review

Lessons in platelet production from inherited thrombocytopenias Alessandro Pecci* and Carlo L. Balduini* Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation – University of Pavia, Pavia, Italy

Summary Our knowledge of the cellular and molecular mechanisms of platelet production has greatly expanded in recent years due to the opportunity to culture in vitro megakaryocytes and to create transgenic animals with specific genetic defects that interfere with platelet biogenesis. However, in vitro models do not reproduce the complexity of the bone marrow microenvironment where megakaryopoiesis takes place, and experience shows that what is seen in animals does not always happen in humans. So, these experimental models tell us what might happen in humans, but does not assure us that these events really occur. In contrast, inherited thrombocytopenias offer the unique opportunity to verify in humans the actual effects of abnormalities in specific molecules on platelet production. There are currently 20 genes whose defects are known to result in thrombocytopenia and, on this basis, this review tries to outline a model of megakaryopoiesis based on firm evidence. Inherited thrombocytopenias have not yet yielded all the information they can provide, because nearly half of patients have forms that do not fit with any known disorder. So, further investigation of inherited thrombocytopenias will advance not only the knowledge of human illnesses, but also our understanding of human platelet production. Keywords: inherited thrombocytopenias, platelets, megakaryopoiesis, genetic disorders, bleeding disorders. The cloning of thrombopoietin (TPO) in 1994 (Metcalf, 1994) represented a turning point in the study of megakaryopoiesis, as it made the in vitro culture of megakaryocytes (Mks) feasible and enabled detailed analyses of the events that transform a haemopoietic progenitor into a cell with the unique capacities to become hyperdiploid, move from the stem cell niche to the vascular niche of bone marrow (BM), elongate proplatelets

Correspondence: Carlo L. Balduini, Medicina Interna III, IRCCS Policlinico San Matteo Foundation, piazzale Golgi, 27100 Pavia, Italy. E-mail: [email protected] *These authors contributed equally to the writing of this paper. ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

through the endothelial cells of BM sinusoids and, finally, release platelets directly in the flowing blood. Also, the possibility of genetically manipulating animals to mimic inherited thrombocytopenias (ITs) greatly contributed to the understanding of megakaryopoiesis, enabling researchers to study in depth the functional consequences of changes in specific proteins, further increasing the capacity of dissecting the biological events involved in platelet production. As a result of these technological advances, megakaryopoiesis has changed, in a few years, from an unknown phenomenon to a biological process that is the object of intensive investigation and whose knowledge advances day by day.

Limitations of in vitro and animal models of megakaryopoiesis Despite their undeniable usefulness, in vitro culture of Mks has important limitations as they do not reproduce the complex microenvironment where megakaryopoiesis physiologically takes place (Deutsch & Tomer, 2013). First, BM contains a variety of cells and molecules that are not included in the in vitro models. In addition, BM has a complex architecture, and different cells and molecules have specific locations instead of being distributed randomly. Moreover, the presence of vessels creates gradients of blood-derived molecules as well as of oxygen tension. Attempts to develop in vitro models that more closely reproduce the BM microenvironment are ongoing (Pallotta et al, 2011), but we are still far from this goal. Finally, in vitro cultures of Mks are often derived from cord blood progenitors and, therefore, reproduce fetal haemopoiesis, which differs from adult haemopoiesis in many aspects (Liu & Sola-Visner, 2011). Concerning animal models, we cannot be sure that a phenomenon observed, for instance, in mice or zebrafishes occurs also in humans. This is well illustrated by the observation that the animal models developed to mimic ITs did not always recapitulate patients’ phenotypes (Alexander et al, 1996; Hachet & Ephrussi, 2001; Boulet & Capecchi, 2004; Albers et al, 2012).

Inherited thrombocytopenias as a tool to understand human megakaryopoiesis ITs are caused by naturally occurring changes in the human genome that result in abnormally low platelet counts. Thus,

First published online 30 January 2014 doi:10.1111/bjh.12752

Review these disorders unequivocally demonstrate that the involved genes play an essential role in maintaining a normal platelet count. We presently know at least 20 genes whose abnormalities affect the platelet count (Balduini & Savoia, 2012), mainly by hindering platelet biogenesis. These genetic defects operate in the context of human BM, and therefore the results obtained by investigation of affected subjects do not have the limitations of those obtained in in vitro or animal models. Moreover, comparison of patients’ phenotype with the abnormalities identified in transgenic animals or in cell cultures may be used to validate these models. So, combining the study of patients with that of the experimental models that reproduce their disorders represents the most powerful tool to improve the knowledge of platelet production. This review uses the information that has emerged from the study of ITs to draw a picture of megakaryopoiesis based on reliable evidence. Describing in detail ITs, therefore, is not the aim of this paper, and we refer the readers to recent reviews (Balduini & Savoia, 2012; Geddis, 2013; Pecci, 2013). However, to make reading this review easier, Table I describes the essential features of ITs.

Lessons in megakaryocytic differentiation Three ITs are characterized by the absence or a severely reduced amount of Mks in the BM: congenital amegakaryocytic thrombocytopenia (CAMT), thrombocytopenia with absent radii (TAR) and radio-ulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT), a disorder previously named congenital-amegakaryocytic thrombocytopenia with radio-ulnar synostosis (CTRUS or ATRUS).

Congenital amegakaryocytic thrombocytopenia The best-known amegakaryocytic thrombocytopenia is CAMT, an autosomal recessive disease caused by mutations in MPL, the gene for the TPO receptor (MPL) (Ballmaier & Germeshausen, 2011). This molecule is expressed in haematopoietic stem cells, Mks and platelets, and, when bound by TPO, activates multiple signalling pathways including Jak2/ STAT, Ras/MPK, and PI3K (Deutsch & Tomer, 2013). Thrombocytopenia of CAMT is severe from birth. Moreover, it always evolves into BM aplasia within the first years of life and results in death whenever patients do not undergo successful haematopoietic stem cell transplantation. Nonsense mutations and deletions resulting in premature stop codons are predicted to cause complete loss of the MPL function, while missense and splice-site mutations may be compatible with some residual receptor function. There is evidence for genotype/phenotype correlation in that patients with complete loss of MPL function have consistently very low platelet counts, while those with residual activity have less severe thrombocytopenia (Ballmaier & Germeshausen, 2011). Thus, platelet count and TPO-MPL functional activity 180

