Diagnosis of inherited bleeding disorders in the genomic era

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1 review Diagnosis of inherited bleeding disorders in the genomic era Suthesh Sivapalaratnam, 1,2 Janine Collins 1,2 and Keith Gomez 3 1 Department of Haematology, University of Cambridge, Cambridge, 2 The Royal London Haemophilia Centre, The Royal London Hospital, London and 3 Katherine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London, UK Summary Inherited bleeding disorders affect between 1 in 1000 individuals for the most common disorder, von Willebrand Disease, to only 8 reported cases worldwide of alpha-2-antiplasmin deficiency. Those with an identifiable abnormality can be divided into disorders of coagulation factors (87%), platelet count and function (8%) and the fibrinolytic system (3%). Of the patients registered in the UK with a bleeding disorder, the remaining 2% are unclassifiable. In addition to bleeding symptoms, patients with an inherited bleeding disorder can manifest other abnormalities, making an accurate and complete diagnosis that reflects the underlying molecular pathology important. Although some inherited bleeding disorders can still be easily diagnosed through a combination of careful clinical assessment and laboratory assays of varying degrees of complexity, there are many where conventional approaches are inadequate. Improvements in phenotyping assays have enhanced our diagnostic armoury but genotyping now offers the most accurate and complete diagnosis for some of these conditions. The advent of next generation sequencing technology has meant that many genes can now be analysed routinely in clinical practice. Here, we discuss the different diagnostic tools currently available for inherited bleeding disorders and suggest that genotyping should be incorporated at an early stage in the diagnostic pathway. Keywords: genetics, diagnostic haematology, platelet disorders, bleeding disorders. Inherited bleeding disorders (IBDs) have a wide range of frequencies from 1 in 1000 live births for von Willebrand Disease (VWD) and 1 in 5000 male live births in Haemophilia A, to only 8 cases worldwide of alpha-2-antiplasmin deficiency (Favier et al, 2001; Palla et al, 2015) ( Correspondence: Dr K Gomez, Katherine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, Pond Street, London NW3 2QG, UK. k.gomez@ucl.ac.uk (Fig 1). Whilst those that manifest bleeding symptoms are often referred to haematologists, many of these disorders present to other specialists with non-haematological features. The underlying molecular abnormality can be the result of variation in any of the genes encoding components of the pathways for platelet-dependent haemostasis or fibrin clot generation and breakdown (Fig 2). Following vessel wall injury, haemostasis is achieved through two main processes: activation of platelets is followed by their adhesion and aggregation, and the plasma coagulation proteins create a fibrin network to which platelets adhere. Subsequently, the plasma coagulation factors are also involved in clot lysis (fibrinolysis), which is a key part of the haemostatic process required for wound healing. Deficiencies of coagulation factors impair normal fibrin clot generation. Platelet disorders may be due to abnormalities in count, function or both. At a molecular level these can be due to abnormalities in membrane proteins (including cell surface receptors), cytoplasmic proteins, granules (alpha, dense or lysosomal) and proteins regulating transcription and translation. Most of these disorders are associated with abnormalities in laboratory assays, although many of the tests are only available in specialist coagulation laboratories. Whilst these tests are generally able to accurately identify the molecular causes of coagulation factor defects, they largely fail to do so for platelet dysfunctions. The latter are therefore grouped together. It is increasingly clear that this is unsatisfactory as it hinders recognition of potentially serious haematological and non-haematological sequelae. Furthermore, there are patients whose abnormal bleeding symptoms are not associated with any laboratory abnormalities. By exclusion, these patients are categorized as having an unspecified bleeding tendency and represent 2% of registrants in the National Haemophilia Database of the UK Haemophilia Centre Doctors Organisation (UKHCDO) ( org/annual-reports/). As their clinical phenotype is similar to those with classifiable bleeding disorders, it is likely that there are undiscovered defects in platelets or the vessel wall. This reflects the inadequacies of current laboratory assays for defining the underlying causes of platelet dysfunction. Defects of the vessel wall are even less well identified. ª 2017 John Wiley & Sons Ltd First published online 14 June 2017 doi: /bjh.14796

