state of the art review

Progress in understanding the diagnosis and molecular genetics of macrothrombocytopenias Remi Favier1,2 and Hana Raslova1,3,4 1

Institut National de la Sant
e et de la Recherche M
edicale, U1170, Equipe Labellis
ee Ligue Contre le Cancer, Villejuif, 2Assistance Publique-H^opitaux de Paris, Armand Trousseau Children Hospital, French Reference Center for Platelet Disorders, Haematological Laboratory, Paris, 3Facult
e de M
edecine, University Paris Saclay and University Paris-Sud 11, Le Kremlin-Bic^etre, and 4Gustave Roussy, Villejuif, France

Summary The inherited macrothrombocytopenias constitute a subgroup of congenital platelet disorders that is the best characterized from the genetic point of view. This clinically heterogeneous subgroup is characterized by a variable degree of bleeding but without predisposition to haematological malignancies, as seen in the two other subgroups. The classification of inherited thrombocytopenia is traditionally based on the description of different clinical and biological features, in particular the measurement of the mean platelet volume. In certain disorders, biochemical platelet components are abnormal, and their analyses are useful in diagnosis. However, these approaches present several limitations, and many cases remain undiagnosed, especially for patients without a clear family history. An analysis of genetic abnormalities was subsequently used for classification, demonstrating that some different clinical entities were, in fact, identical. The genomic approach that was used initially to accurately link some phenotypic diagnoses with the causal genetic alteration was positional cloning and DNA sequencing. More recently, next generation sequencing in the form of whole-genome or -exome sequencing and RNA sequencing has been developed. This review will focus on the progress in understanding the different macrothrombocytopenias that have been identified. Keywords: Inherited macrothrombocytopenia, diagnostic algorithm, next-generation sequencing, animal models.

Correspondence: Hana Raslova, INSERM U1170, Gustave Roussy, 114 rue Edouard Vaillant, Villejuif 94805, France. E-mail: [email protected] and Remi Favier, Assistance Publique-H^ opitaux de Paris, Armand Trousseau Children’s Hospital, Haematological Laboratory, 26 Avenue du Docteur Netter, Paris, France. E-mail: [email protected]

First published online 5 May 2015 doi: 10.1111/bjh.13478

The mechanism of platelet production is more complex than was previously thought. Inherited macrothrombocytopenia may present a unique opportunity to decipher the final mechanism of platelet formation because the increase in platelet size implies a major defect in the process of megakaryocyte fragmentation. To maintain haemostasis, the human body produces 1011 platelets per day, which can increase following injury to blood vessel walls. A process called megakaryopoiesis, taking place in bone marrow, achieves platelet production. This process consists of the differentiation of haematopoietic stem progenitors to bipotent erythro-megakaryocytic progenitors, giving rise to erythroid and megakaryocytic progenitors. Megakaryocytic progenitors first divide by classical mitosis before they switch to endomitosis, a process permitting an increase in DNA content and giving rise to polylobulated megakaryocytes. Following endomitosis, cytoplasmic maturation proceeds with the biogenesis of platelet organelles, the synthesis of platelet proteins and the formation of the demarcation membrane system, which leads to proplatelet extensions. Megakaryopoiesis is achieved by the liberation of platelets into the blood circulation under flow shear. Different cytokines and growth factors, the most important being thrombopoietin, and different transcription factors, such as GATA1, FLI1 and RUNX1, regulate the entire process. One major feature of megakaryocyte (MK) differentiation is the reorganization of the microtubule and acto-myosin cytoskeleton that is necessary for the migration of megakaryocytes and for platelet adhesion and aggregation as well as for platelet generation (reviewed in Bluteau et al, 2009). Molecular defects at any of these steps can lead to aberrant platelet production, resulting in low (thrombocytopenia) or high (thrombocytosis) numbers in circulation, and to defects in platelet function, or thrombopathy, which can result in haemorrhage or, in contrast, acute myocardial infarction or ischaemic stroke. When ubiquitous genes, such as FLNA and MYH9, are genetically altered, defects in platelet production/ function can be associated with other systemic features. Decrease or increase in platelet numbers can be inherited or acquired, such as through infection, inflammation, stress ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

Review and treatment, more particularly for cancers or haematological disorders. Inherited thrombocytopenias (IT) with mild bleeding tendency are not easily distinguishable from immune thrombocytopenia, which is the most common form of acquired thrombocytopenia. Molecular diagnosis now strongly implements the classification of inherited thrombocytopenia based on the clinical profile. In recent years, many advances have been made in the field of inherited disorders, and more than 20 genes have been described as responsible for IT. Inherited thrombocytopenias can be transmitted in an X-linked, autosomal-dominant or autosomal-recessive manner. In the last case, compound heterozygous or homozygous mutations could be at the origin of the pathology. Homozygous mutations are usually identified when consanguinity is present in the family. Classically, IT are divided into three groups according to platelet size (Balduini & Savoia, 2012; Table I). Group 1 comprises IT with giant or large platelets, Group 2 includes IT with normal-sized platelets and Group 3 includes IT with small platelets. In clinical practice, it is easy to use a classification based on the type of inheritance and/or platelet size and/or clinical features. However, recent studies of genes that are altered in IT have allowed a better understanding of megakaryopoiesis and have permitted a proposed classification based on the defects occurring at different steps of MK differentiation and maturation (Pecci & Balduini, 2014). As an example, the study of macrothrombocytopenias that are characterized by mutations in genes that have been implicated in cytoskeleton reorganization and platelet signalling have been extremely informative in furthering the understanding of platelet generation, the terminal phase of megakaryopoiesis. In this review, we will focus on the diagnosis of inherited macrothrombocytopenias (IMT). The first step in the diagnosis of IMT among all IT is based on two main criteria: the measurement of the mean platelet volume (MPV) above normal values and the measurement of the platelet size. As indicated, the measurement of the MPV may differ from one haematological analyser to another (Latger-Cannard et al, 2012). When the analyser cannot measure MPV (Chaquin et al, 2014), abnormal platelet morphology may be observed by standard microscopy on May-Gr€ unwald–Giemsa stained peripheral blood smears. The presence of large (20% platelets >4 lm) and giant platelets (2–16% platelets >8 lm) serves for diagnosis (Balduini et al, 2003; Noris et al, 2014). However, some cases of IMT are not diagnosed due to the presence of a low percentage of macrocytic platelets (5–10%), a feature that is also observed in acquired macrothrombocytopenias. Some macrothrombocytopenias are associated with variable bleeding tendency (Table I). A powerful diagnosis algorithm of inherited platelet function disorders was recently proposed by the Platelet Physiology Subcommittee of the International Society on Thrombosis and Haemostasis (Gresele, 2015). According to this algorithm, the second screening test would be based on platelet function studies, such as light transmission aggregometry, granule release and ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

