Inherited Platelet Function Disorders: Algorithms for ...

Inherited Platelet Function Disorders: Algorithms for Phenotypic and Genetic Investigation Paolo Gresele, MD, PhD1

Loredana Bury, PhD1

Emanuela Falcinelli, PhD1

1 Division of Internal and Cardiovascular Medicine, Department of

Medicine, University of Perugia, Perugia, Italy Semin Thromb Hemost

Abstract

Keywords

► ► ► ►

bleeding diagnostic algorithm genetic testing inherited platelet function disorders ► laboratory investigation

Address for correspondence Paolo Gresele, MD, PhD, Division of Internal and Cardiovascular Medicine, Department of Medicine, University of Perugia, Via E. dal Pozzo, 06126 Perugia, Italy (e-mail: [email protected]).

Inherited platelet function disorders (IPFDs) manifest with mucocutaneous bleeding and are frequently difficult to diagnose due to their heterogeneity, the complexity of the platelet activation pathways and a lack of standardization of the platelet function laboratory assays and of their use for this purpose. A rational diagnostic approach to IPFDs should follow an algorithm where clinical examination and a stepwise laboratory evaluation play a crucial role. A streamlined panel of laboratory tests, with consecutive steps of increasing level of complexity, allows the phenotypic characterization of most IPFDs. A first-line diagnosis of a significant fraction of the IPFD may be made also at nonspecialized centers by using relatively simple tests, including platelet count, peripheral blood smear, light transmission aggregometry, measurement of platelet granule content and release, and the expression of glycoproteins by flow cytometry. Some of the most complex, second- and third-step tests may be performed only in highly specialized laboratories. Genotyping, including the widespread application of next-generation sequencing, has enabled discovery in the last few years of several novel genes associated with platelet disorders and this method may eventually become a firstline diagnostic approach; however, a preliminary clinical and laboratory phenotypic characterization nowadays still remains crucial for diagnosis of IPFDs.

Inherited platelet function disorders (IPFDs) are a heterogeneous group of relatively uncommon hemorrhagic diseases characterized by a lifelong mild to moderate bleeding diathesis. The complexity of the platelet activation pathways and the need to perform laboratory assays on fresh samples represent an intricacy in the investigation of platelet function disorders, and contribute to explain why IPFDs are probably underdiagnosed.1 Lack of agreement about their classification, poor standardization of the laboratory tests, and a large heterogeneity in the diagnostic approaches used represent serious limitations to the identification of IPFDs, especially of the mild cases, and consequently to the clear understanding of their

Issue Theme Platelet Function in Thrombosis and Hemostasis; Guest Editor: Anne-Mette Hvas, MD, PhD.

prevalence, to the possible characterization of new forms and ultimately to advancements in their treatment.1 A few molecularly well-characterized IPFDs, such as Bernard–Soulier syndrome and Glanzmann thrombasthenia, are now diagnosed rather easily based on a few simple tests, while other forms are still rather difficult to diagnose, since the molecular and genetic alterations are unknown and diagnosis requires a complex array of laboratory assays.1–4 Moreover, evolution in the laboratory assays and enhanced knowledge of the diagnostic sensitivity of the established assays should lead to an update of the diagnostic algorithms of IPFDs.5–12 Finally, the widespread introduction in the last few years of high throughput molecular biology techniques has resulted

Copyright © by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI http://dx.doi.org/ 10.1055/s-0035-1570078. ISSN 0094-6176.

Diagnosis of Inherited Platelet Function Disorders in the discovery of several novel genes causing IPFDs and this may revolutionize the diagnostic approach to these disorders. This review summarizes the clinical and laboratory steps for the diagnosis of IPFDs, with a focus on the current challenges and recommendations.

Diagnosis of Platelet Function Disorders A rational diagnostic approach to IPFDs should follow a stepwise algorithm. First of all, it should always be considered that acquired disorders of platelet function (due to drugs, liver or kidney disease, hematologic disorders, and other systemic diseases) are much more frequent than IPFDs.13 Moreover, some syndromic inherited platelet disorders include alteration of liver enzymes and progressive kidney dysfunction among their manifestations,14,15 and differential diagnosis requires clinical experience and a careful assessment of the patient’s personal and family history.5,16,17 The first, important clinical decision is which patients should undergo a thorough laboratory investigation for a suspected IPFD.1,3,5,16

Clinical Evaluation An assessment of the type, time trend, and severity of bleeding symptoms is the first, critical step in the evaluation of patients with bleeding disorders. An accurate interview exploring personal and family bleeding history, concomitant disorders, and medications should always precede laboratory studies. Spontaneous or provoked bleeding, single or multiple bleeding sites, including bruising at not exposed areas, frequency, duration of bleeding and tendency to recurrence, with estimation of volume of blood lost (e.g., for menstrual bleeding), disproportionate bleeding with mild injury, need of transfusion or recurrent iron deficiency anemia should be investigated, keeping in mind that bleeding triggers are influenced by age and sex. First- and second-degree family history of excessive bleeding should also be collected. Platelet function disorders manifest generally with bleeding occurring immediately after injury, primarily in the skin, mucous membranes, nose, and the gynecological tract but also in the gastrointestinal and urinary tracts. Bleeding, generally, does not involve joints and muscles. However, rare exceptions exist and should always be considered. In fact, platelet disorders can sometimes coexist with coagulation factor defects or von Willebrand disease (VWD) that can determine bleeding manifestations such as deep muscle hematomas or hemartrosis.3,18,19 Moreover, some specific IPFDs show unusual clinical features: for example, the Quebec platelet disorder is associated with delayed bleeding, typically starting 12 to 24 hours after surgery or trauma, that can be controlled with antifibrinolytics but not with platelet transfusions20; in the Scott syndrome, affected persons have mild postoperative or postpartum bleeding, trauma-related hematoma, and bleeding after tooth extraction but rarely spontaneous hemorrage.21 The use of a bleeding assessment tool is strongly encouraged to standardize the evaluation of bleeding severity,

Gresele et al.

improve diagnostic accuracy and sensitivity, and possibly predict the future risk of bleeding.22,23 However, the currently available bleeding assessment tools have not been validated for IPFDs and no information of their predictive value for clinical outcomes in this clinical setting is available.24,25 Information on drug history, including herbal remedies, antibiotics, nonsteroidal anti-inflammatory drugs, or on peculiar food intake (that are the commonest causes of platelet dysfunction), history of bleeding-unrelated symptoms, such as eczema or recurrent infection, and family clustering of myelodysplastic syndromes or acute myeloid leukemia, should be collected. The next step is a complete physical examination, with attention not only to the bleeding manifestations but also to the potential involvement of other systems or organs, such as hearing loss, heart, face, or bone dysmorphisms, ocular involvement, mental retardation, skin discoloration, and alteration of liver enzymes, to assess for possible syndromic forms (►Table 1). When the clinical evaluation is suggestive of an IPFD, the proband should then undergo preliminary laboratory investigations aimed at excluding other causes of heritable bleeding disorders.

Preliminary Laboratory Evaluation Patients suspected to have a platelet function disorder should undergo a first laboratory screening, including a full blood count, blood clotting screening (prothrombin time, partial thromboplastin time, fibrinogen), to detect defects of intrinsic or extrinsic clotting pathways, and von Willebrand factor (VWF) screening (VWF antigen, ristocetin cofactor activity and factor VIII coagulant activity) to rule out VWD. It should be borne in mind, however, that rare cases of combined IPFD and blood clotting disorders have been reported; therefore, in patients diagnosed with mild VWD but with disproportionate bleeding, laboratory investigations of platelet function are still advisable.3,19,26 If the above screening tests exclude other bleeding disorders, laboratory investigations for possible IPFDs should be undertaken. Reduced platelet count, especially if mild, does not preclude further testing given that several IPFDs are associated with thrombocytopenia (►Table 2).

