia

Hana Raslova et al. J Clin Invest. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Comparison of FLI1 and ETS1 expression in different human hematopoietic lineages: NK cells, lymphocytes T and B (LyT and LyB), and MKs. The absolute expression level of FLI1 and ETS1 in 100 ng of total cell RNA was measured relative to different dilutions (corresponding to 102–106 copies) of plasmids harboring either FLI1 or ETS1 cDNA. (A) The graph represents the relative expression between FLI1 and ETS1 in NK (CD56), T (CD3), and B (CD19) cells and in MKs. The value indicated above or under the columns is the ratio between the FLI1 and the ETS1 copy numbers in cDNA prepared from 100 ng of total cell RNA. (B) The absolute expression level of FLI1 and ETS1 in MKs measured as the number of mRNA copies per 100 ng of total RNA. The error bars represent the SD of the mean of three experiments, each performed in triplicate wells.
Figure 2
Figure 2
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).
Figure 3
Figure 3
Restoration of megakaryocytic phenotype in two PTS patients after FLI1 cDNA transfer in vitro. (A) Flow-cytometry analysis of CD41 and CD42 expression in MKs from a control and two patients (P1 and P2) 11 days after transduction of their peripheral blood CD34+ cells with PGK-IRES-eGFP or with PGK-Fli1-IRES-eGFP. Gate R3 and numbers indicated below the gates represent the percentage of mature MKs with high levels of CD41 and CD42 expression. (B) Immunolabeling of FLI1 (red staining) and vWF (green staining) in patient 1 and 3 (P1 and P3) MKs grown from CD34+ cells transduced (T), or not (NT), with PGK-Fli1-IRES-eGFP. Images were captured using epifluorescence microscope (Nikon Eclipse 600) with a ×40 objective. Large, presumably polyploid MKs characterized by a typical polylobulated nucleus (DAPI staining) and by a unique, continuous cytoplasmic membrane (vWF staining) in patient 1 and 3 after FLI1 transduction are indicated by a white arrowhead.
Figure 4
Figure 4
Fli1 expression throughout normal MK differentiation. CD34+ cells were cultured in the presence of TPO and analyzed at day 6. (A) Fli1 expression level determined by real-time RT-PCR. Diploid MKs were sorted according to their differentiation stages. Fli1 and two housekeeping β2-M and β5-tubulin5-T) genes (constant throughout MK differentiation; our unpublished data) were amplified in separate wells in five independent experiments (four using β2-M and one using β5-T). The relative expression level of Fli1 in each fraction was normalized to the corresponding values for β2-M or β5-T. A representative experiment is shown with error bars representing the SD of the mean of triplicate wells. (B) Representative picture of TPO-cultured cells showing the distribution of Fli1 nuclear RNA (green) and chromosome 12 (red) visualized by FISH. Cell with a monoallelic expression of Fli1 is on the left, and cell with its biallelic expression is on the right.The number of green spots indicates the number of transcribed Fli1 alleles. The number of Fli1 alleles in the same MK is determined by the number of red spots. Chromatin is counterstained with DAPI (blue). (C) Statistical analysis of monoallelic/biallelic expression of Fli1 in diploid MKs during their differentiation. Cells were sorted as described in A, and RNA-FISH was performed as described in B. One hundred cells of each population were analyzed on an epifluorescence microscope (Nikon Eclipse 600) using a ×60 objective for the presence of Fli1 pre-mRNA. The error bars represent the SD of the mean of three independent experiments.
Figure 5
Figure 5
Single-cell RT-PCR analysis of FLI1 expression in peripheral blood CD34+ cells from three controls and two patients, cultured in presence of TPO, IL-3, IL-6, SCF, and FLT3-L. On day 4, the CD34+CD41CD42, CD34+CD41+CD42, and CD34+CD41+CD42+ populations were sorted after immunolabeling at one cell per well in a 96-well plate. Single-cell RT-PCR was performed on 100 individual cells in each population. β2-M was used as an internal control of mRNA integrity, and only cells positive for β2-M were analyzed. (A) The graphs represent the number of FLI1–expressing cells out of 100 analyzed cells from different populations of three healthy individuals (Control) (the error bars represent the SD of the mean of three independent experiments) and of two Paris-Trousseau patients. (B) RT-PCR analysis in a representative 18 (of 100) CD34+CD41+CD42 cells from one healthy individual and two patients. Safety control of experiment was performed in absence of sorted cell (lane 20: Control).

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