an analysis of the platelet surface glycoprotein by flow cytometry. Although it is challenging to perform these studies when the platelet count is low, this step is particularly important for the diagnosis of IMT with high bleeding tendency, such as Bernard Soulier syndrome (BSS) and Gray Platelet syndrome (GPS) at the biallelic state. In addition to these criteria, genetic alterations in genes that are associated with the corresponding IMT should be investigated in a third step. However, for approximately 40% of IMT (unpublished data from a French cohort), the genetic alterations remain unknown. Genome linkage assays and, more recently, next-generation sequencing (NGS) are proposed to identify the causative genetic alteration.

Classical inherited macrothrombocytopenias that are identified based on clinical, biological and cytogenetic futures Bernard-Soulier syndrome [BSS, Online Mendelian Inheritance in Man (OMIM) number 231200] In 1948, Jean Bernard and Jean Pierre Soulier identified a platelet disorder that was characterized by a bleeding tendency, the presence of giant platelets and thrombocytopenia (Bernard & Soulier, 1948). The disorder was accompanied by an inability of platelets to interact with bovine factor VIII or with the antibiotic ristocetin and by a decreased level in surface sialic acid (Bernard & Soulier, 1948). Weiss et al (1974) showed that BSS platelets have a defective flow-dependent attachment to rabbit aortic subendothelium. Following pioneering studies that were performed in Glanzmann thrombasthenia (GT) (Nurden & Caen, 1974), it appeared that the BSS platelets lacked glycoprotein (GP)Ib (also termed GP1BA), while GPIIb (a IIb, ITGA2B) and GPIIIa (b3, ITGB3) were absent in GT but were normally present in BSS (Nurden et al, 1981). Studies combining lactoperoxidase labelling by crossed immunoelectrophoresis (CIE) with oneand two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis procedures definitively established that GPIb was a main component of the platelet surface and a component of a glycoprotein complex that is indispensable for haemostasis, named GPIb-IX-V, a receptor of the von Willebrand factor (VWF). For a long time, BSS diagnosis was confirmed by the decreased expression of GPIb on the platelet surface and by the lack of platelet agglutination when exposed to ristocetin, while aggregation was normal after exposure to other platelet agonists. More recently, flow cytometry assay with anti-GPIba, GPIbb or GPIX monoclonal antibodies was developed. In most cases, the transmission of BSS is autosomal recessive (biallelic BSS) and accompanied by severe bleeding; however, transmission may be autosomal dominant (monoallelic BSS) in a few cases and is characterized by mild bleeding. The first mutation was detected in one family with autosomal-dominant transmission. This mutation was a nonsense 627

Review Table I. Inherited thrombocytopenias classified according to platelet size. Inherited thrombocytopenias (IT) Group 1 IT with large and giant platelets

Disease name (abbreviation)

Bernard- Soulier syndrome (BSS) Biallelic Monoallelic Di Georges/velocardiofacial (VC) syndrome Platelet type-von Willebrand disease (PT-VWD) MYH9-related disease (MYH9-RD) ITGA2B/ITGB3-related thrombocytopenia (ITGA2B/ITGB3-RT) FLNA-related disease (FLNA-RD) Thombocytopenia associated with sitosterolemia (STSL) GATA1-related disease (GATA1-RD) Paris-Trousseau thrombocytopenia (TCPT) TUBB1-related thrombocytopenia (TUBB1-RT) Gray Platelet Syndrome (GPS) Biallelic Monoallelic a–actinin-related disease (ACTN1-RD) PRKACG-related disease (PRKACG-RD)

Group 2 IT with normal-sized platelets

Group 3 IT with small platelets

Congenital amegakaryocytic thrombocytopenia (CAMT) Thrombocytopenia with absent radii (TAR) Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) ANKRD26-related thrombocytopenia (ANRKD26-RT or THC2) ETV6-related thrombocytopenia (ETV6-RT) CYCS-related thrombocytopenia (THC4) Amegakaryocytic thrombocytopenia with radio-ulnar synostosis (CTRUS) Wiskott-Aldrich syndrome (WAS) X-linked thrombocytopenia (XLT)

W343X (W359X if starting from ATG) mutation in the gene coding for GPIba, leading to a truncated protein (Ware et al, 1990). Using intragenic restriction fragment polymorphism (RFLP) analysis in another family with autosomal-recessive transmission, Finch et al (1990) demonstrated that GP1BA is not the only gene causing BSS and that mutation(s) in other gene(s) could be at the origin of BSS. This assumption was confirmed by the identification of two different missense mutations in the GP9 gene (also termed GPIX), D21G and N45S, in one family with autosomal recessive transmission (Wright et al, 1993). The first mutations in the GP1BB gene were identified in 1997 as compound heterozygous mutations Y88C and A108P (Kunishima et al, 1997). Currently, more than 50 missense, nonsense or frame-shift mutations have been reported in GP1BA, GP1BB or GP9, mainly in biallelic BSS. Nevertheless, A156V (Bolzano mutation), N57H, N41H, Y70D and L73F GP1BA mutations have been reported in 628