2 Fig 1. Incidence of non-acquired bleeding and platelet disorders registered by the UK Haemophilia Centres Doctors Organisation up to April Incidence of non-acquired bleeding and platelet disorders as a proportion of the patients registered by the UK Haemophilia Centres Doctors Organisation up to April 2015 ( ports/). Of note, von Willebrand Disease (VWD) includes probable cases (15% of VWD cases); Haemophilia A and B categories include low-level carriers. Platelet disorder registrations break down as Glanzmann Thrombasthaenia 54%, Bernard Soulier Syndrome 37% and other platelet defects 901%. Data for rarer coagulation factor deficiencies includes prothrombin, factor (F)V, FX and FXIII. The aim of this review is to provide a framework for clinicians to use in the diagnosis of IBDs in an evolving clinical climate requiring more accurate definition of molecular aetiology. We will discuss each step of the diagnostic process, as illustrated in Fig 3. We will also suggest in this review how to incorporate state of the art genetics into the diagnostic pathway. Clinical history The initial step of the diagnostic process is the clinical appreciation of the presence and severity of bleeding symptoms. In an attempt to improve usability of the bleeding history, the International Society on Thrombosis and Haemostasis (ISTH) promotes the Bleeding Assessment Tool (BAT) to generate numerical scores from the bleeding history. The aim is to reduce the subjectivity inherent in assessing clinical symptoms and provide a quantifiable measure of bleeding symptoms. The score was initially developed as a diagnostic tool to identify patients who have a high likelihood of VWD requiring factor assay measurement, but can be useful in all bleeding disorders (Rodeghiero et al, 2011). The ISTH-BAT is validated in Haemophilia A carriers and patients with suspected platelet function disorders (James et al, 2016). Another study evaluated 79 patients with suspected inherited platelet function disorders, and 21 healthy volunteers (Lowe et al, 2013). The median ISTH-BAT score in clinically affected participants was 12 [interquartile range (IQR) 8 16] compared to 0 in healthy volunteers (IQR 0 0). In patients with unexplained bleeding symptoms there was no difference between those with a platelet defect detected by lumi-aggregometry (median 11; IQR 8 16) and those with normal platelet function (median 12; IQR 8 14) who would be considered by a coagulation specialist to have an unclassifiable bleeding disorder. Kaur et al (2016) compared the ISTH-BAT with the condensed form of the electronic bleeding questionnaire (ebq), the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD questionnaire (MCMDM-1) and World Health Organisation BATs, in the assessment of bleeding in 38 patients previously diagnosed with Glanzmann thrombasthenia and 10 with Bernard-Soulier syndrome. Bleeding scores were significantly higher than controls with no significant difference between the tools. The ISTH-BAT had a sensitivity, specificity, positive predictive value and negative predictive value of 100%, 762%, 09 and 1 (Kaur et al, 2016). Unfortunately the negative predictive value for more common, less severe platelet disorders is less good, particularly in children and males who 364 ª 2017 John Wiley & Sons Ltd

3 Fig 2. A schematic overview of the different components of platelet-dependent haemostasis and fibrin clot generation/breakdown. Left panel: The process of platelet formation starting with the haematopoietic stem cell (HSC) in the bone marrow, the generation of megakaryocytes and platelets. The genes implicated in these steps are given. Middle upper panel: The process of primary haemostasis. Right panel: the process of fibrin clot generation and breakdown. For details and an up to date gene list see: GP, G-protein; GPCR, G-protein coupled receptor; PAI-1, Plasminogen activator inhibitor 1; t-pa, tissue plasminogen activator; u-pa, urokinase plasminogen activator. Megakaryopoiesis Transcription factors GATA1 FLI1* GFI1B RUNX1 ETV6 HOXA11* MECOM* TPO/MPL signalling THPO RBM8A* MPL ANKRD26 Pro-platelet formation Cytoskeleton Intracellular regulation CYCS MYH9* FLNA* SRC* WAS* TUBB1 GNE* Platelet Function Haematopoetic stem cell Megakaryocyte ACTN1 DIAPH1* SLFN14 Calcium signalling ORAI1* STIM1* GP signalling GP1BA&B ITGA2B & ITGB3 GP9 VWF Granule biogenesis & trafficking