Laboratory Investigation of Platelet Function A diagnostic algorithm based on a streamlined panel of laboratory tests following steps of increasing levels of complexity should facilitate the phenotypic characterization of IPFDs.1,5,12 Although some of the most complex platelet studies may be performed only in specialized laboratories, a first-step diagnosis of a significant fraction of the platelet function disorders may be made also at nonspecialized centers by using a few, relatively simple tests.5

First-Step Tests The first screening should include an evaluation of a blood smear, light transmission aggregometry (LTA), the measurement of platelet granules content and release, and the Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

Table 1 Syndromic inherited platelet function disorders Disorder

Structural defect

Platelet defect

Associates abnormalities

Arthrogryposis renal dysfunction and cholestasis syndrome

VPS33B; VIPAS39

Abnormal α-granule release

Arthrogryposis, renal dysfunction, cholestasis, cerebral malformations, dysmorphic features

Filaminopathy-related macrothrombocytopenia

Filamin A

Abnormal aggregation, secretion and adhesion

Skeletal dysplasia, mental retardation, cardiac valvular dystrophy, congenital intestinal pseudo-obstr uction, terminal osseous dysplasia

Gsα platelet defect

Increased Gs expression and function Gsα deficiency

Abnormal aggregationinhibition test Decreased cAMP formation on Gαs receptor activation

Short stature, mental disability, brachydactyly Pseudohypoparathyroidism Ib (PHPIb)

Hermansky–Pudlak syndrome, Chediak–Higashi syndrome

Proteins involved in vesicle formation and trafficking

Reduced aggregation and secretion

Skin ocular and hair hypopigmentation, nystagmus

Leukocyte adhesion deficiency III

Kindlin-3

Glanzmann-type defective aggregation

Leukocytosis, recurrent bacterial infections

Paris–Trousseau syndrome

Defective megakaryopoiesis

Abnormal aggregation and secretion (giant α-granules)

Psychomotor retardation, facial and cardiac abnormalities

Stormorken syndrome

Ca2þ release-activated Ca2þ (CRAC) channel

Procoagulant activity (exposure of membrane phosphatidylserine on resting platelets)

Miosis, muscle weakness, dyslexia, ichthyosis, asplenia

Velocardiofacial syndrome

GPIbβ

Abnormal adhesion

Conotruncal cardiac abnormalities, learning disabilities, velopharyngeal insufficiency, immunodeficiency, facial dysmorphisms, and thymic hypoplasia

Wiskott–Aldrich syndrome

WASP: Wiskott-Aldrich Syndrome protein

Abnormal granule release

Eczema, immunodeficiency

evaluation of the main surface glycoproteins (GPs) by flow cytometry. The PFA-100 (Siemens, Malvern, PA) and the skin bleeding time test are not recommended for the initial laboratory screening because of insufficient specificity and sensitivity and/or low reproducibility5; however, they (especially the skin bleeding time) may provide supportive evidence of an impaired primary hemostasis if performed by experienced operators in well-standardized conditions. The evaluation of a Wright- or May–Grünwald–Giemsastained peripheral blood smear allows to gain information on platelet size and structure (e.g., granularity) and on possible associated abnormalities of other blood cells which, if present, may indicate specific inherited platelet disorders1 (►Table 3). The analysis of a peripheral smear provides an estimate of the platelet count, which is important because many cell counters will underestimate platelet number in macro- or microthrombocytopenias.27 In fact, although alterations of platelet diameter are more typical of inherited thrombocytopenias, 28 several IPFDs also present abnormalities of platelet size, such as Bernard–Soulier Seminars in Thrombosis & Hemostasis

syndrome, Gray platelet syndrome, Glanzmann thrombasthenia variants, platelet-type VWD (►Table 3). The absence of α-granules results in large pale platelets characteristic of the Gray platelet syndrome and arthrogryposis, renal dysfunction, and cholestasis syndrome29; leukocyte inclusions suggest MYH9-related disorder30; abnormal red blood cell morphology may point toward mutations in the GATA1 gene. 31 The most widely used method for the study of platelet function in the clinical laboratory is LTA,1 and although problems of standardization exist,32 LTA remains the gold standard for platelet function testing.5,7 Agonists to be used for the initial LTA evaluation should include epinephrine, adenosine diphosphate (ADP), collagen, arachidonic acid, and ristocetin, using concentrations and methodologies indicated in the recently released ISTH guidelines.32 The pattern of impairment of platelet aggregation is characteristic of several specific IPFDs (►Fig. 1). Glanzmann thrombasthenia patients show absent aggregation to all agonists except high-dose ristocetin (which may be

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

Table 2 Inherited platelet function disorders associated with thrombocytopenia and/or alterations of platelet size Disorders

Platelet count reduction

Platelet size

Bernard–Soulier syndrome

Moderate to severe

Giant

Filaminopathy-related macrothrombocytopenia

Mild to moderate

Large

Familial platelet disorder associated with acute myeloid leukemia

Mild to moderate

Normal

GATA1-related disease

Severe

Large

Gray platelet syndrome

Mild

Large

Glanzmann thrombasthenia variant

Mild to moderate

Large

Medich platelet syndrome

Mild

Large

Paris–Trousseau syndrome

Moderate to severe

Normal or slightly increased

Platelet type von Willebrand disease

Mild

Normal or slightly increased

Stormorken syndrome

Mild to moderate

Normal

Velocardiofacial syndrome

Mild

Large

Wiskott–Aldrich

Severe

Small

White platelet syndrome

Mild

Large

Note: Small (< 2.6 μm); large (> 3.2 μm); giant (> 4 μm).27

Table 3 Morphological abnormalities seen on blood smear or TEM in patients with IPFD Disorder

Filaminopathy-related macrothrombocytopenia

Blood smear (platelet size/morphology/other cells’ abnormalities)

TEM (platelet granules/other structural alterations)

Large/normal/normal

Decreased/giant α granules

GATA1-related disease

Large/normal/dyserythropoiesis

Not reported

Gray platelet syndrome/arthrogryposis renal dysfunction and cholestasis syndrome

Large/“gray” or “pale” platelets/dyserythropoiesis (gray platelet syndrome), poorly granulated polymorphonuclear neutrophils

Decreased α granules

Hermansky–Pudlak syndrome/ Chediak–Higashi syndrome

Normal/normal/abnormal giant granules in eosinophils, basophils, and monocytes (Chediak–Higashi syndrome)

Decreased δ-granules

Medich platelet disorder

Large/normal/normal

Decreased α-granules/membranous tube-like scroll inclusions stretching across the cytoplasm

Paris–Trousseau syndrome

Large/normal/normal

Giant α-granules

Stormorken syndrome

Normal/normal/Howell–Jolly bodies in red blood cells

Not reported

White platelet syndrome

Large/normal/normal

Reduced or absent α-granules/presence of Golgi complexes, centrioles (usually in megakaryocytes), dense tubular system channels fused into tubular masses

York platelet syndrome

Large/normal/normal

Normal/giant electron-opaque organelles, giant target organelles with alternating layers of dark and light matrixes, coils/scrolls of smooth endoplasmic reticulum

α2β 1

Not reported

Decreased α-granules

αδ SPD

Normal/“gray” or “pale” platelets/ normal

Decreased α- and δ-granules

δ SPD

Not reported

Decreased δ-granules

Abbreviation: IPFD, inherited platelet function disorder; SPD, storage pool disease; TEM, transmission electron microscopy.

Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

reversible), while patients with Bernard–Soulier syndrome do not respond to ristocetin but show normal aggregation to all other agonists; GPVI or α2β1 deficiency gives no response to collagen but normal aggregation with other agonists; a P2Y12 defect shows a reduced and reversible response to ADP and impaired aggregation in response to collagen and arachidonic acid; patients with storage pool disease (SPD) or platelet secretion defects display a decreased secondary aggregation in response to epinephrine and ADP and decreased aggregation in response to collagen; increased response to a low dose of ristocetin is suggestive of platelet-type VWD, although it may alternatively identify type 2B VWD.5 Platelets from patients with abnormalities of thromboxane (Tx)A2 synthesis, in which cyclooxygenase (COX) or Tx synthase are defective, do not aggregate in response to arachidonic acid but respond to the synthetic Tx analogue U46619, while defects of TPα, the TxA2 receptor, will show absence of aggregation to arachidonic acid and to U46619.5 Inherited deficiency of cytosolic phospholipase A2 is associated with impaired aggregation in response to ADP and collagen but normal response to arachidonic acid.33

It should be noted that a significant proportion of healthy individuals may have abnormal aggregation in response to epinephrine,3 in part due to natural variations in α2 adrenoreceptor numbers34; thus, the role of impaired response to epinephrine as a cause of bleeding remains undetermined, although isolated impaired aggregation to epinephrine has been reported in the Quebec platelet disorder.35 LTA can be combined with the monitoring of adenosine triphosphate (ATP) secretion, either in real time with a lumiaggregometer or by the measurement by luminometry or high-performance liquid chromatography36 of the ATP present in the supernatant of a platelet-rich plasma (PRP) sample centrifuged at the end of LTA recording; defective ATP secretion is suggestive of platelet secretion defects.5,36 The distinction between a deficiency of dense granules (SPD) and impairment of granule release (the so-called primary secretion defect) requires measurement of the total adenine nucleotide platelet content. To this end, adenine nucleotides should be measured in platelet lysates, after the conversion of ADP to ATP with pyruvate kinase. There are two nucleotide pools in platelets: the metabolic and the storage pool, with pronounced differences in their ATP/ADP ratio. Storage pool

Fig. 1 Diagnoses made using a step-by-step approach. Phenotypic and genetic investigation algorithm for IPFDs. The current streamlined panel of laboratory tests may possibly be replaced in the future by the application of DNA chips or NGS combined with the analysis of IPFD genes. ARC, arthrogryposis renal dysfunction and cholestasis syndrome; BSS, Bernard-Soulier syndrome; CHS, Chediak-Higashi syndrome; COX-1, cyclooxygenase-1; cPLA 2, phospholipase A2 ; FPD/AML, familial platelet disorder with propensity to acute myeloid leukemia and myelodysplastic syndrome; GATA1, Macrothrombocytopenia with dyserythropoiesis/anemia/beta-thalassemia; GPS, Gray platelet syndrome; GT, Glanzmann thrombasthenia; HPS, Hermansky Pudlak syndrome; LADIII, leukocyte adhesion deficiency III; LTA, light transmission aggregometry; PSD, primary secretion defects; PTS, Paris-Trousseau syndrome; PT-VWD, platelet type von Willebrand disease; SPD, storage pool disease; TEM, transmission electron microscopy; TP, thromboxane receptor; QPD, Quebec platelet syndrome; VCF, Velo-Cardio-Facial syndrome; WAS, Wiskott-Aldrich syndrome.

Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders defects will show an increased ATP/ADP ratio, while normal ATP/ADP ratio suggests a release defect.10 The measurement of granule content and release should also include one marker of α-granules that may be assessed in platelet lysates and in the supernatant of a PRP sample at the end of LTA recording by enzyme-linked immunosorbent assay (ELISA) (e.g., platelet factor-4, fibrinogen), or by flow cytometry on the surface of activated platelets (e.g., P-selectin). Otherwise, granule contents can be assessed by immunofluorescence (Thrombospondin-1, VWF, fibrinogen, P-selectin) on a blood37 or PRP smear.38 Inherited defects of platelet granules (SPD) encompass a series of disorders characterized by deficiency in dense granules (δ-SPD), α-granules (α-SPD, Gray platelet syndrome), or both (αδ-SPD). Defective α-granular content is also described in Quebec platelet disorder, Wiskott–Aldrich syndrome, arthrogryposis renal dysfunction and cholestasis syndrome, Stormorken or GATA1-related disease.5 Flow cytometry for the measurement of glycoprotein receptor expression is crucial for the diagnosis of platelet adhesive protein defects, such as Glanzmann thrombasthenia and Bernard–Soulier syndrome, and should be performed with resting platelets using antibodies toward GPIIb/IIIa (CD41), GPIIIa (CD61), GPIb (CD42b), and GPIb/IX (CD42a). Decrease in platelet surface expression of GPIb has also been reported in the velocardiofacial syndrome.39 Flow cytometry on activated platelets with an antibody recognizing a GPIIb/IIIa-activation epitope (PAC-1) will confirm the diagnosis of Glanzmann thrombasthenia. Defective GPIIb/IIIa activation, however, has also been found in Stormorken syndrome and protein kinase C defects.5

Second-Step Tests A second set of laboratory tests should be performed in patients for whom the first-step tests have not yielded a diagnosis or have provided abnormal but not conclusive results. These should include LTA, expanding the range of agonists, flow cytometry, expanding the range of antibodies, clot retraction, the measurement of serum TxB2, and transmission electron microscopy (TEM). An extended panel of agonists for LTA should include α-thrombin, thrombin receptor-activating peptides (TRAP-6 and protease-activated receptor-4-activating peptide), collagen-related peptide, convulxin, U46619, platelet-activating factor, phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187. This approach allows to confirm platelet dysfunction and/or to identify impaired aggregation with the additional agonists orienting toward rarer IPFDs forms. For instance, an impaired aggregation with PMA, a direct chemical stimulator of protein kinase C, and platelet-activating factor is suggestive of a defect in a protein kinase C isozyme; defective platelet response to thrombin and TRAP-6 agonists is indicative of gray platelet syndrome; impaired response to collagen-related peptide and convulxin suggests defects of GPVI receptor.5 A test based on the inhibition of collagen-induced platelet aggregation by prostacyclin or prostaglandin E1, which acts on the Gsα-coupled receptor, reflects Gsα activity and allows

Gresele et al.

the identification of GNAS (Guanine Nucleotide binding protein alpha stimulating) defects.40 Expanded flow cytometry should include antibodies toward GPIa/IIa (CD31 and CD49b), GPIV (CD36), and GPVI and may identify alterations of these platelet surface GPs. There are several reports of patients with congenital GPVI deficiency whose platelets have reduced or not detectable GPVI on their surface, while only few cases of defective GPIa/ IIa (α2β1). Platelets lacking platelet GPIV, prevalently found in the Japanese population, have been reported to have reduced adhesion to collagen in flowing whole blood despite normal collagen-induced platelet aggregation, but these alterations do not seem to be associated with bleeding.4 Platelets should also be tested with high affinity probes for phosphatidylserine (such as annexin V, lactadherin), both in resting conditions and after activation with calcium ionophore or the combinations thrombin and collagen, to detect impaired (Scott syndrome) or enhanced (Stormorken syndrome) procoagulant activity.21,41,42 In case enhanced ristocetin-induced platelet aggregation was detected during the first-step laboratory investigations, mixing tests with patient, and control platelets and plasma should be performed, either by LTA or by flow cytometry, to differentiate a plasmatic (VWD type 2B) from a platelet (platelet-type VWD) defect.5,43 Impaired clot retraction, obtained by incubating nonanticoagulated whole blood for 60 minutes at 37°C in glass tubes, points toward Glanzmann thrombasthenia, Stormorken syndrome, or Wiskott–Aldrich syndrome.5 The measurement of TxB2, the stable TxA2 metabolite, by immunoassay (ELISA or radioimmunoassay) in serum (to this end, the supernatant serum deriving from the clot retraction test can be used) provides a global measure of the platelet arachidonic acid-metabolic pathway. TxB2 can be assessed also in the supernatant of a PRP sample following platelet activation with arachidonic acid at the end of LTA recording. Reduced TxB2 may orient toward defects of arachidonic acid release or metabolism (cPLA2, COX-1 or Tx synthase) but is also a clue for the possible intake of aspirin or nonsteroidal anti-inflammatory drugs. Finally, TEM is recommended at this stage for counting platelet α- or δ-granules and/or for the identification of platelet structural alterations (►Table 2). Given the electron density of δ-granules, the morphometric evaluation of electron micrographs of resting platelets allows a reliable count of these structures. A reduced number of dense granules have been reported as a constitutive trait not only of δ- and αδ-SPD but also of some syndromic forms (Hermansky–Pudlak syndrome, Chediak– Higashi syndrome, Wiskott–Aldrich syndrome, and thrombocytopenia with absent radii). Gray platelet syndrome platelets lack α-granules, normally visualized as round-to-oval structures with variable electron density, often with a nucleoid of greater density, that are replaced by empty vacuoles without granule contents. Also, platelets from patients with arthrogryposis renal dysfunction and cholestasis syndrome have abnormal α-granules.44 In contrast, the ultrastructure of α-granules of Quebec platelet disorder platelets, in which the Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