Gene

Bleeding tendency

GP1BA, GP1BB, GP9 GP1BA, GP1BB GP1BA MYH9 ITGA2B, ITGB3

Moderate to Severe Mild Moderate to Severe Mild to Moderate Mild Mild to Moderate

FLNA ABCG5, ABCG8 GATA1 11q23-ter deletion including FLI1 TUBB1

Moderate to Severe Mild Moderate to Severe Moderate Mild

NBEAL2 GFI1B ACTN1 PRKACG

Moderate to Severe Moderate to Severe Mild Severe

MPL RBM8A RUNX1

Severe Moderate to Severe Moderate

50 UTR of ANKRD26

Mild

ETV6 CYCS HOXA11

unknown Mild Mild

WAS WAS

Moderate to Severe Mild

monoallelic BSS and induce a less severe macrothrombocytopenia. No mutation has been identified in the GP5 gene, encoding GPV (Savoia et al, 2011; Balduini & Savoia, 2012; Andrews & Berndt, 2013). Two rare BSS variants have also been described. In the first variant, a mutation in the 50 UTR region of GP1BB impairs GATA1 binding (Ludlow et al, 1996) and leads to a decrease in the GP1BB transcript level. In the second variant, a biallelic N110E mutation in GP1BA modifies the proper maturation of the transcript (Vettore et al, 2011). Although the precise mechanisms of the physiopathology of BSS remain to be understood, reduced platelet number, giant platelet production and increased bleeding could be related to a defect in proplatelet formation of patient MK (Balduini et al, 2009), a defect in interaction with the cytoskeleton and a reduced affinity of VWF for the GPIb/GPIX/ GPV complex. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

Review

Di George (OMIM188400) or velocardiofacial (VCF, OMIM192430) syndromes A 15- to 30-Mb hemizygous deletion on chromosome 22q112 is responsible for both Di George and VCF syndromes, also known as 22q112 deletion syndromes. The deletion includes the GP1BB gene, and the observed phenotypic diversity, including velopharyngeal dysfunction, cranio facial defects, congenital conotruncal heart malformation, hypotonia and immune deficiency, reflects the size of the deletion. The platelet count is not always decreased (Rosa et al, 2011), as in the case with monoallelic GP1BB mutations. However, if a second mutation occurs on the wild type GP1BB allele, a BSS will be induced.

Platelet type-von Willebrand disease (PT-VWD) (OMIM177820) First described by Weiss et al (1982), PT-VWD is an autosomal-dominant bleeding disorder that is very similar to type 2B VWD. However, while type 2B VWD results from a functionally abnormal VW molecule, PT-VWD is due to a mutation in the GP1BA gene, encoding the platelet glycoprotein GPIba. The first mutation causing PT-VWD disease (G233V) was described by Miller et al (1991). Other mutations in the GP1BA gene have been described so far, M239V (Russell & Roth, 1993), G233S (Matsubara et al, 2003), a 27-bp in-frame deletion (Othman et al, 2005), D235Y (Enayat et al, 2012) and W246L (Woods et al, 2014). All of these mutations induce a gain of function resulting in an enhanced affinity for VWF and enhanced platelet aggregation to a low dose of ristocetin (05 mg/ml), which normally does not induce platelet agglutination.

MYH9-related disease (MYH9-RD, OMIM155100) Previously described as a May (in 1909) or Hegglin (in 1945) anomaly, Epstein (in 1972) and Fetchner (in 1985) syndromes or Sebastian platelet syndrome (in 1990), these distinct clinical entities reflect the different clinical signs that are associated with macrothrombocytopenia and mild bleeding tendency. May–Hegglin anomaly and Sebastian platelet syndrome share ultrastructural leucocyte inclusions called D€ ohle-like Bodies of different sizes, whereas Epstein and Fechner syndromes are distinguished by hearing loss, nephritis and/or cataracts. Recently, the chronic alteration of hepatic enzymes has been detected in some patients (Favier et al, 2013). Before the identification of the mutated causative gene, a diagnosis was made regarding the detection of protein aggregates (D€ ohle-like bodies) in the cytoplasm of neutrophils by the microscopy of blood smears and, later on, by immunofluorescence analysis using an anti-MYH9 antibody. The absence of aggregates excludes MYH9-RD (Savoia et al, 2010). In 1999, the disease locus was mapped to chromosome 22q12.3-q13.2 by linkage analysis (Kunishima et al, 1999). ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