are directly related, and this definitely establishes that TPO plays a non-redundant role in platelet production through its interaction with MPL. This role of the TPO-MPL axis in humans is further supported by the observations that MPL mutations that result in a constitutively activated receptor or reduced TPO clearance, as well as TPHO mutations resulting in increased TPO translation, have been identified in some families with familial forms of essential thrombocythaemia (Skoda, 2010; Teofili & Larocca, 2011). The finding that BM Mks are absent or severely reduced in CAMT clearly indicates that TPO is essential for commitment-differentiation of multipotent stem cells to the Mk lineage. However, the finding that the residual Mks observed in CAMT patients are often small and immature supports a role of the TPO-MPL axis also in the subsequent phases of Mk maturation. Another important lesson from CAMT is that the TPO-MPL pathway is essential also for erythrocyte and leucocyte production in humans, because the disease evolves to BM aplasia. The non-synchrony between the onset of thrombocytopenia and that of anaemia and leucopenia indicates that individual haematopoietic lineages have different dependencies on MPL signalling and points to the existence of agedependent differences in the regulation of haematopoiesis by TPO-MPL. Mpl / mice did not reproduce the clinical course of CAMT patients because they were thrombocytopenic but did not develop pancytopenia (Alexander et al, 1996) and therefore cannot be used to investigate the mechanisms of BM failure. In contrast, induced pluripotent stem cells from a CAMT patient reproduced defective megakaryopoiesis of patients and demonstrated that MPL signalling is essential for the maintenance of multipotent haematopoietic and common Mk/erythrocyte progenitors (Hirata et al, 2013). This role of the TPO-MPL axis provides an explanation for the trilineage haematopoietic responses observed in a recent clinical trial evaluating the TPO-mimetic eltrombopag in patients with severe aplastic anaemia refractory to immunosuppression (Olnes et al, 2012).

Thrombocytopenia with absent radii As with CAMT, TAR also presents at birth with thrombocytopenia and reduced number of BM Mks, but infants have the additional finding of bilateral absent radii, sometime associated with other malformations. At variance with CAMT, the platelet count ameliorates during the first years of life and patients usually do not develop additional cytopenias (Geddis, 2009). TAR is caused by the compound inheritance of a low-frequency noncoding single nucleotide polymorphism and a rare null allele in RBM8A, a gene encoding the exon-junction complex subunit member Y14 (Albers et al, 2013). The recessive inheritance of the phenotype and the finding that the level of Y14 is significantly lower in patients’ platelets (Albers ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Review Table I. Main features of inherited thrombocytopenias classified according to the main mechanism of defective platelet production.

Inheritance

Gene (chromosome localization)

AR

MPL (1p34)

AR

RBM8A (1q21
1)

AD

HOXA11 (7p15-14)

AD

CBFA2 (21q22)

AD

Large deletion (11q23-ter)

XL

GATA1 (Xp11)

AD

GFI1B (9q34
13)

ANKRD26-related thrombocytopenia (THC2, 313900)

AD

ANKRD26 (10p2)

Gray platelet syndrome (GPS, 139090)

AR

NBEAL2 (3p21
1)

Defective proplatelet formation and/or platelet release MYH9-related disease (MYH9-RD, nd)

AD

MYH9 (22q12-13)

ACTN1-related thrombocytopenia (ACTN1-RT, nd) FLNA-related thrombocytopenia (FLNA-RT, nd)

AD XL

ACTN1 (14q24) FLNA (Xq28)

Wiskott–Aldrich syndrome (WAS, 301000)

XL

WAS (Xp11)

AR

GP1BA (17p13), GP1BB (22q11), GP9 (3q21) GP1BA (17p13) ITGA2B (17q21
31), ITGB3 (17q21
32) TUBB1 (6p21
3) CYCS (7p15
3)

Disease (abbreviation in this paper, OMIM entry) Defective megakaryocytic differentiation Congenital amegakaryocytic thrombocytopenia (CAMT, 604498) Thrombocytopenia with absent radii (TAR, 274000)

Congenital thrombocytopenia with radio-ulnar synostosis (CTRUS, 605432)

Defective megakaryocyte maturation Familial platelet disorder and predisposition to acute myeloid leukaemia (FPD/AML, 601399) Paris-Trousseau thrombocytopenia (TCPT, 188025/600588), Jacobsen syndrome (JBS, 147791) GATA1-related diseases (GATA1-RDs, Dyserythropoietic anaemia with thrombocytopenia, 300367 – X-linked thrombocytopenia with thalassaemia, 314050) GFI1B-related thrombocytopenia (GFI1B-RT, nd)

X-linked thrombocytopenia (XLT, 313900) Bernard-Soulier syndrome (BSS, 231200) Biallelic

Monoallelic ITGA2B/ITGB3-related thrombocytopenia (ITGA2B/ITGB3-RT, nd) TUBB1-related thrombocytopenia (TUBB1-RT, nd) CYCS-related thrombocytopenia (CYCS-RT, 612004)

AD AD AD AD

Other features

Evolves into bone marrow aplasia in infancy. Normal-sized platelets Platelet count tends to rise and often normalizes in adulthood. Reduced megakaryocytes. Normalsized platelets. Bilateral radial aplasia +/ other malformations Radio-ulnar synostosis +/ other defects. Possible evolution into aplastic anaemia. Normal-sized platelets Possible development of leukaemia or MDS. Normal-sized platelets Cardiac and facial defects, developmental delay +/ other defects. Large platelets Haemolytic anaemia, possible unbalanced globin chain synthesis, possible congenital erythropoietic porphyria. Large platelets Red blood cells anisocytosis. Large platelets Possible development of leukaemia or MDS. Normal-sized platelets Thrombocytopenia worsens with age. Evolutive myelofibrosis and splenomegaly. Giant platelets Cataracts, nephropathy and/or deafness. Liver enzymes may be elevated. Giant platelets Large platelets Periventricular nodular heterotopia (MIM 300049). Large platelets Severe immunodeficiency leading to death in infancy. Small platelets Mild immunodeficiency. Small platelets Giant platelets