HPS1* DTNBP1* VIPAS39* APHPS3* NBEAL2 STXBP23B1* HPS4* BLOC1S3* NBEA* HPS5* LYST* PLAU HPS6* VPS33B* BLOC1S6 GPCR signalling P2YR12 TBXAS1* TBXA2R PLA2G4A GP receptor signalling ITGA2B GP1BB VWF ITGB3 GP9 FERMT3 GP1BA RASGRP2 GP6 ANO6 Platelet Glycoprotein Ib receptor * Presence of phenotypes outside the blood system Endothelium von Willebrand factor Fibrin Collagen Glycoprotein IIb/IIIa receptor Platelets 1. Coagulation cascade Contact activation (intrisic) pathway Damaged surface Tissue factor XII XIIa (extrinsic) pathway XI XIa Trauma IX IXa VIIa VII Common VIIIa Tissue factor pathway X Xa X Trauma Va Prothrombin Thrombin (II) (IIa) Fibrinogen Fibrin XIIIa Cross-linked fibrin clot 2. Inhibitors of coagulation IX IXa VIIIa VII TF TFPI Protein C X Xa Va X ATIII Prothrombin Thrombin 3. Fibrinolysis Fibrinogen Plasminogen PAI-1 t-pa u-pa Plasmin Fibrindegradation Fibrin products TAF1 Alpha-2- antiplasmin Fibrin have had fewer haemostatic challenges. This means that while BATs can usefully distinguish the severity of bleeding disorders, a negative score does not remove the need for further laboratory assays. It maybe that larger studies of other platelet defects will enhance the utility of BATs and support their integration into a universal diagnostic approach with standardized laboratory testing for patients with suspected bleeding disorders. These larger studies are currently being conducted by the ISTH. Bleeding Assessment Tools aim to quantify the bleeding symptoms resulting from haemostatic challenges but these can be modified by medications and diet. In addition to anticoagulants, non-steroidal anti-inflammatory drugs and platelet receptor antagonists, there are also certain cardiovascular agents (beta-blockers, diuretics and calcium channel blockers), antimicrobials (beta-lactams, antifungals, nitrofurantoin), psychotropics (tricyclic antidepressants) and antiepileptics that can impair platelet function and result in easy bruising or other bleeding symptoms. Frequent dietary culprits are alcohol, caffeine, garlic, onion and ginger, although usually these would need to be consumed in much larger amounts than would be found in a normal diet. A full list can be found in Table I of the laboratory assessment of platelet function guideline of the British Society of Haematology (Harrison et al, 2011). Malnutrition and subsequent vitamin K deficiency can cause dysfunction of the vitamin K dependent coagulation factors: II, VII, IX and X. In particular, vitamin K deficiency should be considered in patients with inflammatory bowel disease, short bowel syndrome or long hospitalization. The timeline of symptoms and construction of a family tree are useful in indicating whether the patient is likely to have an inherited or acquired disorder. Early age of onset with multiple family members affected make an inherited cause more likely. However, it is important to consider autosomal recessive inheritance of disorders with asymptomatic carriers, especially when parents are consanguineous. Table I details the known IBDs and their mode of inheritance. Some have multiple modes of inheritance reported, such as VWD. Variants that result in a null allele, such as a deletion, are generally inherited recessively because up-regulation of the normal allele compensates for the defect in heterozygous individuals. Missense variants that result in a polypeptide with reduced function can have a dominant negative effect on the product of the normal transcript as both are incorporated into the multimers of the mature protein. The presence of syndromic features also favours an inheritable cause and, in particular, the presence of albinism, bone abnormalities, recurrent infections, behavioural disorders, kidney impairment and deafness should be assessed as these are now wellrecognised associations of some platelet disorders. An overview of non-haematological clinical features associated with IBDs is shown in Table II. The type of bleeding symptoms can differentiate between coagulation factor disorders on one hand and platelet disorders or VWD on the other (Table III). Bruising or bleeding ª 2017 John Wiley & Sons Ltd 365