One year later, the May Hegglin/Fetchner syndrome consortium identified mutations in the MYH9 gene (Seri et al, 2000). Mutations in MYH9 were then found in the separate entities that are now designated as MYH9-related disease (MYH9-RD). Today, more than 40 mutations have been described, and although MYH9-RD is a congenital macrothrombocytopenia that is transmitted in an autosomal-dominant manner, MYH9 mutations are sporadic in 35% of the cases (Balduini & Savoia, 2012). MYH9 or non-muscle myosin IIA (NMMHC-IIA) is a heavy chain subunit of non-muscle myosin II. MYH9 contains two main domains, the N-terminal and tail domains. The Nterminal domain is composed of the head (motor) domain, interacting with actin and binding ATP, and the neck domain, binding lights chains. The tail domain is necessary for the dimerization of heavy chains and for phosphorylation. MYH9 belongs to a superfamily of motor cytoskeleton proteins that play an important role in cellular processes that require mechanical force for the reorganization of the actin cytoskeleton, such as the migration (adhesion and polarity), cytokinesis and maintenance of cell shape and is the only non-muscle myosin II that is present in platelets (Pecci et al, 2014). Studies were undertaken to correlate the clinical phenotype and the genotype of the different MYH9-RD patients. Pecci et al (2008) provided evidence that mutations in the motor domain of the MYH9 protein were associated with nephritis and hearing loss development before the age of 40 years. In contrast, mutations in the tail domain of the protein were associated with moderate thrombocytopenia and the absence of renal disease or cataracts (Pecci et al, 2008). A recent study including 255 patients from 121 families was performed to define disease evolution as associated with seven different MYH9 genotypes. The D1424H substitution is associated with a high risk of developing delayed manifestations of the disease, while E1841K and D1424N mutations and C-terminal deletions are associated with a low risk of delayed manifestations (Pecci et al, 2014). The mechanisms underlying the MYH9-related thrombocytopenia were rather linked to a defect in proplatelet formation than bone marrow dysmegakaryopoiesis. Indeed, a reduced number of proplatelet-forming MK with decreased branching and increased tip size was detected in cultures from patient progenitors (Pecci et al, 2009; Chen et al, 2013). A defect in the migration of MK and the abnormal release of platelets has also been suggested (Pecci et al, 2009). Interestingly, a recent study of MYH9 tail mutants (D1424N and E1841K) showed that MYH9 activity is critical to pre/proplatelet fission to platelets. Indeed, normal MK suppresses myosin IIa activity by enhancing phosphorylation on S1943 to increase ploidy and to ensure fragmentation. Myosin IIA re-activation occurs under shear and is necessary for platelet fission, while the D1424N and E1841K MYH9 mutants are not sensitive to reactivation. This result suggests that the macrothrombocytopenia in patients with tail mutants arises from a defect in fission of pre/ proplatelets to small platelets (Spinler et al, 2015). 629

Review

ITGA2B/ITGB3-related thrombocytopenia (ITGA2B/ ITGB3-RT, OMIM18780) Biallelic mutations in the ITGA2B or ITGB3 genes encoding GPIIb and GPIIIa, respectively, which lead to a loss of function are the causal origin of Glanzmann thrombasthenia (GT), a thrombopathy without thrombocytopenia. Recently, the new monoallelic mutations R995Q (Peyruchaud et al, 1998) and R995W (Kunishima et al, 2011) in ITGA2B and D723H (Schaffner-Reckinger et al, 2009) (Ghevaert et al, 2008) and L718P (Jayo et al, 2010) in ITGB3, as well as a 120-bp deletion leading to the loss of amino acids 647-686 in GPIIIa (Gresele et al, 2009), have been described. These mutations induce a gain of platelet function and thrombocytopenia. These mutations, which are located either in the tail cytoplasmic domain or the membrane-proximal region of the cytoplasmic domain of GPIIb or GPIIIa proteins, disrupt a potential salt bridge between the membrane-proximal portions of the alpha (GPIIb) and beta (GPIIIa) cytoplasmic domains. This disruption increases the activation state of GPIIb/IIIa and affects platelet formation. Diagnosis is suspected by a decrease in the GPIIb/IIIa complex on the platelet surface associated with a platelet defect that is characterized by a spontaneous and abnormal fixation of fibrinogen and PAC1 antibody. The pathophysiology may be related to a defect in proplatelet formation. A decreased Rho A activity, as mediated by integrin activation, was suggested to induce premature proplatelet formation (Schaffner-Reckinger et al, 2009). More recently, a negative regulation of proplatelet formation as a result of the constitutive activation of the complex-mediated outside-in-signalling was reported (Bury et al, 2012).

FLNA-related disease (FLNA-RD) Mutations in the FLNA gene, located on chromosome Xq28 and encoding filamin A (FLNA), are at the origin of a wide spectrum of rare diseases, including familial or sporadic cases. The two main phenotypes that are linked to FLNA mutations are periventricular nodular heterotopia (PNH) and the otopalatodigital syndrome spectrum disorders, including skeletal dysplasia, mental retardation, and congenital malformations. Cardiac valvular dystrophy, congenital intestinal pseudo-obstruction and terminal osseous dysplasia could also be associated with FLNA-RD (Parrini et al, 2006). The importance of filamin A in megakaryopoiesis was underlined in mouse models (Falet et al, 2010); (Jurak Begonja et al, 2011). The knockout of filamin A specifically in MK led to severe macrothrombocytopenia due to the premature release of large and fragile platelets into the circulation with accelerated clearance (Jurak Begonja et al, 2011). Subsequently, Nurden et al (2011) described macrothrombocytopenia in three distinct female patients, only two of them displaying classical signs of FLNA-RD, with

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monoallelic Y1525X and E1803K mutations and the deletion of exons 31–32 to 48. Heterogeneity in platelet size with normal, large and giant platelets and the heterogeneous distribution of a-granules within platelets with the occasional presence of enlarged forms were observed. In vitro megakaryopoiesis was normal, but proplatelet formation was decreased. Interestingly, macroplatelets were devoid of filamin A, suggesting that they were generated from MKs with an inactivated X allele harbouring wild type FLNA. A more recent study performed on patient platelets clearly showed that filamin A is essential for their adhesive functions (Berrou et al, 2013). Filamin A binds to actin and interacts with integrins to form the branching network of filaments that make up the cytoskeleton. How FLNA mutations cause macrothrombocytopenia remains to be elucidated. However, one should keep in mind that GPIbafilamin A interaction is important for the regulation of platelet size (Kanaji et al, 2012) and that the Syk kinase-filamin A interaction plays an important role in the regulation of a granules (Falet et al, 2010).