Large platelets Large platelets Giant platelets Normal-sized platelets

OMIM, Online Mendelian Inheritance in Man; AD, autosomal dominant; AR, autosomal recessive; XL, linked to chromosome X; MDS, myelodysplastic syndrome; nd, not defined.

et al, 2012) indicates that TAR originates from reduced expression of this molecule below a critical threshold. The exon-junction complex is involved in RNA processing and ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

required for same basic cellular functions as nuclear export and subcellular localization of specific transcripts (Albers et al, 2013). Although how Y14 deficiency affects Mk 181

Review production is still unknown, recent studies in platelets of TAR children showed defects in TPO signal transduction (Fiedler et al, 2011). In particular, a correlation has been found between the lack of phosphorylation of the Jak2 kinase (directly downstream of the TPO receptor) and the number of platelets. TPO signalling corrected with age, as Jak2 phosphorylation after TPO stimulation was normal in nearly all investigated adult samples. Interestingly, the improvement of thrombocytopenia occurring in patients during the first years of life preceded restoration of TPO-MPL signalling, which was detected much later. Thus, these events are not directly connected and an unknown, age-related, mechanism mediates the improvement of platelet production. The animal models created so far did not reproduce the phenotype of TAR (Hachet & Ephrussi, 2001; Albers et al, 2012), and therefore this approach was not useful for understanding the molecular mechanisms of defective Mk production. Nevertheless, as CAMT indicates that the role of the TPO-MPL signalling pathway in haemopoiesis varies according to age, TAR demonstrates the existence of age-related differences in the requirement for Y14 in megakaryopoiesis.

Radio-ulnar synostosis with amegakaryocytic thrombocytopenia This very rare IT shares many features with both CAMT and TAR. Amegakaryocytic thrombocytopenia is present from birth and, as in TAR, there is a defect in radii, in this case consisting in the proximal fusion of ulna and radius. At variance with TAR, platelet count does not improve with age, and, similarly to CAMT, although less frequently, children may develop BM failure (Thompson et al, 2001). Most patients with the clinical phenotype of RUSAT described so far had mutations in HOXA11 (Castillo-Caro et al, 2010), a member of the HOX family of genes encoding for transcription factors whose expression is spatially and temporally regulated during embryonic development. In particular, HOXA11 was previously known to play a role in mice endometrial growth and female fertility (Taylor et al, 1997). The molecular mechanisms that translate HOXA11 mutations in thrombocytopenia and BM aplasia are unknown. Hoxa11-knockout mice were useless to clarify this phenomenon, as they presented with skeletal abnormalities but did not have any haematological phenotypes (Boulet & Capecchi, 2004). So, the only lesson from RUSAT is that HOXA11 in humans is essential from birth for Mk production, but may be required later for the maintenance of haematopoiesis.

Lessons in megakaryocyte maturation In six forms of IT, a defect of Mk maturation seems to be the most important pathogenetic mechanism, although in some cases other mechanisms, such as shortened platelet life 182

span or impaired proplatelet formation (PPF), contribute to the low platelet count.

Thrombocytopenias deriving from mutations affecting transcription factors Familial platelet disorder with predisposition to acute myeloid leukaemia. Familial platelet disorder with predisposition to acute myeloid leukaemia (FDP/AML) derives from mutations in RUNX1, also known as AML1 or CBFA2, encoding for the DNA-binding a subunit of the core binding factor (CBF) transcription complex. Heterodimerization to its partner CBF-b enhances the affinity of RUNX1 to DNA and protects it from proteolytic degradation (Song et al, 1999). The CBF regulates expression of multiple haematopoiesis-specific genes and is essential for the establishment of definitive haematopoiesis (Asou, 2003). Somatic mutations of RUNX1 are frequently identified in subjects with acute myeloid leukaemia (AML) and chronic myelomonocytic leukaemia (Murati et al, 2012). Most mutations identified in FPD/AML patients result in haploinsufficiency, whereas some variations are predicted to act by dominant-negative effects (Matheny et al, 2007). Patients have thrombocytopenia, in some cases associated with a platelet aspirin-like functional defect, and increased risk to develop AML or myelodysplastic syndromes, which occur in about 40% of cases (Liew & Owen, 2011). The latter phenotype reflects the role of RUNX1 in regulating the homeostasis between proliferation and differentiation of stem cells in adult haematopoiesis (Link et al, 2010). Immature haematopoietic progenitors of FDP/AML patients have increased clonogenic potential and, in some cases, aberrant self-renewal capacities (Bluteau et al, 2011). Consistently, Runx1 conditional knockout mice presented expansion of haematopoietic stem cells that could predispose to leukaemia development (Nishimoto et al, 2011). Mks cultured from CD34+ cells of FDP/AML subjects were characterized by profound defects in maturation to glycoprotein (GP)IIb+/GPIX+ cells and in polyploidization. These abnormalities identified in in vitro studies are consistent with the finding of micromegakaryocytes and immature Mks with poorly developed cytoplasm and reduced/absent nuclear lobulation in patients’ BM biopsies. Similar defects could be induced by RUNX1 silencing in human Mks (Bluteau et al, 2012). Moreover, Runx1-deleted mice have thrombocytopenia that is associated with remarkable abnormalities of both polyploidization and cytoplasmic maturation of BM Mks (Ichikawa et al, 2004). In in vitro models of Mk maturation, RUNX1 downregulated the expression of MYH10, the gene for non-muscle myosin heavy chain IIB (NMMHC-IIB), and NMMHC-IIB silencing was shown to be essential for the switch from mitosis to endomitosis and, therefore, for polyploidization (Lordier et al, 2012). In FDP/AML patients, defective Mk polyploidization was associated with persistence of ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Review NMMHC-IIB in mature Mks and platelets, and drug-mediated inhibition of NMMHC-IIB in patients’ Mks rescued the polyploidization defect, strongly suggesting that this mechanism is operative in vivo (Antony-Debr
e et al, 2012; Bluteau et al, 2012). Defective Mk maturation is not the only cause of thrombocytopenia in FDP/AML. In fact, it has been shown that patients’ Mks have a defect of proplatelet extension and maturation, possibly deriving from reduced RUNX1-mediated transactivation of two other cytoskeletal proteins, MYH9 (see below) and its regulator, MYL9. Moreover, a defective MPL expression has been identified in FPD/AML, and it has been suggested that reduced Mk differentiation could also contribute to thrombocytopenia (Heller et al, 2005; Bluteau et al, 2012). Paris-Trousseau thrombocytopenia and Jacobsen syndrome. ParisTrousseau thrombocytopenia (TCPT) and Jacobsen Syndrome (JBS) are overlapping disorders caused by partial deletions of 11q23-ter. The distinction between the two forms lies on the severity of the syndromic picture (Table I), which, in turn, reflects the size and the breakpoints of the causative deletions (Favier et al, 2003; Mattina et al, 2009). The platelet defects of TCTP/JBS are due to the loss of one copy of FLI1, which is included in the deleted region. Different experimental approaches indicated that FLI1 promotes platelet biogenesis by transactivation of many genes associated with Mk development, such as MPL, GP9, GP1BA, ITGA2 and PF4 (Athanasiou et al, 2000; Raslova et al, 2004), but, indeed, TCPT/JBS is the most convincing demonstration of the non-redundant role of this gene in human megakaryopoiesis. BM biopsies of TCPT/JBS patients showed a maturation block in the megakaryocytic linage, with abundance of immature, hypolobulated, dystrophic Mks (Favier et al, 2003). Raslova et al (2004) showed that the lentivirus-mediated FLI1 transduction in the CD34+ cells of three TCPT children rescued the in vitro abnormalities of megakaryopoiesis, by increasing the number of differentiated GPIIb+/GPIX+ or von Willebrand factor (VWF) positive cells, large polylobulated Mks, and proplatelet-bearing Mks. As with FDP/ AML, TCPT is associated with aberrant NMMHC-IIB persistence in mature Mks and platelets, which could be implicated in the defective polyploidization (Antony-Debr
e et al, 2012). GATA1-related thrombocytopenias. Mutations of GATA1 are responsible for inherited forms of thrombocytopenia that are associated with various degrees of anaemia, reflecting the key role of this transcription factor in both megakaryopoiesis and erythropoiesis. In fact, GATA1 regulates not only genes expressed in the Mk lineage, such as GP1BA, GP1BB, PF4, MPL and NFE2, but also genes of the erythroid line, such as HBB, ALAS1 and BCL2L1 (Millikan et al, 2011). Genotype-phenotype correlations provided important information about the significance of variations in different ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