4 Fig 3. A diagnostic process for inherited bleeding disorders incorporating genetic analysis. APTT, activated partial thromboplastin time; LTA, light transmission aggregometry; NGS, next generation sequencing; PT, prothrombin time; ROTEM, rotational thromboelastometry; TEG, Thromboelastography: TT, thrombin time; VWF, von Willebrand factor. immediately after trauma is suggestive of a platelet disorder or VWD, and delayed bleeding is more characteristic of a coagulation factor deficiency. Mucosal bleeding and petechiae are more characteristic of a platelet disorder or VWD and haemarthroses are more prominent in severe coagulation factor disorders (e.g. Haemophilia A). Autosomal inheritance is typical of platelet disorders, although the female:male ratio of diagnosed platelet disorders is not equal but 2:1. This is predominantly because of the frequency of obstetric and gynaecological bleeding symptoms warranting further investigations, which results in easier identification of a platelet disorder in females. By contrast, in mild platelet disorders, if few haemostatic challenges have taken place, men may be asymptomatic and incorrectly assume that they are unaffected. Physical examination Platelet disorders can be part of a multi-system disorder. Therefore, a full physical examination may give clues facilitating diagnosis (Harrison et al, 2011; Nurden et al, 2012; Palla et al, 2015; Lentaigne et al, 2016; Bariana et al, 2017). Some key features to assess for include: albinism, a feature of Hermansky-Pudlak and Chediak Higashi syndromes; eczema, seen in Wiskott-Aldrich, Jacobson and Paris-Trousseau (PT) syndromes; facial abnormalities, associated with Jacobson/PT or DiGeorge syndromes; heart defects associated with Jacobson/ PT or Thrombocytopenia absent radius syndrome and sensorineural deafness, a feature of MYH9-related disease and associated with DIAPH1 variants. A comprehensive summary of these non-haematological features can be found in Table II. 366 ª 2017 John Wiley & Sons Ltd

5 Table I. All genes associated with inherited bleeding disorders that are approved by the International Society on Thrombosis and Haemostasis. Tier 1 genes (causal in at least 4 independent pedigrees) Genes Platelet Disorders MOI SERPINF2 Alpha 2-antiplasmin deficiency AR LMAN1; MCFD2 Combined V and VIII deficiency AR F5 Factor V deficiency AR F7 Factor VII deficiency AR F10 Factor X deficiency AR F11 Factor XI deficiency AR F13A1; F13B Factor XIII deficiency AR FGA; FGB; FGG Fibrinogen deficiency AD; AR; AR F8 Haemophilia A XR F9 Haemophilia B XR GGCX Multiple coagulation factor deficiency type 1 AR VKORC1 Multiple coagulation factor deficiency type 2 AR SERPINE1 Plasminogen activator inhibitor 1 (PAI-1) deficiency AR PLG Plasminogen deficiency AR F2 Prothrombin deficiency AR VWF von Willebrand disease: Type 1; Type 2A; Type 2B, Type 2M Type 2N; Type 3 AD AR Genes Platelet Disorders MOI P2RY12 ADP receptor defect AR HOXA11, MECOM* Amegakaryocytic thrombocytopenia with radioulnar synostosis AD VIPAS39; VPS33B Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome AR ANKRD26; CYCS Autosomal dominant thrombocytopenia AD GP1BA; GP1BB; GP9 Bernard-Soulier syndrome or mild thrombocytopenia AR or AD (e.g. Bolzano variant) GP6 Bleeding diathesis due to glycoprotein VI deficiency AR LYST Chediak-Higashi syndrome AR MPL Congenital amegakaryocytic thrombocytopenia (CAMT) AR PLA2G4A Deficiency of phospholipase A2, group IVA AR NBEA Dense granule abnormality AD CHST14 Ehlers Danlos Syndrome, musculocontractural type AR STXBP2 Familial haemophagocytic lymphohistiocytosis, type 5 AR RUNX1 Familial platelet disorder with predisposition to AML AD TBXAS1 Ghosal syndrome AR ITGA2B; ITGB3 Glanzmann thrombasthenia AR NBEAL2* Grey platelet syndrome AR GFI1B Grey platelet-like syndrome AD HPS1; AP3B1 (HPS2); HPS3; HPS4; HPS5; Hermansky-Pudlak syndrome AR HPS6; DTNBP1 (HPS7); BLOC1S3 (HPS8); BLOC1S6 (HPS9) FERMT3 Leucocyte adhesion deficiency, type III AR ACTN1* Macrothrombocytopenia AD FLNA Macrothrombocytopenia XR TUBB1 Macrothrombocytopenia, beta-tubulin 1 related AD MYH9 May-Hegglin and other MYH9-related disorders AD GNE Myopathy associated with thrombocytopenia AR FLI1 Paris-Trousseau thrombocytopenia and Jacobson syndrome AD or AR RASGRP2* Platelet-type bleeding disorder 18 AR GP1BA Platelet-type von Willebrand disease AD PLAU Quebec platelet disorder AD ANO6 Scott syndrome AR ª 2017 John Wiley & Sons Ltd 367