Sitosterolaemia (OMIM210250) Sitosterolaemia is an autosomal-recessive sterol storage disorder in which plant sterol accumulates in the blood and tissues (Bhattacharyya & Connor, 1974). The major clinical features are xanthomas, premature atherosclerosis and arthritis. Mutations in the ABCG5 and ABCG8 genes, located head-to-head on chromosome band 2p21, result in defective transporter proteins called Sterolin 1 (ABCG5) and 2 (ABCG8) (Patel et al, 1998; Berge et al, 2000). Rees et al (2005) described a macrothrombocytopenia with giant platelets, stomatocytic heamolysis and splenomegaly in pedigrees harbouring ABCG5 E77X and E146X mutations and ABCG8 Q271X and W361X mutations,. In some rare cases, macrothrombocytopenia and haemolysis might be the only signs of the disease. Two mouse models (with an Abcg5 deletion or point mutation) showed a decreased platelet count with an increased platelet size associated with a normal or increased MK number. The disturbed platelet formation is probably caused by plant sterol-induced changes in MK development with a poorly developed demarcation membrane system (Kruit et al, 2008; Chase et al, 2010). Thrombocytopenia is rather associated with a microenvironment abnormality than with dysmegakaryopoiesis. Kanaji et al (2013) demonstrated that the accumulation of plant sterols in platelet membranes induces their hyperactivation, as characterized by the constitutive binding of fibrinogen to GPIIb/GPIIIa, the internalization of the GPIIb/GPIIIa complex, the generation of platelet-derived microparticles, the quantitative changes in and subcellular localization of filamin leading to impaired platelet adhesion to VWF, and the inability to form stable thrombi.

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

Review

Macrothrombocytopenias identified by analogy with different mouse knock-out models GATA1-related disease (GATA1-RD, OMIM300367, 314050) Targeted mutagenesis in embryonic stem cells and mouse models has revealed the role of the X-linked GATA1 transcription factor in erythroid and megakaryocyte lineages. In analogy to these models, the first pedigree with suspicion of GATA1 alteration was reported by Nichols et al (2000). In this pedigree, two male half-siblings were anaemic and presented signs of severe thrombocytopenia since birth. In addition to dyserythropoiesis, numerous small and dysplastic MKs were present in the bone marrow as well as dysplastic changes in platelets. Consistent with an X-linked disorder and the characteristic pattern of a blood defect, GATA1 was sequenced, allowing the identification in both affected boys of the G613A mutation converting valine to methionine at amino acid 205 (V205M) (Nichols et al, 2000). This substitution abrogates the interaction of GATA1 with its partner FOG1 (Friend of GATA, also termed ZFPM1), with the complex acting mainly as a gene transcription repressor. Several other mutations in GATA1 were subsequently found either located in the N-terminal zinc finger domain (G208R, G208S D218G and D218Y), leading to a loss of interaction with FOG1 or not (R216Q, R216W), as well as one mutation in the C-terminal domain (X414R) generating a longer protein. These mutations induce macrothrombocytopenia (Singleton et al, 2013; Pecci & Balduini, 2014). Platelet defects, such as reduced adhesion and aggregate formation on collagen and weak ristocetin-induced agglutination, are observed in patients with R216Q and D218G mutations, respectively (Freson et al, 2001; Hughan et al, 2005).

Paris-Trousseau thrombocytopenia (TCPT, OMIM 188025) or Jacobsen syndrome (JBS, OMIM600588) The mutated gene FLI1 causing macrothrombocytopenia linked to JBS or TCPT was also found by analogy to the mouse model. FLI1 belongs to the ETS family of transcription factors and is one of the main regulators of megakaryopoiesis. JBS (Jacobsen et al, 1973) and its variant, TCPT (Favier et al, 1993), are two rare congenital disorders with overlapping clinical features that include growth and mental retardation, cardiac defects, dysmorphogenesis of the digits and face, pancytopenia and macrothrombocytopenia. A proportion of the platelets are large in size. Some of these platelets contain giant a-granules as revealed by May-Gr€ unwald– Giemsa staining or electron microscopy and/or are deficient in dense granules (Krishnamurti et al, 2001). Hart et al (2000) reported the phenotype of genetically modified mice in which a targeted null mutation in the Fli1 locus was introduced. These mice presented intracranial haemorrhages and ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

dysmegakaryopoiesis similar to features that were observed in JBS patients with an 11q23.3 deletion that includes a FLI1 locus (Hart et al, 2000). These observations suggest that megakaryocytic defects in these patients could be a result of hemizygous loss of FLI1. The implication of FLI1 was confirmed by in vitro studies demonstrating that the overexpression of FLI1 in haematopoietic progenitors from TCPT patients could correct the phenotype (Raslova et al, 2004). In 2012, a new rapid diagnostic test allowing FLI1 genetic alterations was proposed (Antony-Debre et al, 2012). Indeed, this test consists of the detection of MYH10 (non-muscle myosin IIB) in patient platelets. MYH10 is downregulated by the RUNX1/FLI1 transcriptional complex during megakaryopoiesis, leading to the complete absence of MYH10 in normal platelets (Lordier et al, 2012). The presence of MYH10 in platelets could therefore be indicative of FLI1 or RUNX1 alterations (Antony-Debre et al, 2012). The usefulness of this test in the initial screening of inherited platelet disorders was also underscored in the work of Stockley et al (2013), where NGS of candidate genes was performed. Two new FLI1 alterations predicting R337W and Y343C substitutions and a 4bp frameshift deletion (N331Tfs*-4) were identified in 6 non-syndromic patients of three pedigrees by NGS, and the presence of MYH10 in the platelets was confirmed (Stockley et al, 2013).

