In this issue of Blood, Stockley et al describe mutations in FLI1 and RUNX1, identified by next-generation sequencing (NGS) studies, in 6 of 13 patients with excessive bleeding and impaired platelet dense granule secretion, and highlight transcription factor (TF) mutations as an important mechanism for inherited platelet dysfunction.1
In the majority of patients suspected to have an inherited platelet function defect, based on abnormal platelet aggregation and secretion studies, the underlying molecular and genetic mechanisms remain unknown. Platelet aggregation and secretion as monitored in clinical studies are relatively late or “end” responses that follow a sequence of upstream events on platelet activation. Given the complex and redundant platelet pathways, the findings of such studies are generally not predictive with any confidence of the underlying genetic or molecular mechanisms in most patients. Until recently, the focus of studies on the molecular mechanisms has been on postulated candidate proteins and pathways. This is the major paradigm by which most platelet defects have been identified to date; it is driven by existing knowledge and comes with limitations. At the genetic level, the focus has largely been on delineating mutations in the coding sequence of genes encoding the candidate proteins.
It is in this context that the paper by Stockley et al makes an important contribution. First, it focuses attention on genetic defects in TFs that regulate megakaryocytic and platelet genes as a cause of platelet dysfunction. Second, it highlights the power of newer approaches such as NGS technology in unraveling the genetic abnormalities in patients with platelet function defects.
TFs and the cis-regulatory sequences to which they bind are critical players in regulating lineage-specific gene expression. FLI1, a member of the ETS (E-twenty-six) family, and RUNX1, along with GATA-1, GATA-2, TAL/SCL, NF-E2, and others, are key regulators of hematopoietic lineage differentiation, megakaryopoiesis, and platelet production.2 FLI1, RUNX1, and GATA-1 physically interact in a combinatorial manner in regulating megakaryocytic genes.2 A single TF mutation may alter the expression of numerous genes, affect diverse cellular mechanisms, and in the present context, lead to concurrent defects in platelet number and function.
In their manuscript, Stockley et al1 report findings in 13 unrelated families from the UK Genotyping and Phenotyping of Platelets study, a multicenter study of patients with suspected platelet function defect. In all, 366 patients have been evaluated in the study. NGS studies were performed in 13 patients who were selected from a group of 56 patients classified as having a secretion defect; they were also selected on the basis of having an affected relative who was available for study. All 13 patients studied had decreased dense granule secretion on platelet activation with multiple agonists and decreased aggregation in response to some agonists. They found FLI1 or RUNX1 mutations in 6 families, of whom 5 had thrombocytopenia, leading the authors to suggest that there was an enrichment of these mutations in patients with secretion defects. This spotlight on TF abnormalities is revealing.
The association of FLI1 and RUNX1 mutations with thrombocytopenia and platelet dysfunction has been previously documented. It is notable that Stockley et al1 found FLI1 mutations in 3 of 13 families studied. Such mutations have been a part of the Paris Trousseau and Jacobsen syndromes, associated with constitutional deletions that include the FLI1 locus on chromosome 11.3 These patients are characterized by bone marrow dysmegakaryopoiesis, thrombocytopenia with a subpopulation of platelets with giant α-granules, and anomalies affecting various organs. It is unclear if the patients described by Stockley et al1 had manifestations besides platelet and skin abnormalities. Moreover, FLI1 is also a transcriptional regulator of several genes, including GP6, GPIBA, GP9, ITGA2, and c-MPL. FLI1 mutations may therefore impact platelet function as well.
Multiple molecular abnormalities associated with alterations in both platelet number and function are documented in patients with inherited RUNX1 mutations. These patients were initially recognized by an association between autosomal dominant thrombocytopenia, abnormal platelet responses, and an increased predisposition to leukemia, and subsequently linked to a RUNX1 haplodeficiency.3,4 The platelet function and biochemical abnormalities reported with RUNX1 mutations encompass decreased aggregation, secretion, protein phosphorylation (myosin light chain and pleckstrin), production of 12-hydroxyeicosapentaenoic acid and αIIbβ3 activation on platelet activation, dense and α-granule deficiencies, and a selective decrease in 1 protein kinase C isoform (PKC-θ).5-8 These indicate that multiple aspects of platelet structure and function are compromised. Interestingly, several patients described earlier as having storage pool deficiency (dense or α-granules) have been subsequently shown to harbor RUNX1 mutations.5
Most inherited RUNX1 mutations have affected the conserved Runt domain.5 Each TF regulates expression of multiple genes, and consistent with this, platelet expression profiling in a patient with RUNX1 haplodeficiency revealed downregulation of numerous genes, including MYL9 (myosin light chain), ALOX12 (12-lipoxygenase), PF4 (platelet factor 4), and PRKCQ (PKC-θ),7-10 all directly relevant to platelet biology. ALOX12, PRKCQ, PF4, and MYL97,8,10 are direct transcriptional targets of RUNX1. Furthermore, patients with RUNX1 haplodeficiency have impaired megakaryopoiesis and decreased platelet thrombopoietin receptors (Mpl).4,7 Thus, platelet dysfunction in RUNX1 haplodeficiency is driven by alterations in multiple genes and pathways.
Mutations in hematopoietic TF GATA-1 are also associated with platelet defects. They encompass X-linked macrothrombocytopenia and platelet dysfunction, including impaired responses to collagen and ristocetin (related to abnormalities in GPIbß), diminished platelet GαS, and the gray platelet syndrome.7
Stockley et al1 suggest that mutations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia. The prevalence of RUNX1 or FLI1 mutations in the overall heterogeneous population of patients with abnormal platelet responses remains unknown. The relatively high frequency observed may be a function of patient selection for the NGS studies. Because the primary focus was on patients with impaired dense granule secretion, it would be of interest to know how many had storage pool deficiency, either of dense or α-granules, both reported with RUNX1 mutations.5,7
This study effectively draws attention to the fact that TF abnormalities may be far more common than currently considered in the pathogenesis of inherited platelet function defects, particularly in those with thrombocytopenia. Equally important, it elegantly highlights the ability of an approach utilizing NGS to detect genetic abnormalities in platelet disorders. It would be most interesting to know what hitherto unrecognized platelet genetic defects are uncovered in the larger pool of patients with impaired platelet function and secretion.
Conflict-of-interest disclosure: The author declares no competing financial interests.