functional regions of GATA1. Dyserythropoietic anaemia with thrombocytopenia (DAT) is caused by five substitutions (p.V205M, p.G208R or p.G208S, and p.D218G or p.D218Y) impairing the ability of GATA1 to bind with its cofactor FOG1 while preserving its DNA binding capacity. The disease is characterized by thrombocytopenia, platelet morphological and functional defects, and severe dyserythropoietic anaemia. Correlations between the severity of the clinical picture and the degree of impairment of GATA1/FOG1 interaction have been reported (Freson et al, 2002). X-linked thrombocytopenia with thalassaemia (XLTT) derives from the p.R216Q substitution that impairs GATA1 binding with palindromic DNA sites and does not affect the interaction with FOG1. XLTT patients present with milder thrombocytopenia and platelet dysfunction than in DAT, and haemolysis associated with an unbalanced a:b globin chain synthesis that can result in mild anaemia (Balduini et al, 2004). Thus, the p.R216Q has a much lesser impact on erythroid lineage maturation and erythrocyte production than substitutions impairing the FOG1-binding ability, as confirmed by in vitro investigations (Yu et al, 2002). BM examination of patients with DAT or XLTT evidenced an increased number of immature, dystrophic Mks, suggesting the dysmegakaryopoietic nature of GATA1-related thrombocytopenia (Nichols et al, 2000; Freson et al, 2002; Balduini et al, 2004). Investigations on GATA1-deficient mice confirmed that alteration of Mk maturation is the main mechanism of thrombocytopenia. In fact, Mks of these mice exhibited hyperproliferation but evident defects of both polyploidization and cytoplasmic development associated with reduced transcription of key genes in Mk maturation, such as GP1BA, GP1BB, PF4, MPL, and NFE2 (Vyas et al, 1999). GFI1B-related thrombocytopenia. Very recently, two families with autosomal-dominant macrothrombocytopenia, a-granule deficiency and moderate-to-severe bleeding caused by monoallelic mutations GFI1B have been reported. In one family, thrombocytopenia was associated with erythrocyte anisopoikilocytosis (Monteferrario et al, 2014; Stevenson et al, 2013). Animal and cell culture models showed that GFI1B is a transcriptional factor involved in homeostasis of haematopoietic stem cells and development of the megakaryocytic and erythroid lineages. Mice with conditional disruption of Gfi1b had thrombocytopenia and anaemia (Saleque et al, 2002; Khandanpour et al, 2010; Randrianarison-Huetz et al, 2010). The phenotype of patients with GFI1B mutations confirms that its product also has a role in human biogenesis of platelets and erythrocytes. Patients’ BM biopsies showed an increased number of Mks with severe dysplastic features, including nuclear hypolobulation and a-granule deficiency, indicating that the platelet defect derives from altered Mk maturation (Monteferrario et al, 2014). Mild myelofibrosis was also observed. Deranged Mk maturation was associated with defective expression of several platelet-specific proteins, such as fibrinogen, P-selectin, 183

Review and GPIba. Both causative mutations result in the disruption of the highly-conserved DNA-binding regions of zinc-finger domains 5 and/or 6 of GFI1B. Transfection studies of cell lines and mouse Mks demonstrated that GFI1B mutants inhibit the activity of the wild-type protein in a dominant-negative manner (Monteferrario et al, 2014; Stevenson et al, 2013). This finding is consistent with mouse models showing that only complete Gfi1b ablation affects megakaryopoiesis, whereas the loss of a single Gfi1b allele is not sufficient to cause a phenotype (Saleque et al, 2002).