TUBB1-related thrombocytopenia (TUBB1-RT, OMIM613112) Kunishima et al (2009) reported a new congenital autosomal dominant blood disorder that is characterized by large/giant platelets and thrombocytopenia. The platelet function and the MK number and morphology in the bone marrow were normal. Similar to a mouse model in which Tubb1 knockout led to a defect in proplatelet generation with platelets lacking the characteristic discoid shape (Schwer et al, 2001), TUBB1 was sequenced in two affected members of the family, allowing the identification of the heterozygous R318W mutation (Kunishima et al, 2009). Interestingly, in the mouse model, Tubb1 knockout does not lead to the generation of large or giant platelets, while Cavalier King Charles Spaniels dogs have a high prevalence of inherited macrothrombocytopenia caused by a mutation in the canine TUBB1 gene (Davis et al, 2008). In patients, the expression level of b1-tubulin in platelets was half decreased, and MK cultured from CD34+ progenitors displayed impaired cytoplasm fragmentation and the release of large and giant platelets. The plasma concentration of glycocalicin, a proteolytic fragment of GPIba, was reduced, suggesting that the thrombocytopenia is partly due to peripheral platelet destruction (Kunishima et al, 2009). A second mutation (F260S) in TUBB1 was recently identified in patients, with platelets displaying an abnormal b1-tubulin distribution. This mutation abolishes the b-tubulin/a-tubulin 631

Review association in proplatelets and disrupts microtubule assembly, leading to impaired proplatelet formation and the production of fewer but larger proplatelet tips (Kunishima et al, 2014).

New genes detected by genome wild linkage analysis or NGS Gray Platelet Syndrome (GPS, OMIM139090) Gray Platelet Syndrome is an inherited mild-to-moderate bleeding disorder that is characterized by macrothrombocytopenia and a deficiency of a-granules in platelets. GPS patients present abnormal macroplatelets with a typical grey appearance that is caused by the absence of a-granules (Raccuglia, 1971; Gerrard et al, 1980). Secretion-dependent platelet aggregation induced by ADP and collagen is decreased, and bone marrow fibrosis with leucocytes engulfed in megakaryocytes (emperipolesis) is often associated. The transmission of GPS is generally autosomal recessive, but two families have been described with an autosomal-dominant inheritance pattern (Stevenson et al, 2013); (Monteferrario et al, 2014). Gunay-Aygun et al (2010) performed a genome-wide linkage analysis and homozygosity mapping of 25 GPS patients from 14 pedigrees, and mapped the cause of the disease within a 94-megabase interval on chromosome 3p21.1-22.1. The Sanger sequencing of this region (GunayAygun et al, 2011) or whole-exome sequencing (Albers et al, 2011) performed in patients with an autosomal-recessive transmission pattern identified biallelic missense, nonsense, frameshift and consensus splice-site mutations in the NBEAL2 gene. In parallel, the next-generation RNA sequence analysis of platelets from one GPS individual within the targeted region of 27 megabases inside the originally described 94-megabase region identified an abnormal distribution of reads mapping to NBEAL2 (Kahr et al, 2011). NBEAL2 (neurobeachin-like 2) encodes a BEACH/ARM/WD40 domain protein that is involved in the biogenesis of a-granules by a yet unknown mechanism. Nbeal2 knockout mice exhibit splenomegaly, macrothrombocytopenia (Kahr et al, 2013), platelets that are deficient in a-granules and impaired platelet functions, including thrombo-inflammation (Kahr et al, 2013); (Deppermann et al, 2013), thus mimicking human pathology. Genetic linkage analysis combined with sequencing identified the mutation on chromosome 9 that is responsible for moderate thrombocytopenia, abnormal platelet function, decreased a-granule content and red cell anisopoikilocytosis in one family (Stevenson et al, 2013). This single nucleotide insertion c.880-881insC predicts the frameshift mutation H294fsX307 in the fifth zinc finger DNA-binding domain of transcription repressor GFI1B that is necessary for the development and differentiation of erythroid and megakaryocytic lineages (Stevenson et al, 2013). One year later, the 632

linkage analysis of 14 members from another family with autosomal dominant macrothrombocytopenia also revealed a candidate locus on chromosome 9q34 (Monteferrario et al, 2014). Within this locus, the nonsense mutation c.859C>T in the GFI1B gene was identified as generating a truncated Q287* protein. Overexpression in the mouse model of this truncated GFI1B protein acting as dominant negative led to dysmegakaryopoiesis (Monteferrario et al, 2014). However, platelets are not always ‘grey’ in monoallelic GFI1B-mutated cases, and platelets in other clinical entities may have a pale and grey appearance due to the reduced formation of a-granules, such as ARC (arthrogryposis, renal dysfunction, cholestasis) syndrome or GATA1related diseases. Therefore, genetic testing is required to distinguish these cases.

ACTN1-related disease (ACTN1-RD, OMIM615193) In recent years, exome sequencing has become an effective new tool for identifying genes in rare disorders when conventional approaches were unsuccessful. Whole-exome sequencing was used to identify the genetic alteration responsible for a mild form of macrothrombocytopenia that is transmitted with an autosomal-dominant pattern in Japanese pedigrees (Kunishima et al, 2013). Mutations were identified in the ACTN1 gene, the gene encoding a-actinin 1 (ACTN1). Six different variants, V105I, Q32K, R46Q, R738W, E225K and R752Q, were found in seven pedigrees representing 37% of the Japanese cohort (Kunishima et al, 2013). In parallel, one of these mutants, R46Q, was also identified by genome-wide linkage analysis in combination with targeted NGS in one French pedigree (Gueguen et al, 2013). More recently, ACTN1 mutations were screened by whole-exome or Sanger sequencing in a large Italian cohort, and six novel missense heterozygous mutations (D22N, R46W, G251R, T737N, G764S and E769K) were found in 11 pedigrees, representing 42% of the cohort (Bottega et al, 2014). a-Actinin 1 is a member of the actin-cross-linking protein superfamily that is involved in the organization of the cytoskeleton. a-Actinins form dimers with an actin-binding domain (ABD) at the N terminus and cross-link actin filaments in bundles. Because all of the reported ACTN1 mutations are localized within the functional ABD and C-terminal calmodulin-like domain of the protein and can act in a dominant negative manner, they cause the disorganization of actin filament assembly as demonstrated by the overexpression of the mutants (Kunishima et al, 2013); (Bottega et al, 2014). In the megakaryocytic lineage, mutant a-actinin 1 did not change the number of proplatelet-forming MKs but reduced the number of proplatelet tips displaying an increased size (Kunishima et al, 2013). Because a-actinins interact with several cytoskeleton proteins and receptors, such as b-integrins, proplatelet formation may also be affected by altered signalling. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