pathogenic event is the proteolysis of α-granule proteins, is normal.35 Abnormally, large α-granules are present in platelets of patients with the Paris–Trousseau and the Jacobsens syndromes.5 Moreover, patients with filaminopathy show heterogeneity in α-granules, with some platelets having few α-granules and others enlarged forms.45 Other structural abnormalities, such as membranous inclusions, platelet organelles abnormalities, endoplasmic reticulum-derived inclusion bodies or particulate cytoplasmic structures with selective immunoreactivity for polyubiquitinated proteins (PaCSs) and proteasome, have been described.46,47

Third-Step Tests Finally, the very special cases still evading diagnosis and for which a strong clinical suspicion of an IPFD persists may be studied with a series of additional laboratory tests. More specific functional studies, such as spreading assay, or adhesion and thrombus formation under flow conditions, may be conducted to further explore the platelet functional defect. For instance, impaired thrombus formation has been described in patients with Glanzmann thrombasthenia, Hermansky–Pudlak syndrome, and Gray platelet syndrome using a multisurface and multiparameter flow assay in whole blood48 and also in GPIV49 and GPVI50 deficiency; defective outside-in signaling in Glanzmann thrombasthenia, Glanzmann thrombasthenia variants or Wiskott– Aldrich Syndrome; increased spreading in some cases of filaminopathy-related macrothrombocytopenia. Moreover, a wide variety of tests available in specialized research laboratories may provide further diagnostic information, including the analysis of receptor expression, the analysis of intracellular proteins, and the generation of second messengers to assess platelet signal transduction defects. Receptor-binding studies allow measuring receptor copy number expressed on the platelet surface and their binding affinity. These studies may detect patients with defective or missing receptors on their platelets, such as defects of the P2Y12 receptor for ADP or of the TPα receptor for TxA25. Inherited platelet signal transduction defects are a poorly defined group of disorders and it has been suggested that they may represent a significant fraction of the patients with abnormal secondary wave of aggregation and decreased granule release but a normal α- and δ-granules content. These disorders include defects of platelet cytosolic phospholipase A2, COX-1, Tx synthase, phospholipase C, calcium mobilization, pleckstrin, or tyrosine phosphorylation. Abnormalities of the GTP-binding proteins that link surface G protein-coupled receptors to intracellular enzymes have also been described, such as Gαq, Gαi2, and Gαs49,51 (►Table 4). Identification of these defects requires detailed biochemical studies, not available to most laboratories, including western blotting for surface GPs or intracellular proteins (e.g., to determine the presence of MYH10, recently proposed as a biomarker for familial platelet disorder associated with Seminars in Thrombosis & Hemostasis

acute myeloid leukemia and Paris–Trousseau syndrome; expression of COX-1, cytosolic phospholipase A2, phospholipase C for the identification of deficiencies of these proteins, etc.), the study of protein phosphorylation using prelabeled P32-platelets or antibodies recognizing the phosphorylated forms of proteins, the measurement of second messengers (Caþ2, cAMP, IP3) using flow cytometry, ELISA, or specific radio-binding assays (►Table 4).

Genetic Testing Genotyping, mainly restricted to the analysis of candidate genes, has played in the past mainly a confirmatory role in the diagnosis of IPFD but nowadays it has become increasingly important and is being proposed as a first-line diagnostic investigation by some authors. In fact, the number of genes known to be involved in platelet function and causing an IPFD has steadily increased after the introduction of novel high throughput molecular biology techniques. However, excluding the forms for which a candidate gene is known, the identification of new genetic defects underlying an IPFD is challenging and made particularly difficult by the complexity of the mechanisms regulating platelet activation, with many proteins not yet known to be involved in platelet function. Moreover, platelet disorders are heterogeneous and sometimes are coinherited with other genetic defects, such as the association of VWD and Glanzmann thrombasthenia,18,52 or VWD and heterozygous mutations in platelet proteins (P2Y12, TP, and GPVI).19 Finally, complex genetic conditions such as compound heterozygosity, large chromosome deletions (del11q23-ter in Paris Trousseau syndrome), and compound inheritance of a null allele and one point mutation (the 1q21.1 deletion associated with a point mutation in RBM8A described in thrombocytopenia absent radii53), render the unraveling of an underlying genetic defect particularly complex. Candidate gene analysis and Sanger sequencing have been used in the field of platelet disorders starting from the early 1990s, when mutations in the genes coding for αIIbβ3 and GPIb/IX/V have been first described in Glanzmann thrombasthenia and Bernard–Soulier syndrome.54,55 Sanger sequencing, associated with genome-wide linkage analysis, has allowed to identify the DNA variations associated with P2RY12,56 MYH9,57 and PLAU1.58 However, many IPFDs, including well-characterized forms such as the Gray platelet syndrome remained without an associated mutant gene for years. The application of next-generation sequencing (NGS) to IPFDs has allowed to discover in a few years several novel genes associated with platelet disorders, such as NBEAL259–61 and GFI1B62,63 for gray platelet syndrome, ACTN164 for autosomal dominant thrombocytopenia, RBM8A53 for thrombocytopenia absent radii, STIM1 and ORAI1 for Stormorken and York syndromes, 65,66 very recently ETV667 for thrombocytopenia with predisposition to hematologic malignancy, FYB68 for small-platelet thrombocytopenia, and SLFN14 for thrombocytopenia and decreased ATP secretion. 69

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

Table 4 Inherited platelet function disorders requiring complex laboratory testing Disorders

Platelet abnormality

Third level diagnostic test

P2Y12 deficiency

P2Y12 receptor

Receptor-binding studies

Thromboxane A2 receptor defect

TP receptor

Receptor-binding studies

Rarer adhesive proteins receptor defects

α2β1, GPVI, or GPIV

Genetic testing and adhesion assay

Glanzmann thrombasthenia variant

GPIIb/IIIa

Spreading and genetic testing

Velocardiofacial syndrome

GPIbβ

Genetic testing

GATA1-related disease

Transcription factor involved in MK development

Genetic testing

Quebec platelet disorder

Urokinase-type activator

Genetic testing

Familial platelet disorder associated with acute myeloid leukemia

Transcription factor involved in megakaryopoiesis and platelet formation

Genetic testing and presence of MYH-10 by WB

Filaminopathy-related macrothrombocytopenia

Filamin A

Genetic testing

Paris–Trousseau syndrome

Primary protein abnormality unknown

Genetic testing and presence of MYH-10 by WB

Gαq deficiency

Decrease in platelet membrane Gαq

GTPase activity, presence of Gαq by WB

Gαs abnormalities

Increased platelet Gαs protein;

Presence of Gαs by WB, sensitivity to inhibition by agents that elevate cAMP; Absence of Gαs by WB, cAMP formation upon Gαs receptor activation

Gαs deficiency Gαi1 deficiency

Decrease in platelet membrane Gαi1

Absent inhibition of forskolin-stimulated cAMP increase by ADP, thrombin, or epinephrine

Phospholipase C-β2 deficiency

Decreased platelet PLC-β2 protein

Diminished formation of IP3 and DAG

Phospholipase A2 deficiency

PLA2 enzyme

Absence of enzyme at WB, release of arachidonic acid from phospholipids

COX-1 deficiency

COX-1 enzyme

Absence of enzyme at WB

Tx synthase deficiency

Tx synthase enzyme

Absence of enzyme at WB

Defects in protein phosphorylation

PKC-θ deficiency

Phosphorylation of pleckstrin and MLC

Defects in phosphatidylinositol metabolism

Primary protein abnormality unknown

Formation of IP3 by radio-binding assay

Defects in calcium mobilization

Primary protein abnormality unknown

Ca2þ mobilization

Abbreviations: cAMP, cyclic adenosine monophosphate; COX-1, cyclooxygenase-1; DAG, diacylglycerol; IP3, inositol trisphosphate; MLC, myosin light chain; MYH-10, myosin heavy chain 10; WB, western blotting.