Other inherited thrombocytopenias with defective megakaryocyte maturation ANKRD26-related thrombocytopenia. ANKRD26-related thrombocytopenia (ANKRD26-RT) is caused by monoallelic point substitutions in the 5′UTR of ANKRD26 that are likely to result in overexpression of the gene during megakaryopoiesis (Pippucci et al, 2011). The first reported series of patients suggested that ANKRD26-RT predisposes to myeloid malignancies (Noris et al, 2011), and a recently published additional case series confirmed this finding (Noris et al, 2013). In both reports, malignancies occurred in about 8% of patients. Moreover, some subjects presented with leucocytosis and increased haemoglobin levels, which suggest a deregulation at the level of the common myeloid progenitor that, in some cases, evolves into malignancies. Mks and platelets of ANKRD26-RT patients are characterized by cytoplasmic inclusions containing components of the ubiquitin/proteasome system, a phenomenon that has been described in other dysplastic conditions, as well as in cancer (Necchi et al, 2013). Moreover, examination of BM of ANKRD26-RT patients showed increased number of Mks with marked dysmegakaryopoiesis and micromegakaryocytes (Noris et al, 2012). Altogether, these observations suggest the dysmegakaryopoietic nature of ANKRD26-RT. ANKRD26 was recently implicated in the regulation of adipogenesis in mouse embryonic fibroblasts by interacting with some pathways that have a prominent role also in Mks and platelets, such as ERK or Akt pathways (Fei et al, 2011). However, both the exact role of ANKRD26 in physiological megakaryopoiesis and the molecular pathogenesis of ANKRD26-RT remain unknown. Gray platelet syndrome. Gray platelet syndrome (GPS) is characterized by macrothrombocytopenia, absence of platelet a-granules, splenomegaly and myelofibrosis. Thrombocytopenia and myelofibrosis worsen with age (Gunay-Aygun et al, 2010). This disorder derives from biallelic loss-of-function mutations of NBEAL2, coding for neurobeachin-like protein 2, which probably plays a role in protein-protein interactions, membrane dynamics and vesicle trafficking (Balduini & Savoia, 2012). The number of BM Mks is normal, and until a short time ago, reduced platelet survival and BM fibrosis were held 184

responsible for thrombocytopenia (Gunay-Aygun et al, 2010). Kahr et al (2013) developed an Nbeal2 / mouse model that reproduced macrothrombocytopenia, profound deficiency of platelet and Mk a-granules and splenomegaly of GPS patients, although it did not show myelofibrosis. In vivo and in vitro investigations revealed that Mks of these mice were characterized by abnormal maturation, with abundance of immature forms, reduced proportion of mature and proplatelet-forming Mks, and reduced polyploidization. In addition to the lack of a-granules, ultrastructural abnormalities of mature Mks included cytoplasmic vacuolization and abnormal localization of VWF (Kahr et al, 2013). On this basis, it has been suggested that normal development of a-granules and proper package of their cargo proteins are required for normal Mk maturation. Deppermann et al (2013) developed another Nbeal2 / mouse model that reproduced macrothrombocytopenia and a-granule deficiency, whereas splenomegaly and myelofibrosis were not reported. Although Mks of these latter knock-out mice presented some morphological features similar to those reported by Kahr et al (2013), the production of mature, proplateletforming Mks was unaffected in this model. Differently to what observed in some GPS patients, platelet survival was normal (Deppermann et al, 2013). Given that both mouse models differ in some aspects from GPS patients, further studies are required to confirm that conclusions deriving from their investigation apply also to humans.

Lessons in proplatelet formation and platelet release In eight forms of ITs, the main pathogenetic mechanism is the alteration of PPF, whereas Mk differentiation and maturation are preserved. This group of disorders includes thrombocytopenias deriving from mutations of key cytoskeleton components or of membrane integrins expressed in the late phases of Mk differentiation, as well as two rare forms deriving from mutation of b1 tubulin or cytochrome c. Figure 1 outlines the cytoskeletal molecules and the membrane glycoproteins (GPs) whose mutations result in ITs.

Thrombocytopenias deriving from mutations affecting the cytoskeleton MYH9-related disease. MYH9-related disease (MYH9-RD) is a syndromic disorder deriving from mutations of MYH9, the gene for the heavy chain of non-muscle myosin IIA (NMMHC-IIA) (Savoia et al, 2010; Balduini et al, 2011). Myosin IIA belongs to a superfamily of cytoskeletal proteins that slide along actin filaments and generate mechanical forces by ATP hydrolysis. It is ubiquitously expressed in mammalians and participates in many basic cellular functions, such as cytokinesis, cell motility and polarization, and maintenance of cell shape (Vicente-Manzanares et al, 2009). The observation that macrothrombocytopenia is the only ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Review

Fig 1. Cytoskeleton components and related molecules (in colour) that are affected by mutations in inherited thrombocytopenias. A cytoskeleton structurally intact and functional, as well as a proper interaction with the cell membrane, are required in order for megakaryocytes to efficiently form proplatelets and release platelets in the lumen of bone marrow sinusoids. The defective cytoskeleton anchoring to the membrane caused by mutations affecting the GPIb-IX-V complex (Bernard-Soulier syndrome) or filamin 1 (FLNA-related thrombocytopenia), as well as the deranged cytoskeleton structure/ function induced by mutations in a-actinin (ACTN1-related thrombocytopenia) or MYH9 (MYH9-related disease) hinder proplatelet formation and/or result in ectopic release of platelets outside the vascular lumen. Also, the constitutive activation of the GPIIb-IIIa complex induced by monoallelic mutations in ITGA2 or ITGB3 (ITGA2/ ITGB3-related thrombocytopenia) hampers proplatelet formation and platelet release by affecting cytoskeleton function.

phenotype presented from birth by all MYH9-RD patients pointed for the first time toward a non-redundant role of NMMHC-IIA in platelet biogenesis. Patients with MYH9-RD have a normal or increased number of Mks with normal morphology (Heynen et al, 1988). Accordingly, Mks cultured from haematopoietic progenitors of MYH9-RD subjects showed a preserved response to TPO with normal output of mature forms (Pecci et al, 2009), and mouse models of MYH9-RD showed an increased presence of mature Mks in the BM (Zhang et al, 2012; Suzuki et al, 2013). Moreover, an in vitro study found that even Mks differentiated from Myh9 / mouse embryonic stem cells present no overt maturation defects, thus supporting the conclusion that NMMHC-IIA is totally dispensable for the production of mature Mks (Chen et al, 2007). In contrast, there is consistent evidence that MYH9 mutations affect PPF. Mks cultured from MYH9-RD patients presented an obvious defect in branching of proplatelets resulting in reduced complexity of their architecture, as well as enlarged proplatelet tips with thicker microtubule bands (Pecci et al, 2009). Accordingly, clearly defective PPF has been observed in two mouse models of MYH9-RD closely reproducing the phenotype of the human disease (Zhang et al, 2012; Suzuki et al, 2013). These abnormalities were demonstrated not only in Mks cultured in vitro, but also in ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Fig 2. Myosin IIA-mediated inhibition of proplatelet formation. Interaction of megakaryocyte with collagen type I through GPIa-IIa activates the small GTPase Rho and the Rho-kinase ROCK, which in turn phosphorylates the regulatory light chains of myosin IIA resulting in the inhibition of proplatelet formation. Type I collagen is abundant in the stem cell niche of BM and, thus, this pathway serves to prevent megakaryocytes from a premature release of platelets in this space before they reach the bone marrow sinusoids (Chen et al, 2007). This inhibitory mechanism is affected in MYH9-related disease due to defective myosin IIA.

