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Clonal hematopoiesis and platelet traits
tion in FANCC. Such patients suffer from progressive bone marrow failure, pancytopenia and predisposition to cancer.50 Knockout mouse studies revealed that FANCA is needed for normal megakaryopoiesis and platelet produc- tion. Megakaryocytes in the deficient mice were found to be in a senescent state.50
In humans, heterozygous mutations of FANCA are observed in a proportion of patients with AML.51 However, carriers of such mutations do not seem to have a significant risk of developing cancer.52 On the other hand, FANCA deletion mutations, especially in combina- tion with other germline mutations, might contribute to breast cancer susceptibility.53 The phenotype coupled to somatic mutations in FANCA and/or FANCC is probably linked to genomic instability caused by defective FANC proteins.51 How these somatic mutations contribute to CHIP-related CVD needs to be established.
GATA2
In immature hematopoietic stem cells, the transcription factor GATA-binding factor 2 (GATA2) is expressed earlier than GATA1, and becomes down-regulated upon differen- tiation.54 This has also been observed in Gata2-knockout mice, revealing that GATA2 is required for hematopoietic stem cell and progenitor cell development.55 In humans, congenital GATA2 deficiency is accompanied by a hypocellular and dysplastic bone marrow, resulting in low platelet counts.55 Furthermore, germline deletion muta- tions in the GATA2 gene are associated with an increased predisposition to infection, AML, CMML or MDS.
On the other hand, a somatic mutation (L359V) in GATA2 has been identified in approximately 10% of patients in the progression phase of CML.56 This concerns a gain-of-function resulting in increased transcription fac- tor activity, in contrast to gene deletion. Reports indicate that in approximately 50% of patients with any GATA2 mutation, the megakaryocyte development is abnormal.57 Unlike GATA1, GATA2 regulates platelet GPIIb rather than GPIb expression.54 Variants of GATA2 have also been associated with increased susceptibility for coronary artery disease,58 linking this gene to CHIP.
GFI1B
Another transcription regulator crucial for erythroid and megakaryocytic differentiation is growth factor independent 1B transcription repressor (GFI1B). As a DNA-binding pro- tein, it regulates the dormancy and mobilization of hematopoietic stem cells.59 Next to the full-size protein of 330 amino acids implicated in megakaryopoiesis, a shorter form is expressed that may rather regulate erythroid development.60 The longer protein modulates the expres- sion of several proto-oncogenes and tumor suppressor genes.59 Accordingly, a functional disturbance of GFI1B can contribute to leukemia development. In mice, genetic deletion of Gfi1b resulted in early lethality, where the embryos showed failed megakaryocyte development.61
For human GFI1B, both germline and somatic muta- tions have been identified. These generally result in a trun- cated or a dysfunctional form of the protein, thereby reducing DNA binding and transcription repressor activi- ty.62 In the inherited disorder gray platelet syndrome, patients with a GFI1B mutation display (macro)thrombo- cytopenia with platelets that are reduced in a-granules.63 The patient's platelets were also found to be reduced in GPIb and GPIIb/IIIa expression, whereas that of the
hematopoietic precursor marker CD34 was markedly increased. The suggestion that, in these and other patients with a truncating mutation of GFI1B, megakaryocyte development is impaired was recently supported by platelet proteome analysis.64 In mice, a megakaryocyte- specific deletion of Gfi1b enhanced the expansion of megakaryocytes, but resulted in severe thrombocytope- nia.65 Here, the (tubulin) cytoskeleton appeared to be underdeveloped in the mutant megakaryocytes, explain- ing an inadequate proplatelet formation.
Whole-exome sequencing efforts have revealed the presence of alternative GFI1B splice variants, which is accompanied by impaired megakaryocyte differentiation and thrombopoiesis.60 However, in heterozygous carriers, platelet counts and function were in normal ranges. Little is known about clonal hematopoiesis. A somatic mutation of GFI1B has been identified in patients with AML.59
SMAD4
The 'vascular' transcription factor SMAD family member 4 (SMAD4) acts as a tumor suppressor, triggered by signal- ing pathways evoked by transforming growth factor-b or bone morphogenetic protein.66 Within the cellular nucleus, SMAD4 forms a complex with other SMAD isoforms to control gene expression. In mice, SMAD4 was found to play a role especially in vascular development.66 On the other hand, a megakaryocyte-specific deficiency of SMAD4 is described, causing mild thrombocytopenia with partially dysfunctioning platelets, likely as a conse- quence of altered promotor activities.67
In humans, both somatic and inherited mutations of SMAD4 are known. Inherited mutations of the gene pres- ent with distinct phenotypes, ranging from a vascular bleeding disorder (hereditary hemorrhagic telangiectasia) to gastro-intestinal and bone marrow abnormalities.68 Somatic mutations of the 358-515 amino-acid region are linked to pancreatic carcinoma.69 During the screening for somatic driver mutations linked to clonal hematopoiesis, a similar mutation of SMAD4 was found.16 Mutations in SMAD4 are related to a bleeding rather than thrombotic phenotype.
TP53
The tumor suppressor tumor protein p53 (TP53 or p53) is a critical player in cell cycle progression and apoptosis. Herein, TP53 maintains the quiescent state of hematopoi- etic stem cells, and controls DNA damage responses upon cellular stress.70 In megakaryocytic cells derived from Tp53 knockout mice, cell size and polyploidization were increased due to higher DNA synthesis and decreased apoptosis. In human cell cultures, TP53 knockdown affected the expression of platelet integrins, granule com- ponents and cytoskeletal proteins, which was accompa- nied by functional platelet defects.71
Regarding human disease, the TP53 deletion occurring in multiple myeloma is accompanied by a lowering in platelet count.72 In this context, mutant TP53 forms are considered to drive clonal hematopoiesis via the epigenet- ic regulator EZH2, leading to overmethylation of histone H3. This can down-regulated several genes associated with self-renewal and differentiation of hematopoietic stem cells.70 A common consequence is expansion of the affected hematopoietic cell clones. Markedly, the TP53 gene is top ranking in mutated genes found in CHIP.73 How mutated TP53 in hematopoietic cells contributes to
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