Despite these advances, for many IPFDs, especially the mild forms, the genetic molecular defect is still unknown. This is the case for very rare forms of IPFDs, such as the Medich or White platelet syndromes, but also for patients with relatively frequent conditions such as the platelet secretion defects.1,5,12,17 It is expected that the widespread application of NGS will identify in the near future several novel genes associated with IPFDs. Indeed, despite recent progress, a definite diagnosis is still not attained in almost half of IPFD patients: a recent worldwide survey on the diagnosis of suspected platelet function disorders revealed that only 53.9% of patients with a bleeding disorder of confirmed platelet origin turned out to have a known IPFD.1 However, especially when a candidate gene is not known, the identification of the causative defect by NGS is cumbersome and requires the exclusion of many gene variations that are

unlikely to contribute to bleeding, a procedure for which the knowledge of the platelet phenotype can be of great help.70,71 Therefore, despite progress in genome/exome sequencing technologies, laboratory testing of platelet function is likely to remain an important step in the diagnosis of IPFDs for the foreseeable future. The remainder of this review will focus on the application of Sanger sequencing and NGS for the diagnosis of IPFDs and on the identification of patients that deserve genetic testing.

Sanger Sequencing of Candidate Genes The Sanger method has been the standard DNA sequencing technique until recently. Candidate genes are amplified by polymerase chain reaction as short DNA segments (200– 1,000 nucleotides), sequenced after purification and the Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders

Gresele et al.

resulting sequences are aligned with the human genome consensus sequence using dedicated software, such as the NCBI BLAST (Basic Local Alignment Search Tool).72 Sanger sequencing can still be the first-line approach for IPFDs with known causative mutant genes when the laboratory investigations orient toward a specific disorder and for which the identification of the exact mutation may have prognostic relevance. However, sometimes Sanger sequencing may not be the optimal approach even when the clinical picture and platelet function testing strongly orient toward a specific disorder, like in inherited diseases for which many molecular defects in different genes may be causative, such as the Hermansky– Pudlak syndrome.73 Finally, Sanger sequencing remains the method of choice for confirming mutant genotypes discovered by NGS.72

Next-Generation Sequencing-Based Approaches The term NGS refers to a set of methods including DNA sequencing (whole genome or exome sequencing), RNA sequencing and epigenome characterization. Millions to billions of short sequence reads are produced and aligned to a reference sequence to allow genetic variant discovery.72 NGS enables the simultaneous analysis of large groups of candidate genes, thus it may allow rapid identification of a known genetic mutation; moreover, it is the method of choice to identify novel underlying genetic variations. The cost of whole exome sequencing, the NGS method most frequently applied for the study of inherited bleeding disorders, has steadily decreased and is now affordable for most laboratories and not much higher than an extensive set of platelet function tests.70,71 The recent development of chips allowing the simultaneous sequencing of a predefined panel of genes implicated in IPFDs, such as the ThromboGenomics platform (https://thrombogenomics. org.uk), makes this approach a realistic first-line diagnostic option for the future.

NGS for IPFDs with Known Candidate Genes Each patient with a suspected IPFD for which one or more candidate genes are known can undergo genotyping using NGS and restricting the analysis of results to a subgroup of genes. This is faster compared with Sanger sequencing, and even less expensive when the investigated disorder has a high number of candidate genes. For instance, the Thrombo Genomics platform allows the exome sequencing of 75 selected genes involved in platelet and coagulation disorders, and a dedicated software permits the sequence analysis and the rapid identification of the gene variation(s) detected. This approach can solve the problem of uncertain diagnosis of many IPFDs, can shorten the time of genotyping, and it will soon become available for the routine diagnosis of patients with bleeding disorders, possibly replacing candidate gene sequencing and platelet function tests. Another example comes from the UK GAPP (Genotyping and Phenotyping of Platelets) study group that have successfully used NGS combined with the selective analysis Seminars in Thrombosis & Hemostasis

of the nine Hermansky–Pudlak syndrome candidate genes to obtain a rapid genetic diagnosis of Hermansky–Pudlak syndrome, an approach that has also allowed to identify a novel HPS4 mutation.74 The same problem applies to Bernard–Soulier syndrome (three candidate genes, approximately 110 different mutations described), Glanzmann thrombasthenia (two large candidate genes, 3,333 þ 3,995 bp coding sequence, approximately 200 different mutations described), and even MYH9- RD (one large candidate gene, 7,501 bp coding sequence, approximately 35 different mutations described). However, accessibility to NGS and to dedicated software still limits the widespread application of this approach.

NGS for IPFDs with Unknown Candidate Genes In patients with a strong clinical and laboratory suspicion of an IPFD but for which no mutation of the known candidate genes is detected, a different approach involving whole exome/genome sequencing must be applied. To identify the responsible mutant gene from the approximately 4,000 DNA variations that will be found in each subject, the variations unlikely to contribute to bleeding (e.g., common gene variations, intronic and synonymous variations) must be eliminated. Then, the remaining variations that are of potential relevance will be further screened based on the clinical history, result of platelet function tests, inheritance of the disorder, and segregation of the variation with the disease in different family members.71 This approach has been employed by several research groups in the last few years leading to identification of several novel gene mutations causing platelet disorders.59–69 Moreover, potentially damaging variants in platelet G proteincoupled receptors genes51 and the association of FLI1 and RUNX1 mutations with platelet dense granule secretion defects have been described.75 However, this approach is complex, cumbersome and requires great expertise in platelet disorders, molecular biology, and bioinformatics to predict the clinical significance of novel missense variants.76 When a novel gene causing IPFD has been identified by NGS, expression of the mutant protein in a cellular model should be performed to confirm the detrimental functional consequences of the mutation. Therefore, although NGS-based approaches may potentially revolutionize the diagnosis of IPFDs and in perspective become the first diagnostic approach, a preliminary clinical and laboratory phenotypic characterization still remains crucial. Of note, recently the BRIDGE-BPD consortium proposed the application of human phenotype ontology terms to annotate bleeding phenotypes showing that phenotypic characteristics of IPFDs cluster and may guide the discovery of the mutant gene.77

Which Patients Should Undergo Genetic Testing? For some IPFDs, a combination of clinical and laboratory criteria is sufficient for a conclusive diagnosis (e.g., Glanzmann thrombasthenia or Bernard–Soulier syndrome). For others, genotyping is not mandatory but it is advisable

614201

612840 609821

GPS BDPLT17 nd HPS

LADIII BDPLT8 TCPT PLA2G4A VWDP nd

Gray platelet syndrome associated with thrombocytopenia

Gs platelet defect

Hermansky–Pudlak syndrome

Leukocyte adhesion deficiency, type III

P2Y12 deficiency

Paris–Trousseau syndrome

Phospholipase A2 (cPLA2) deficiency

Platelet-type von Willebrand disease

Primary secretion defect

nd

177820

600522

188025

203300

nd

187900

139090

187800

273800

300367

BDPLT11

XLTDA

GATA1-related diseases

nd

GPVI deficiency

nd

Filaminopathy-related macrothrombocytopenia

185050 601399

Gray platelet syndrome

FPDMM

Familial platelet disorder and predisposition to acute myelogenous leukemia

608404

nd

Delta granule deficiency

nd

BDPLT10

nd

Combined α-delta granule deficiency

214500

GPIV deficiency

CHS

Cediak–Higashi syndrome

153670

GT

BSSA2

Bernard–Soulier syndrome monoallelic

231200

BDPLT16

BSS

Bernard–Soulier syndrome biallelic

208085, 613404

Autosomal dominant Glanzmann thrombasthenia

ARCS

Arthrogryposis, renal dysfunction, and cholestasis

OMIM entry

Glanzmann thrombasthenia

OMIM abbreviation

Disease

Table 5 Genotyping of patients with a suspected IPFD

AR/AD

AD

AR (?)