Review

PRKACG- and GNE-related macrothrombocytopenias Whole-exome sequencing led to the identification of a c.222C>G mutation in the PRKACG gene in a severe form of autosomal recessive macrothrombocytopenia that is associated with thrombopathy with a high bleeding tendency (Manchev et al, 2014). PRKACG encodes the c-catalytic subunit of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). PKA phosphorylates different platelet substrates, including filamin A and GPIbb. Genetic alterations in FLNA and GP1BB are responsible for the two above-mentioned macrothrombocytopenias, FLNA-RD and BSS, respectively. Homozygous PRKACG mutation c.222C>G, leading to a missense substitution of isoleucine for methionine at position 74 (I74M), is at the origin of a marked defect in proplatelet formation with an increased size of the tips. Indeed, the overexpression of wild-type PRKACG in patient haematopoietic progenitors could correct this defect. Interestingly, this mutation led to the degradation of filamin A in patient MK and platelets, while no defect in phosphorylation of GPIbb was found, suggesting that filamin A is a major target of PRKACG in normal megakaryopoiesis and that the c-subunit

of PRKACG is responsible for the phosphorylation of filamin A at Ser2152, protecting the protein from proteolysis. Patients carrying the PRKACG mutation also have a homozygous GNE mutation. The c.1675G>A mutation led to the substitution of glycine for arginine at position 559 (G559R) (Manchev et al, 2014). GNE encodes a bifunctional enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE/ MNK), which initiates and regulates the biosynthesis of Nacetylneuraminic acid, a precursor of sialic acids (Stasche et al, 1997). The compound heterozygous mutations in this gene generally result in myopathy or sialuria. Although neither myopathy nor sialuria have been reported in patients with a homozygous G559R mutation, two other studies reported the implication of Y217H and D515Qfs*2 (Zhen et al, 2014), and V603L and G739S (Izumi et al, 2014) compound heterozygous GNE mutations in severe macrothrombocytopenia in patients presenting myopathy. In the second study, whole-exome sequencing also revealed mutations in the CPEB2 and FLNB genes, but their implication in the pathology remains unknown. GNE mutations could lead to reduced GNE activity, failure to catalyse sialic acid biosynthesis and therefore

Fig 1. Diagnostic algorithm for syndromic inherited macrothrombocytopenias. AD, autosomal dominant, AR, autosomal recessive, XLT, X-linked transmission, MYH9-RD, MYH9-related disease, GPS, Gray platelet syndrome, TCPT, thrombocytopenia Paris Trousseau, FLNA-RD, FLNArelated disease, CGH, comparative genomic hydridization. *Only 25% of platelets are large. **Some rare cases of sitosterolaemia are associated only with thrombocytopenia without any other clinical signs. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

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Review decreased sialic acid content on the platelet surface, consequently accelerating its elimination by the liver. A defect in specific a 2,3-sialyation leading to platelet binding to the liver ASPG-receptor was described in a case of macrothrombocytopenia in association with variable neutropenia (Jones et al, 2011). The real implication of GNE mutations in macrothrombocytopenia associated with myopathy should be further investigated because a majority of patients with myopathy do not present thrombocytopenia (Mori-Yoshimura et al, 2014).

New candidate genes for unexplained macrothrombocytopenias in humans highlighted by animal models Although mouse models do not reflect always the pathophysiology of human disease, some might be helpful in the identification of gene alterations in still unexplained

macrothrombocytopenias in humans. Here, we review mouse models with genetic alterations in Wdr1, G6b, Ptpn6/Ptpn 11 (previously termed Shp1/Shp2), Rhoa, Rac1, Cdc42, Dnm2 and Sp1/3 genes, leading to macrothrombocytopenia. The first model was described by Kile et al (2007). Mice carrying a mutation in Wdr1, the cofilin partner, develop both macrothrombocytopenia and autoinflammatory disease. Thrombocytopenia is due to maturation defects in MK leading to a failure in platelet shedding, and autoinflammatory disease is characterized by a massive infiltration of neutrophils into inflammatory lesions (Kile et al, 2007). Mazharian et al (2012, 2013) generated knock-out mice in which the lack of the ITIM (immunoreceptor tyrosine kinase-based inhibition motif)-containing receptor G6b or the simultaneous lack of both SHP1 (PTPN6) and SHP2 (PTPN11) (tyrosine phosphatase proteins) caused a macrothrombocytopenia and susceptibility to bleeding resulting

Fig 2. Diagnostic algorithm for non-syndromic inherited macrothrombocytopenias. AD, autosomal dominant, AR, autosomal recessive, XLT, X-linked transmission, BSS, Bernard-Soulier syndrome, MYH9-RD, MYH9-related disease, GPS, Gray platelet syndrome, GATA1-RT, GATA1related thrombocytopenia, GFI1B-RT, GFI1B-related thrombocytopenia, FLNA-RD, FLNA-related disease, ITGA2B/B3-RT, ITGA2B/B3-related thrombocytopenia, PT-VWD, Platelet type von Willebrand disease, PRKACG-RD, PRKACG-related disease; TUBB1-RT-related thrombocytopenia; ACTN1-RT, ACTN1-related thrombocytopenia; CGH array, comparative genomic hybridization array. Grey arrow indicates that giant platelets could be found in macrothrombocytopenias with large platelets, this is the case particularly for TUBB1-RT. *The detection of MYH10 in platelets of patients with GATA1 was not reported, but as GATA1 is present in the same regulator transcriptional complex with RUNX1 and FLI1, and the presence of MYH10 in platelets of patients with GATA1 mutations could not be excluded.