the animals’ BM examined by live imaging (Zhang et al, 2012). Proplatelet branching serves to multiply the number of free proplatelet ends, and, therefore, to increase the number of platelets released by each Mk. So, a defect in this process can be directly associated with thrombocytopenia (Italiano et al, 1999; Thon & Italiano, 2012). On the other hand, the increased size of proplatelet tips observed in animal and in in vitro models reproduced the platelet macrocytosis that is always observed in patients (Noris et al, 2009; Pecci et al, 2009). These observations indicate that NMMHC-IIA determines platelet size and number by regulating the cytoplasmatic remodelling and the fission events that result in PPF and platelet release (Thon et al, 2010; Thon & Italiano, 2012). Investigations on MYH9-RD pathogenesis suggested that NMMHC-IIA also regulates the timing of PPF. In particular, in vitro studies both mouse and human Mks indicated that functional NMMHC-IIA is required for suppression of PPF upon Mk adhesion to type I collagen through GPIa-IIa (Chang et al, 2007; Chen et al, 2007; Balduini et al, 2008) This mechanism is considered essential for spatio-temporal regulation of PPF in vivo (Fig 2). Consistently, Mks cultured from blood progenitors of MYH9-RD patients extended proplatelets even in adhesion to type I collagen, supporting the hypothesis that a premature, ectopic release of platelets contributes to thrombocytopenia (Pecci et al, 2009). ACTN1-related thrombocytopenia. Mutations in ACTN1, the gene for the isoform 1 of a-actinin, were recently identified 185

Review as responsible for a non-syndromic autosomal-dominant thrombocytopenia with platelet macrocytosis (Kunishima et al, 2013). ACTN1 is a member of a superfamily of proteins involved in the organization of acto-myosin cytoskeleton: in non-muscle cells, ACTN1 stabilizes the filamentous actin network by cross-linking actin filaments in bundles. All causative mutations hit the functional actin-binding or calmodulin-like ACTN1 domains, and deranged organization of actin filaments upon adhesion to immobilized extracellular matrix proteins was observed in cells transfected with ACTN1 mutants. Interestingly, mouse Mks transduced with these ACTN1 variants showed qualitative abnormalities of PPF, such as defective proplatelet branching, reduced number of proplatelet free ends and enlarged tips (Kunishima et al, 2013), which therefore resembled the alterations observed in MYH9-RD patients and mouse models of this disorder. FLNA-related thrombocytopenia. Filamin A, encoded by the gene FLNA, is a ubiquitously expressed component of the cytoskeleton. Filamin A functions in Mks and platelets include the binding of the cytoplasmic tail of GPIba to the filamentous actin network, cross-linking of actin filaments, and regulation of Syk kinase activation (Nakamura et al, 2006; Falet et al, 2010). Nurden et al (2011a) reported four unrelated females with platelet macrocytosis with or without thrombocytopenia resulting from monoallelic FLNA mutations. Mks of these subjects showed preserved in vitro development despite the fact that a proportion of these cells (as well as a proportion of platelets) were completely negative upon FLNA immunostaining (Nurden et al, 2011a; Berrou et al, 2013). Thus, FLNA is dispensable for Mk production, consistent with the increased number of mature Mks observed in Flna-null mice (Jurak Begonja et al, 2011). However, Mks of patients had both quantitative and qualitative defects in PPF. In particular, some mature Mks presented ultrastructural abnormalities consistent with an aberrant cytoplasmic fragmentation rather than a typical proplatelet extension, possibly deriving from instability of the submembranous actin cytoskeleton (Nurden et al, 2011a). Wiskott–Aldrich syndrome and X-linked thrombocytopenia. Wiskott–Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are two X-linked disorders characterized by moderate to severe thrombocytopenia deriving from mutations in the WAS gene, encoding for the WAS protein (WASp). WAS mutations resulting in truncated or not expressed WASp cause WAS, which has the additional feature of severe immune deregulation, while missense mutations causing a reduced expression of normal sized WASp result in XLT, which is mainly characterized by thrombocytopenia. A peculiar characteristic of both WAS and XLT is the reduced size of platelets (Massaad et al, 2013). WASp is selectively expressed in haematopoietic cells, where it plays a key role as regulator of the actin cytoskele186

ton. WAS mutations do not affect either differentiation or maturation of Mks, because these cells are normally represented in the BM of patients. Moreover, in vitro liquid cultures of human WAS Mks showed that they normally produce platelets (Haddad et al, 1999). A Was / mouse model confirmed that Mks regularly extend proplatelets. It also showed that WASp-deficient Mks have a profound defect in chemotactic migration and in the inhibition of PPF mediated by Mk adhesion to type I collagen (Sabri et al, 2006). These defects resulted in an ectopic shedding of platelets within the BM space. However, the Was / mice had only mild thrombocytopenia and did not reproduce the immunodeficiency of WAS. So, it is uncertain whether similar defects are also operative in patients with WAS mutations. Moreover, it is well known that platelet life span is reduced in WAS/XLT patients and that splenectomy normalizes or significantly increases platelet count (Litzman et al, 1996). In conclusion, it is presently unknown whether and to what extent a defect of platelet production contributes to thrombocytopenia in WAS/XLT.