AD

AR

AR

AR

AD (if paternally inherited)

AD

AR

AR

AR

AD

AR

XL

XL

AD

AR/AD

AR/AD

AR

AD

AR

AR

Inheritance

Unknown

GP1BA (17p13.2)

PLA2G4A (1q31.1)

del11q23-ter

P2RY12 (3q24-q25)

FERMT3 (11q13.1)

HPS1 (10q24.2), ADTB3A (5q14.1), HPS3 (3q24), HPS4 (22q12.1), HPS5 (11p15.1), HPS6 (10q24.32), DTNBP1 (6p22.3), BLOC1S3 (19q13.32)

GNAS (20q13.32)

GFI1B (9q34.13)

NBEAL2 (3p21.1)

GP6 (19q13.42)

GP4 (7q21.11)

ITGA2B, ITGB3 (17q21.32)

GATA1 (Xp11)

FLNA (Xq28)

RUNX1 (21q22)

Unknown

Unknown

CHS1 (1q42.1–42.2)

GP1BA (17p13)

GP1BA (17p13), GP1BB (22q11), GP9 (3q21)

VPS33B (15q26.1), VIPAS39 (14q24.3)

Gene (chromosome localization)

(Continued)

For research

Not necessary

Recommended

Recommended

Advisable

Not necessary

Advisable (G/P)

Recommended

Advisable

Advisable (G/P)

Not necessary

Not necessary

Recommended

Not necessary

Recommended

Recommended

Recommended

For research

For research

Advisable (G/P)

Recommended

Not necessary

Advisable (G/P)

Recommendation for genotyping

Diagnosis of Inherited Platelet Function Disorders Gresele et al.

Seminars in Thrombosis & Hemostasis

STIM1 (11p15.4) AD nd York platelet syndrome

Seminars in Thrombosis & Hemostasis

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; GP, glycoprotein; G/P, genotype/phenotype correlation; IPFD, inherited platelet function disorder; nd, not defined; XL: X-linked; (?) not sure because too few cases reported.

Advisable

WAS (Xp11) XL WAS Wiskott–Aldrich syndrome

nd

Not necessary

Del22q11.2 AD VCF Velocardiofacial syndrome

192430

Recommended

Recommended TBXAS1 (7q34) AD (?) Tx synthase deficiency

277970

Advisable TBXA2R (19p13.3) AD BDPLT13

BDPLT14

Thromboxane A2 receptor defect

614009

STIM1 (11p15.4) ORAI1 (12q24.31) AD STRMK Stormorken syndrome

614158

Advisable

TMEM16F (12q12) AR

185070

Recommended

Advisable

PLAU (10q24) AD

SCTS Scott syndrome

262890

QPD Quebec platelet disorder

601709

Recommendation for genotyping Gene (chromosome localization) OMIM abbreviation Disease

Table 5 (Continued)

OMIM entry

Inheritance

Diagnosis of Inherited Platelet Function Disorders

Gresele et al. because a genotype/phenotype correlation has been established. In all other cases, genotyping is recommended.

IPFDs for Which Genotyping Is Not Necessary Biallelic Bernard–Soulier syndrome, Glanzmann thrombasthenia, Wiskott–Aldrich syndrome, leukocyte adhesion deficiency III, platelet-type VWD, gray platelet syndrome, GPIV and GPVI deficiency are disorders that can be clearly diagnosed without the need of genetic testing5 (►Table 5). Of course, genetic analysis if available can be of interest because it confirms diagnosis, may allow discovering novel mutations in a known underlying gene, and enlarges the available genetic databases to allow genotype/phenotype correlation studies.

IPFDs for Which Genotyping Is Not Necessary but Is Advisable Some IPFDs can be diagnosed without genetic testing but, given that a genotype/phenotype correlation has been described, genotyping is advisable for a more accurate prognostic evaluation: MYH9-RD,17,78 arthrogryposis renal dysfunction and cholestasis syndrome,79 Chediak–Higashi syndrome,80 Hermansky–Pudlak syndrome, 73 and gray platelet syndrome.81 Finally, Stormorken and Scott syndromes, P2Y12, and TP receptor defects are IPFDs where the platelet phenotype may not be undisputedly attributed to that specific disorder, thus genetic testing is advisable (►Table 5).

IPFDs for Which Genotyping Is Recommended The rare monoallelic, dominant variant forms of Bernard– Soulier syndrome and Glanzmann thrombasthenia require genetic analysis to be diagnosed. In these patients, the clinical and laboratory picture may be confusing, for example, their familial transmission is different from the classical forms which are recessive, GPIIb/IIIa or GPIb/IX/V are reduced and not absent, platelet dysfunction may be of variable severity, platelet volume is usually increased, thus genetic testing is recommended.82–85 Genetic testing should also be performed for suspected filaminopathy-related macrothrombocytopenia, GATA1related disease, Gsα platelet defects, Paris–Trousseau Syndrome, cPLA2 deficiency, Quebec platelet disorder, Tx synthase deficiency because results of platelet function studies may be inconclusive due to the heterogeneity of functional alterations, redundancy in signaling pathways or uncertain characterization for the only few cases described in the literature. Finally, genetic analysis is highly recommended for familial platelet disorder associated with acute myeloid leukemia. Indeed, this IPFD is associated with a predisposition to acute myeloid leukemia, as RUNX1 is a gene frequently involved in the pathogenesis of sporadic leukemia and myelodysplastic syndromes, and evolution to leukemia depends on the type of mutation, where mutations that generate dominant-negative proteins favor leukemic evolution while mutations inducing haploinsufficiency only rarely evolve in leukemia.86

Diagnosis of Inherited Platelet Function Disorders

Conclusions IPFDs represent a large and heterogeneous group of bleeding diseases that range in severity from mild to severe and that can be associated with defects in bone marrow megakaryopoiesis and with somatic defects. An accurate clinical evaluation should be the first step in determining whether a patient requires laboratory testing. The laboratory approach to the differential diagnosis of IPFDs should follow a rational algorithm based on a streamlined panel of laboratory tests following steps of increasing levels of complexity. First-line exome analysis is a conceivable alternative to extensive platelet function testing in the investigation of suspected IPFDs. However, except for a few well-defined forms, the identification of a specific DNA variation as causative of an IPFD in the absence of phenotypic information is very challenging. Platelet phenotyping by a step-by-step approach, therefore, remains for the time being an essential element in the diagnosis of IPFD and in the identification of still unrecognized forms. Indeed, the application of a previously developed diagnostic algorithm for inherited thrombocytopenias87 together with the application of NGS has significantly reduced the number of patients without a definite diagnosis.88 It is thus foreseeable that the systematic application of standardized diagnostic algorithms to patients with mucocutaneous bleeding of probable platelet origin on the one hand will increase the fraction of known IPFDs diagnosed, thus expanding the clinical knowledge on these rare disorders, and on the other will allow to select the cases on which performing deep investigations, thus allowing to unravel novel forms.

Funding This work was supported in part by grants to P.G. from Telethon foundation (protocol # GGP10155 and protocol # GGP15063) and by a fellowship to E.F. from Fondazione Umberto Veronesi.

