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.

Platelet alpha granules in BLOC-2 and BLOC-3 subtypes of Hermansky-Pudlak syndrome

“…Several human and murine genes have been linked to delta granule biogenesis; some encode known vesicle trafficking proteins whereas others encode components of BLOC protein complexes, whose exact functions are unknown [4,10,13,14,19]. Similarly, little is known about the biogenesis of alpha granules, although a few genes (e.g., GATA1, FLI1, NFE2) [27][28][29], glycoprotein Ib-beta [30] and VPS33B [31]) have been implicated in this process. A mutation in RABGGTA, coding for subunit A of rab-geranylgeranyl transferase [32], causes deficiency of both alpha and delta granules in the gunmetal mouse.…”

Molecular mechanisms of megakaryopoiesis

“…Thus, these patients have only one copy of these Ets genes due to a heterozygous loss of regions in chromosome 11 and display normal and small, immature, lysing MKs. Lentivirus-mediated Fli-1 expression in CD34 + cells restored normal megakaryopoiesis [135]. It is significant that Fli-1 expression is monoallelic in the intermediately mature CD41 + CD42 -cells, but bi-allelic before and after this stage of megakaryopoiesis [135].…”

Methods for genetic modification of megakaryocytes and platelets

“…1 Bernard-Soulier syndrome: GPIb KO [18], GPIb KO [17]; Glanzmann thrombasthenia, GPIIIa integrin KO [19,96]; Hermansky-Pudlak syndrome [20]. 2 Embryonic stem cell line [60]; K562 cell line [97]; 3 Human embryonic stem cell line [98]; 4 UT7 cell line [99]; 5 Dami [36], K562 [39,40] cell lines; 6 [7,100]; 7 [63,64,66]; 8 [45]; 9 [101]; 10 [102]; 11 [85,86,[89][90][91][93][94][95]. Table I.…”

“…This disorder results in patients having two distinct populations of megakaryocytes as a consequence of dysmegakaryopoiesis, caused by a deficiency of the FLI-1 transcription factor [62]. Raslova et al [63] expressed FLI-1 in CD34þ cells taken from three Paris-Trousseau syndrome patients using a lentiviral vector; two showed a significant increase in the total number of cells surviving and a corresponding increase in the percentage of megakaryocytes, whilst in the third patient they observed a decrease in the amount of initial cell death, an improvement in megakaryocyte maturation, and an increase in the number of proplatelet-producing megakaryocytes.…”