Thrombocytopenias deriving from mutations of integrins Bernard-Soulier syndrome. Bernard-Soulier Syndrome (BSS) is caused by biallelic mutations in GP1BA, GP1BB or GP9 affecting the assembly of GPIba, GPIbb and GPIX in the GPIb-IX-V complex in Mk endoplasmic reticulum and its subsequent expression on plasma membrane (Berndt & Andrews, 2011; Savoia et al, 2011). Most patients present with thrombocytopenia, giant platelets and a bleeding tendency that is more severe than expected on the basis of platelet count. This discrepancy is explained by the inability of GPIb/IX/V-deficient platelets to adhere to the vascular subendothelium exposed in damaged vessels. The majority of obligate carriers do not have macrothrombocytopenia, although the diameters of their platelets are in the upper part of the normal range (Strassel et al, 2004; Savoia et al, 2011). However, a few mutations in GPIba, namely p.A156V (Bolzano mutation), p.N41H, p.Y54D and p.L57F, exert a dominant effect that results in mild macrothrombocytopenia also in monoallelic subjects expressing about 50% of the wild-type protein (monoallelic BSS) (Noris et al, 2012). Mks of patients with biallelic BSS show normal in vitro differentiation and maturation (Balduini et al, 2009; Vettore et al, 2011), in agreement with the earlier observation of a normal or increased number of Mks in patients’ BM (Tomer et al, 1994). However, Mks cultured from these subjects do not extend proplatelets, suggesting that thrombocytopenia derives from a defect of the latest phases of megakaryopoiesis. This hypothesis is further supported by the observation that proplatelet extension is reduced, but not abolished, in subjects with monoallelic BSS, suggesting that a correlation exists between the severity of the GPIb/IX/V defect, the degree of thrombocytopenia and the impairment of in vitro PPF. Of note, proplatelet tips of Mks cultured from subjects ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Review with monoallelic BSS recapitulate the morphological abnormalities of circulating platelets, consisting of enlarged size and abnormal a-tubulin distribution. Animal models of BSS reproduced patients’ phenotype and mice with targeted disruption of GPIba or GPIbb confirmed that GPIb-IX-V deficiency induces macrothrombocytopenia by hampering PPF (Kanaji et al, 2002; Strassel et al, 2009). An important issue is which of the properties of the GPIb-IX-V complex is essential for platelet biogenesis. In Mks of BSS patients, the defects in PPF were evident not only upon adhesion to VWF, but also when Mks were cultured in suspension or upon adhesion to fibrinogen (Balduini et al, 2009; Vettore et al, 2011). Similar observations were made with Mks of GPIbb-null mice, which showed the same PPF abnormalities when observed within animal BM or when cultured in suspension (Strassel et al, 2009). These observations suggest that the alteration of PPF is intrinsic to Mks rather than due to a defective interaction with components of the extracellular matrix. The finding that patients or mice with severe deficiency of VWF or P-selectin, another ligand of GPIb-IX-V, have normal platelet counts further supports this hypothesis (Subramaniam et al, 1996; Denis et al, 1998). Furthermore, macrothrombocytopenia of GPIba-null mice could be rescued by transduction of the intracellular domain of GPIba alone, indicating a role in PPF for this portion of the integrin, which binds the acto-myosin cytoskeleton though Filamin A (Fig 1) (Kanaji et al, 2002). Indeed, the abnormalities of proplatelet morphology of BSS patients and mouse models have many similarities with those of patients and mouse models of macrothrombocytopenias deriving from mutations of cytoskeletal proteins (Balduini et al, 2009; Pecci et al, 2009; Strassel et al, 2009; Zhang et al, 2012). This growing body of evidence suggests that macrothrombocytopenia of BSS derives from alteration of Mk cytoskeletal functions due to the defective anchoring of the acto-myosin network to plasma membrane. ITGA2B/ITGB3-related thrombocytopenia. Glanzmann Thromboasthenia (GT) is characterized by a severely defective GPIIb/ IIIa complex in Mks and platelets that derives from biallelic mutations in ITGA2 or ITGB3. Affected subjects have normal platelet count and morphology, but present with severe bleeding diathesis due to defective binding to platelets of fibrinogen and other soluble adhesive proteins because of the GPIIb-IIIa defect (Nurden et al, 2013). Interestingly, five different ITGA2 or ITGB3 mutations causing macrothrombocytopenia in a dominant manner have been reported recently. Affected subjects had some functional defects of platelets, but did not present with the severe GT phenotype (Nurden et al, 2011b; Bury et al, 2012). Patients’ Mks and platelets, as well as cell lines transfected with these mutants, showed spontaneous binding of PAC1 (a monoclonal antibody that binds only the activated GPIIb/IIIa complex) under resting conditions. This indicates that mutations ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

result in a constitutive, albeit partial, activation of GPIIb-IIIa, with a constitutive activation of the downstream effectors of GPIIb-IIIa signalling, such as FAK and c-Src (Ghevaert et al, 2008; Kunishima et al, 2011; Bury et al, 2012). Thus, the GPIIb-IIIa complex has a crucial role in platelet function, but is dispensable for platelet production in humans; however, an inappropriate activation of this complex results in macrothrombocytopenia. A few evidences in humans and mice suggested that mutations of ITGA2B/ITGB3-related thrombocytopenia affect platelet count by impairing PPF. Mks differentiated from patients, in the face of preserved Mk maturation, showed qualitative defects of in vitro PPF, consisting of defective branching, reduced number and increased size of proplatelet tips (Ghevaert et al, 2008; Bury et al, 2012). Abnormalities of proplatelet maturation in peripheral blood were also reported in two patients (Bury et al, 2012). Moreover, transduction of the GPIIb R995W variant in fetal liver mouse Mks resulted in defects of proplatelet architecture very similar to those observed in patients (Liew & Owen, 2011). These PPF abnormalities were attributed to defective remodelling of the actomyosin cytoskeleton consequent to the constitutive activation of c-Src/FAK (Bury et al, 2012). Other observations suggested that inappropriate activation of GPIIb-IIIa signalling is associated with reduced activity of RhoA, which is implicated in myosin light chain kinase-dependent activation of NMMHC-IIA (Schaffner-Reckinger et al, 2009). Therefore, even the hyperfunction of GPIIb-IIIa is expected to affect cytoskeletal function.