References

7

8

9

10

11

12

13

14

15

16

17

18

19

1 Gresele P, Harrison P, Bury L, et al. Diagnosis of suspected inherited

2

3

4

5

6

platelet function disorders: results of a worldwide survey. J Thromb Haemost 2014;12(9):1562–1569 Israels SJ, El-Ekiaby M, Quiroga T, Mezzano D. Inherited disorders of platelet function and challenges to diagnosis of mucocutaneous bleeding. Haemophilia 2010;16(Suppl 5):152–159 Quiroga T, Goycoolea M, Panes O, et al. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007;92(3):357–365 Hayward CP, Rao AK, Cattaneo M. Congenital platelet disorders: overview of their mechanisms, diagnostic evaluation and treatment. Haemophilia 2006;12(Suppl 3):128–136 Gresele P; Subcommittee on Platelet Physiology. Diagnosis of inherited platelet function disorders: guidance from the SSC of the ISTH. J Thromb Haemost 2015;13(2):314–322 Streif W, Oliveri M, Weickardt S, Eberl W, Knoefler R; Thromkid Study Group of GTH. Testing for inherited platelet defects in

20

21

22

23

24

Gresele et al.

clinical laboratories in Germany, Austria and Switzerland. Results of a survey carried out by the Permanent Paediatric Group of the German Thrombosis and Haemostasis Research Society (GTH). Platelets 2010;21(6):470–478 Hayward CP, Moffat KA, Plumhoff E, Van Cott EM. Approaches to investigating common bleeding disorders: an evaluation of North American coagulation laboratory practices. Am J Hematol 2012;87 (Suppl 1):S45–S50 Bolton-Maggs PH, Chalmers EA, Collins PW, et al; UKHCDO. A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. Br J Haematol 2006; 135(5):603–633 Hayward CP, Moffat KA, Raby A, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol 2010;134(6):955–963 Harrison P, Mackie I, Mumford A, et al; British Committee for Standards in Haematology. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br J Haematol 2011;155(1):30–44 Mezzano D, Quiroga T, Pereira J. The level of laboratory testing required for diagnosis or exclusion of a platelet function disorder using platelet aggregation and secretion assays. Semin Thromb Hemost 2009;35(2):242–254 Dawood BB, Lowe GC, Lordkipanidzé M, et al. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel. Blood 2012;120(25):5041–5049 Gresele P, Van Geet C, Scharf RE, Makris M, Rao K. Acquired non immune thrombocytopenia and acquired disorders of platelet function. Haematologica 2015; In press Pecci A, Biino G, Fierro T, et al; Italian Registry for MYH9-releated diseases. Alteration of liver enzymes is a feature of the MYH9related disease syndrome. PLoS ONE 2012;7(4):e35986 Savoia A, Kunishima S, De Rocco D, et al. Spectrum of the mutations in Bernard-Soulier syndrome. Hum Mutat 2014; 35(9):1033–1045 Quiroga T, Mezzano D. Is my patient a bleeder? A diagnostic framework for mild bleeding disorders. Hematology Am Soc Hematol Educ Program 2012;2012:466–474 Pecci A, Klersy C, Gresele P, et al. MYH9-related disease: a novel prognostic model to predict the clinical evolution of the disease based on genotype-phenotype correlations. Hum Mutat 2014; 35(2):236–247 Pontara E, Gresele P, Cattini MG, et al. Spontaneous hemarthrosis in combined Glanzmann thrombasthenia and type 2N von Willebrand disease. Blood Coagul Fibrinolysis 2014;25(4): 401–404 Watson S, Daly M, Dawood B, et al. Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors. Hamostaseologie 2010;30(1):29–38 McKay H, Derome F, Haq MA, et al. Bleeding risks associated with inheritance of the Quebec platelet disorder. Blood 2004;104(1): 159–165 Zwaal RF, Comfurius P, Bevers EM. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids. Biochim Biophys Acta 2004;1636(2–3):119–128 Rodeghiero F, Tosetto A, Abshire T, et al; ISTH/SSC joint VWF and Perinatal/Pediatric Hemostasis Subcommittees Working Group. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. J Thromb Haemost 2010;8(9):2063–2065 Federici AB, Bucciarelli P, Castaman G, et al. The bleeding score predicts clinical outcomes and replacement therapy in adults with von Willebrand disease. Blood 2014;123(26):4037–4044 Bidlingmaier C, Grote V, Budde U, Olivieri M, Kurnik K. Prospective evaluation of a pediatric bleeding questionnaire and the ISTH Seminars in Thrombosis & Hemostasis

Diagnosis of Inherited Platelet Function Disorders

25

26

27

28

29 30

31

32

33

34

35

36

37

38

39

40

41

42

Gresele et al.

bleeding assessment tool in children and parents in routine clinical practice. J Thromb Haemost 2012;10(7):1335–1341 Lowe GC, Lordkipanidzé M, Watson SP; UK GAPP study group. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013;11(9):1663–1668 Daly ME, Dawood BB, Lester WA, et al. Identification and characterization of a novel P2Y 12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study. Blood 2009;113(17):4110–4113 Noris P, Klersy C, Gresele P, et al; Italian Gruppo di Studio delle Piastrine. Platelet size for distinguishing between inherited thrombocytopenias and immune thrombocytopenia: a multicentric, real life study. Br J Haematol 2013;162(1):112–119 Noris P, Biino G, Pecci A, et al. Platelet diameters in inherited thrombocytopenias: analysis of 376 patients with all known disorders. Blood 2014;124(6):e4–e10 Nurden AT, Nurden P. The gray platelet syndrome: clinical spectrum of the disease. Blood Rev 2007;21(1):21–36 Pecci A, Noris P, Invernizzi R, et al. Immunocytochemistry for the heavy chain of the non-muscle myosin IIA as a diagnostic tool for MYH9-related disorders. Br J Haematol 2002;117(1):164–167 Freson K, Devriendt K, Matthijs G, et al. Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1 mutation. Blood 2001;98(1):85–92 Cattaneo M, Cerletti C, Harrison P, et al. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost 2013;11:1183–1189 Faioni EM, Razzari C, Zulueta A, et al. Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency. Thromb Haemost 2014;112(6):1182–1189 Lin TM, Lin JS, Tseng JY, Wu SY, Chen TY. Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese. Platelets 2015;•••:1–6(e-pub ahead of print) Hayward CP, Rivard GE, Kane WH, et al. An autosomal dominant, qualitative platelet disorder associated with multimerin deficiency, abnormalities in platelet factor V, thrombospondin, von Willebrand factor, and fibrinogen and an epinephrine aggregation defect. Blood 1996;87(12):4967–4978 Mumford AD, Frelinger AL III, Gachet C, et al. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders. Thromb Haemost 2015;114(1):14–25 De Candia E, Pecci A, Ciabattoni G, et al. Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives. J Thromb Haemost 2007;5(3): 551–559 Villeneuve J, Block A, Le Bousse-Kerdilès MC, et al. Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: a novel organization for these secreted proteins. Exp Hematol 2009; 37(7):849–856 Van Geet C, Devriendt K, Eyskens B, Vermylen J, Hoylaerts MF. Velocardiofacial syndrome patients with a heterozygous chromosome 22q11 deletion have giant platelets. Pediatr Res 1998;44(4): 607–611 Freson K, Izzi B, Labarque V, et al. GNAS defects identified by stimulatory G protein alpha-subunit signalling studies in platelets. J Clin Endocrinol Metab 2008;93(12):4851–4859 Stormorken H, Holmsen H, Sund R, et al. Studies on the haemostatic defect in a complicated syndrome. An inverse Scott syndrome platelet membrane abnormality? Thromb Haemost 1995; 74(5):1244–1251 Halliez M, Fouassier M, Robillard N, et al. Detection of phosphatidyl serine on activated platelets’ surface by flow cytometry in