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Review from reduced platelet production and function (Mazharian et al, 2012, 2013). SHP1/SHP2-deficient mice were severely macrothrombopenic and had a reduced platelet surface expression of GPVI, GPIba and GPIIb/GPIIIa. Another model, in which G6b was deleted, phenocopies several features of Shp1/2-deficient mice suggesting that Shp1 and Shp2 are downstream of the G6b receptor (Mazharian et al, 2013). The multiple roles of small GTPases, RhoA, Rac1 and CDC42, in megakaryopoiesis, platelet shedding and platelet activation are highlighted by genetic inactivation. A conditional knockout mouse model to ablate RhoA specifically in MKs was created. RhoA knockout led to macrothrombocytopenia, and RhoA-null MK were enlarged and displayed a higher mean ploidy level, but no defect in proplatelet formation was observed. To explain the thrombocytopenia, the authors suggested that RhoA null MK could release prematurely aberrant platelets that were rapidly cleared from circulation (Suzuki et al, 2013). Mice with double Rac1- and Cdc42-knockout specifically in MK were created. These mice developed thrombocytopenia with abnormal platelet morphology and platelet function. No apparent defect in MK maturation in bone marrow was observed. However, MK showed abnormal morphology and uncontrolled fragmentation. Proplatelet formation was abrogated in vitro, and severely defective tubulin organization was noted with no major defects in actin assembly or structure (Pleines et al, 2013). The specific knockout of dynamin 2 (DNM2), a large ubiquitous GTPase, also led to severe macrothrombocytopenia with moderately accelerated platelet clearance (Bender et al, 2014). DNM2dependent endocytosis plays a major role in the formation of the membrane demarcation system and thrombopoiesis (Bender et al, 2014). Finally, a recent study showed that the simultaneous loss of Sp1 and Sp3 (Specificity proteins (Sp)/ Kr€ uppel-like 1 and 3) transcription factors in MK resulted in severe macrothrombocytopenia. The MK numbers were normal in the bone marrow and spleen but displayed a striking inability to form proplatelets. The down regulation of several cytoskeleton-related proteins and kinases upon Sp1/Sp3 deletion, such as the downstream effector kinase Mylk, could explain the observed defects in megakaryopoiesis (Meinders et al, 2014). Together, these mouse models show that genetic alterations in transcription factors, signalling molecules or cytoskeleton proteins that have been implicated in cytoskeleton reorganization at terminal stages of megakaryocyte maturation, i.e., proplatelet formation, could lead to macrothrombocytopenia. These various genes might be candidates for the diagnosis of a new form of macrothrombocytopenia in humans.

Which approach for the future? We propose two different diagnostic algorithms (Figs 1, 2) that share some common analyses, including a careful clinical ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 170, 626–639

and biological evaluation and genetic screening. We must also keep in mind that some patients with a heterozygous mutation may have giant platelets in the peripheral blood without thrombocytopenia. In syndromic IMT, the phenotypic data allow the targeting of the sequencing of one or of several candidate genes. However, some syndromic and non-syndromic macrothrombocytopenias remain undiagnosed when sporadic and/ or when the candidate gene is not known. We suggest that all of the previously described candidate genes are first screened by targeted NGS. In the case of negative results, whole-exome/genome sequencing or RNA sequencing should be performed. The initial analysis could focus on all of the genes that are known to play a role in the regulation of platelet formation, i.e., their number and size. Indeed, the new powerful technologies that have been used during recent years provide evidence of new candidate genes regulating platelet biogenesis, such as TPM4 (Gieger et al, 2011), which was very recently found mutated in three pedigrees with macrothrombocytopenia (Pleines et al, 2014). Other genes remain to be identified, and no leads should be ruled out. However, it should be noted that the cost of these new technologies remains high, and that they have been developed by only a few reference centres. Therefore, all patients cannot be appropriately diagnosed, which often leads to a misdiagnosis of inherited thrombocytopenia. At this time, data obtained from phenotypic analyses and targeted NGS remain essential and determine the strategy to be employed by the reference centre. The current downward trend in the cost of NGS will, in the future, allow this methodology to be included in the first line of diagnosis.

Conclusions A new horizon is opening in the molecular approach for the diagnosis of IT, as shown by the effectiveness of NGS of pedigrees with unclassified macrothrombocytopenias. This approach could be helpful not only for the diagnosis of IMT, but also for the identification of new genes that could be involved in the regulation of normal platelet production. The combination of patient clinical features, identification of new genes and the study of their function in in vitro (megakaryopoiesis derived from patient blood progenitors or induced pluripotent stem cell models) or in vivo models will contribute in a major way to understanding the physiopathology of IMT and the development of new treatments.

Acknowledgements This work was supported by the Ligue contre le Cancer [equipe labellisee 2013 (to H. Raslova)], by the Centre de Reference des pathologies plaquettaires, by the European grant ERA-NET (to C. Balduini, 2013). We are grateful to Dr. W. Vainchenker and Dr. F. Wendling for the critical 635

Review reading of the manuscript and helpful suggestions. We thank all patients for their support and their participation.

Conflict of interests The authors have no competing interests.

Author contributions Both authors contributed equally to the writing of this paper.

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