Other inherited thrombocytopenias with defects of platelet formation or platelet release TUBB1-related thrombocytopenia. Kunishima et al (2009) reported one family with autosomal-dominant macrothrombocytopenia caused by the mutation p.R318W in TUBB1, the gene for b1 tubulin, whose expression in mammals is restricted to mature Mks and platelets. This mutation was predicted to dominantly affect microtubule assembly by disrupting interaction of a and b tubulin in heterodimers. Mks were normally represented in proband BM biopsy, and in vitro development of patient Mks was normal. However, Mks presented an abnormal morphology consistent with impaired proplatelet extension and release (Kunishima et al, 2009). In addition to the in vitro studies on PPF referenced above, other in vivo evidences support the role of b1 tubulin in regulating platelet number and size. Tubb1-null mice have macrothrombocytopenia deriving from defective PPF, and the p.D249N missense mutation is responsible for autosomal-dominant macrothrombocytopenia in dogs (Schwer et al, 2001; Davis et al, 2008). Moreover, the p.Q43P TUBB1 variant in humans is associated with enlarged platelets carrying subtle morphological abnormalities (Freson et al, 2005). Finally, a genome-wide association study in up to 66 867 individuals of European ancestry identified the 187

Review TUBB1 locus as a major determinant of the mean platelet volume (Gieger et al, 2011). CYCS-related thrombocytopenia. Cytochrome c is an evolutionarily well-conserved small protein of the mitochondrial intermembrane space that participates in two key cellular functions: mitochondrial respiration and initiation of the intrinsic pathway of apoptosis. Two pedigrees with autosomal-dominant thrombocytopenia caused by heterozygous cytocrome c mutations, p.G41S or p.Y48H, have been described (Morison et al, 2008; De Rocco et al, 2014). These variations are the only naturally occurring mutations reported for this molecule so far. In vitro studies of the purified proteins showed that both mutants increase the proapoptotic activity of the cytochrome, whereas only the p.Y48H affects the respiratory chain (De Rocco et al, 2014). A mild thrombocytopenia was the only phenotype of patients with CYCS mutations, who were otherwise healthy and long-lived. These observations suggest that, at least in its heterozygous form, the increased proapoptotic activity of cytochrome c does not significantly affect the homeostasis of most tissues and organs during development or adult life, and that platelet biogenesis is particularly sensitive to enhanced activation of the intrinsic apoptotic pathway (Morison et al, 2008). Several studies, mainly based on in vitro culture systems, supported the notion that a localized activation of the intrinsic apoptotic pathway is required for triggering PPF in mature Mks (De Botton et al, 2002; Kaluzhny et al, 2002). Mks differentiated in vitro from two individuals with the cytocrome c p.G41S demonstrated increased propensity to release platelets in the culture medium. Accordingly, ultrastructural analysis of BM biopsy of another family member showed signs of ectopic release of platelets within the BM (Morison et al, 2008). Therefore, CYCS-related thrombocytopenia was explained with an ectopic, premature proplatelet release within the BM osteoblastic niche promoted by enhanced activation of the intrinsic apoptosis. However, more recently, a series of elegant experiments in mice knockout for key components of the apoptotic pathways argued against a role of apoptosis in promoting PPF, and instead suggested that Mks must restrain the intrinsic pathway to progress to PPF (Josefsson et al, 2011; White et al, 2012). These conflicting results may derive in part from the different experimental models and the different stimuli used to induce or suppress apoptosis (Josefsson et al, 2011). Further studies on patients with CYCS-related thrombocytopenia could be of great value to clarify the actual role of cytochrome c in human megakaryopoiesis.

Final considerations A scheme of megakaryopoiesis based on the findings obtained by the study of patients with ITs as well as by the analysis of experimental models reproducing their clinical 188

Fig 3. Schematic view of human megakaryopoiesis based on the findings obtained in patients with inherited thrombocytopenias and in experimental models that successfully reproduced their disorders. Haematopoietic stem cell (HSC) differentiation into megakaryocyte (Mk) is induced by the binding of thrombopoietin (TPO) to its MPL receptor and the consequent activation of multiple signaling pathways. A defect in MPL (congenital amegakaryocytic thrombocytopenia) or in the TPO/MPL-dependent stimulation of Mks (thrombocytopenia with absent radii) results in amegakaryocytic thrombocytopenias. Mk maturation requires the timely activation of transcription factors that regulate the expression of a number of proteins, often specific to Mks and platelets. A defect in the transcription factors RUNX1 (familial platelet disorder with predisposition to acute myeloid leukaemia), GATA-1 (GATA-1 related thrombocytopenias), FLI1 (Paris-Trousseau thrombocytopenia and Jacobsen syndrome) or GFI1B (GFI1B related thrombocytopenia) impairs Mk maturation by affecting this ordered sequence of events. Also mutations resulting in loss of function of NBEAL2 (Gray platelet syndrome) and gain of function of ANKRD26 (ANKRD26-related thrombocytopenia) affect Mk maturation. While Mks mature, they move from the stem cell niche to the vascular niche, where they extend proplatelets into vessel lumen, releasing platelets directly in the flowing blood. Mutations affecting cytoskeleton functions result in thrombocytopenia by affecting proplatelet formation (MYH9-related disease, ACTN1-related thrombocytopenia, FLNArelated thrombocytopenia, Bernard-Soulier syndrome, ITGA2B/ ITGB3-related thrombocytopenia, TUBB1-related thrombocytopenia) and/or inducing premature, ectopic release of platelets (MYH9related thrombocytopenia, Wiskott–Aldrich syndrome and X-linked thrombocytopenia, CYCS-related thrombocytopenia).

and laboratory picture is reported in Fig 3. As shown, ITs have provided an essential contribution to understanding the physiological mechanisms of platelet formation in humans. Of note, ITs are expected to provide further valuable information on this matter. In fact, the genes whose mutations are responsible for the forms identified so far are normal in almost half of the patients. So, it is easy to predict that mutations in several new genes are responsible for many additional, not yet identified, types of IT. We are convinced that the next generation sequencing techniques that are now available will identify these genes in the near future and will further advance the knowledge of the mechanisms that regulate platelet production in humans.

Competing interests The authors have no competing interests. ª 2014 John Wiley & Sons Ltd British Journal of Haematology, 2014, 165, 179–192

Review

Acknowledgements The authors would like to thank the Italian Telethon Fondazione Onlus (grant number GGP10089), and the

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