Seminars in Thrombosis & Hemostasis

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

whole blood: a simpler test for the diagnosis of Scott syndrome. Br J Haematol 2015;•••: 10.1111/bjh.13391 (e-pub ahead of print) Giannini S, Cecchetti L, Mezzasoma AM, Gresele P. Diagnosis of platelet-type von Willebrand disease by flow cytometry. Haematologica 2010;95(6):1021–1024 Lo B, Li L, Gissen P, et al. Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet alpha-granule biogenesis. Blood 2005;106(13):4159–4166 Nurden P, Debili N, Coupry I, et al. Thrombocytopenia resulting from mutations in filamin A can be expressed as an isolated syndrome. Blood 2011;118(22):5928–5937 Necchi V, Balduini A, Noris P, et al. Ubiquitin/proteasome-rich particulate cytoplasmic structures (PaCSs) in the platelets and megakaryocytes of ANKRD26-related thrombocytopenia. Thromb Haemost 2013;109(2):263–271 Clauser S, Cramer-Bordé E. Role of platelet electron microscopy in the diagnosis of platelet disorders. Semin Thromb Hemost 2009; 35(2):213–223 de Witt SM, Swieringa F, Cavill R, et al. Identification of platelet function defects by multi-parameter assessment of thrombus formation. Nat Commun 2014;5:4257 Rao AK. Inherited platelet function disorders: overview and disorders of granules, secretion, and signal transduction. Hematol Oncol Clin North Am 2013;27(3):585–611 Hermans C, Wittevrongel C, Thys C, Smethurst PA, Van Geet C, Freson K. A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder. J Thromb Haemost 2009; 7(8):1356–1363 Jones ML, Norman JE, Morgan NV, et al; UK GAPP study group. Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors. Thromb Haemost 2015;113(4): 826–837 Ahmad F, Kannan M, Kishor K, Saxena R. Coinheritance of severe von Willebrand disease with Glanzmann thrombasthenia. Clin Appl Thromb Hemost 2010;16(5):529–532 Albers CA, Paul DS, Schulze H, et al. Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exonjunction complex subunit RBM8A causes TAR syndrome. Nat Genet 2012;44(4):435–439, S1–S2 Newman PJ, Seligsohn U, Lyman S, Coller BS. The molecular genetic basis of Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations in Israel. Proc Natl Acad Sci U S A 1991;88(8):3160–3164 Ware J, Russell SR, Vicente V, et al. Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with BernardSoulier syndrome. Proc Natl Acad Sci U S A 1990;87(5):2026–2030 Hollopeter G, Jantzen HM, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 2001;409(6817):202–207 Seri M, Cusano R, Gangarossa S, et al; The May-Hegglin/Fechtner Syndrome ConsortiumMutations in MYH9 result in the MayHegglin anomaly, and Fechtner and Sebastian syndromes. Nat Genet 2000;26(1):103–105 Paterson AD, Rommens JM, Bharaj B, et al. Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene. Blood 2010;115(6):1264–1266 Albers CA, Cvejic A, Favier R, et al. Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nat Genet 2011;43(8):735–737 Gunay-Aygun M, Falik-Zaccai TC, Vilboux T, et al. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules. Nat Genet 2011;43(8):732–734 Kahr WH, Hinckley J, Li L, et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nat Genet 2011; 43(8):738–740 Stevenson WS, Morel-Kopp MC, Chen Q, et al. GFI1B mutation causes a bleeding disorder with abnormal platelet function. J Thromb Haemost 2013;11(11):2039–2047

Diagnosis of Inherited Platelet Function Disorders 63 Monteferrario D, Bolar NA, Marneth AE, et al. A dominant-negative

64

65

66

67

68

69

70

71

72

73

74

75

76

GFI1B mutation in the gray platelet syndrome. N Engl J Med 2014; 370(3):245–253 Kunishima S, Okuno Y, Yoshida K, et al. ACTN1 mutations cause congenital macrothrombocytopenia. Am J Hum Genet 2013;92(3): 431–438 Nesin V, Wiley G, Kousi M, et al. Activating mutations in STIM1 and ORAI1 cause overlapping syndromes of tubular myopathy and congenital miosis. Proc Natl Acad Sci U S A 2014;111(11): 4197–4202 Markello T, Chen D, Kwan JY, et al. York platelet syndrome is a CRAC channelopathy due to gain-of-function mutations in STIM1. Mol Genet Metab 2015;114(3):474–482 Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nat Genet 2015;47(2):180–185 Levin C, Koren A, Pretorius E, et al. Deleterious mutation in the FYB gene is associated with congenital autosomal recessive smallplatelet thrombocytopenia. J Thromb Haemost 2015;13(7): 1285–1292 Fletcher SJ, Johnson B, Lowe GC, et al. SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects. J Clin Invest 2015;125(9):3600–3605 Daly ME, Leo VC, Lowe GC, Watson SP, Morgan NV. What is the role of genetic testing in the investigation of patients with suspected platelet function disorders? Br J Haematol 2014;165(2):193–203 Watson SP, Lowe GC, Lordkipanidzé M, Morgan NV; GAPP consortium. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 2013;11(Suppl 1):351–363 Dewey FE, Pan S, Wheeler MT, Quake SR, Ashley EA. DNA sequencing: clinical applications of new DNA sequencing technologies. Circulation 2012;125(7):931–944 Sánchez-Guiu I, Torregrosa JM, Velasco F, et al. Hermansky-Pudlak syndrome. Overview of clinical and molecular features and case report of a new HPS-1 variant. Hamostaseologie 2014;34(4):301–309 Jones ML, Murden SL, Bem D, et al; UK GAPP study group. Rapid genetic diagnosis of heritable platelet function disorders with next-generation sequencing: proof-of-principle with HermanskyPudlak syndrome. J Thromb Haemost 2012;10(2):306–309 Stockley J, Morgan NV, Bem D, et al; UK Genotyping and Phenotyping of Platelets Study Group. Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects. Blood 2013;122(25):4090–4093 Buitrago L, Rendon A, Liang Y, et al; ThromboGenomics Consortium. αIIbβ3 variants defined by next-generation sequencing:

77

78

79

80

81

82

83

84

85

86

87

88

Gresele et al.

predicting variants likely to cause Glanzmann thrombasthenia. Proc Natl Acad Sci U S A 2015;112(15):E1898–E1907 Westbury SK, Turro E, Greene D, et al; BRIDGE-BPD Consortium. Human phenotype ontology annotation and cluster analysis to unravel genetic defects in 707 cases with unexplained bleeding and platelet disorders. Genome Med 2015;7(1):36 Verver EJ, Topsakal V, Kunst HP, et al. Nonmuscle Myosin Heavy Chain IIA Mutation Predicts Severity and Progression of Sensorineural Hearing Loss in Patients With MYH9-Related Disease. Ear Hear 2015 (e-pub ahead of print) Smith H, Galmes R, Gogolina E, et al. Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome. Hum Mutat 2012;33(12): 1656–1664 Lozano ML, Rivera J, Sánchez-Guiu I, Vicente V. Towards the targeted management of Chediak-Higashi syndrome. Orphanet J Rare Dis 2014;9:132 Bottega R, Pecci A, De Candia E, et al. Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency. Haematologica 2013;98(6):868–874 Savoia A, Balduini CL, Savino M, et al. Autosomal dominant macrothrombocytopenia in Italy is most frequently a type of heterozygous Bernard-Soulier syndrome. Blood 2001;97(5): 1330–1335 Nurden AT, Pillois X, Fiore M, Heilig R, Nurden P. Glanzmann thrombasthenia-like syndromes associated with Macrothrombocytopenias and mutations in the genes encoding the αIIbβ3 integrin. Semin Thromb Hemost 2011;37(6):698–706 Gresele P, Falcinelli E, Giannini S, et al. Dominant inheritance of a novel integrin beta3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families. Haematologica 2009;94(5):663–669 Bury L, Malara A, Gresele P, Balduini A. Outside-in signalling generated by a constitutively activated integrin αIIbβ3 impairs proplatelet formation in human megakaryocytes. PLoS ONE 2012; 7(4):e34449 Antony-Debré I, Manchev VT, Balayn N, et al. Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. Blood 2015;125(6):930–940 Noris P, Pecci A, Di Bari F, et al. Application of a diagnostic algorithm for inherited thrombocytopenias to 46 consecutive patients. Haematologica 2004;89(10):1219–1225 Savoia A. Molecular basis of inherited thrombocytopenias. Clin Genet 2015 (e-pub ahead of print)

Seminars in Thrombosis & Hemostasis