6 Table I. (Continued) Tier 1 genes (causal in at least 4 independent pedigrees) Genes Platelet Disorders MOI STIM1; ORAI1 Stormorken syndrome AD RBM8A* Thrombocytopenia absent radius (TAR) syndrome AR DIAPH1* Thrombocytopenia and sensorineural hearing loss AD ETV6* Thrombocytopenia and susceptibility to cancer AD THPO Thrombocytopenia and thrombocythaemia 1 AD TBXA2R Thromboxane A2 receptor defect AR WAS Wiskott-Aldrich syndrome XR GATA1 X-linked macrothrombocytopenia with dyserythropoiesis XR Tier 2 gene list (further study required to establish pathogenicity) Genes Disorder References MOI SLFN14* Autosomal dominant thrombocytopenia and Fletcher et al (2015) AD platelet secretion defect AP3D1* Hermansky-Pudlak syndrome (HPS10) Ammann et al (2016) AR TPM4 Macrothrombocytopenia Pleines et al (2017) AD FYB* Recessive microthrombocytopenia Hamamy et al (2014); Levin et al (2015) AR ABCG5; ABCG8 Sitosterolaemia with thrombocytopenia Chase et al (2010); Wang et al (2014); AR Rees et al (2005) SRC* Thrombocytopenia, bleeding and myelofibrosis Turro et al (2016) AD AD, autosomal dominant; AR, autosomal recessive; MOI, mode of inheritance; XR, X-linked recessive. *Molecular basis of inherited bleeding disorder discovered using a next generation sequencing approach. Genes in which Copy Number Variants (CNVs) are described, see text for further information. Furthermore, the distribution of bruising may be useful, as in mild IBDs it is usually limited to the limbs whereas bruising on the face and the trunk suggests a more severe disorder. Screening bloods and investigations for coagulation disorders The initial blood tests should include a full blood count (FBC), blood film microscopy, coagulation screen and von Willebrand factor (VWF) assays. On the FBC, note should be made of platelet count and mean platelet volume (MPV). Both thrombocytopenia and thrombocythemia (counts > /l) can be associated with bleeding. The film should be examined for signs of macrothrombocytopenia, grey appearance of platelets (indicative of Grey Platelet Syndrome) and neutrophil inclusion bodies (indicative of MYH9-related disorders). A coagulation screen should include the activated partial thromboplastin time (APTT), Prothrombin Time (PT), Thrombin Time and Fibrinogen. The Clauss assay more reliably identifies dysfibrinogenaemias than the PT-derived fibrinogen. VWF assays should measure both antigen and activity, ideally both platelet binding (ristocetin co-factor or GpIb binding assay) and collagen binding, as well as the factor VIII level. The interpretation of these results is described elsewhere in more detail (Mumford et al, 2014). In brief, if there is an abnormality in the APTT or PT, mixing studies are warranted. Normalisation on mixing studies suggests a coagulation factor deficiency. Isolated prolongation of the PT suggests a factor VII deficiency, while isolated prolongation of the APTT suggests a deficiency of factor VIII, IX or XI. Deficiencies of contact factors, such as factor XII, also prolong the APTT but do not result in a bleeding phenotype. When APTT and PT are both prolonged, factor II, V, X or combined (e.g. combined factor V and factor VIII) deficiencies should be considered. Thrombocytopenia and coagulopathy due to liver disease should be excluded by consideration of the clinical context and measuring liver enzymes. If the initial coagulation factor assays are normal, then the extremely rare disorders, including factor XIII deficiency and alpha-2-antiplasmin deficiency should be excluded. However, it is worth noting that these tend to be severe bleeding disorders that present in childhood to consanguineous parents. Mild deficiencies of these factors at the level seen in carrier parents are generally not associated with symptoms. Therefore, there is little value in applying these tests for mild bleeding symptoms presenting in adults. Specific assays for factor XIII and a2-antiplasmin are available and global haemostatic tests (see below) may be abnormal. (Dieval et al, 1991; Muszbek et al, 2011). There are anecdotal reports of other coagulation factor abnormalities, such as plasminogen activator inhibitor- 1 deficiency, but a clear causative association with bleeding has 368 ª 2017 John Wiley & Sons Ltd

7 Table II. Additional features associated with inherited bleeding disorders. System Feature Disease Skin Albinism Hermansky-Pudlak syndrome Chediak-Higashi Eczema Wiskott-Alrich Jacobson/Paris-Trousseau Impaired wound healing Factor XIII deficiency Musculoskeletal Upper extremities Absence radius, presence thumb Thrombocytopenia Absent Radius Limited pronation and supination forearm Amegakaryocyte thrombocytopenia with ulnar synostosis Hand abnormalities Jacobson/Paris-Trousseau Lower extremities Various anomalies Thrombocytopenia Absent Radius Hip dysplasia Amegakaryocyte thrombocytopenia with ulnar synostosis Facial abnormalities Jacobson/Paris-Trousseau DiGeorge/Velocardialfacial syndrome Short stature Mediterranean macrothrombocytosis Noonan related Skeletal abnormalities Homozygous Pelger-Huet Vitamin K-dependant factor deficiency Joint contractures Arthrogryposis renal dysfunction cholestasis syndrome Mitochondrial myopathy York syndrome Skeletal and bone marrow Edentulism, large forehead, SRC1 Ocular hypotelorism, Deep set eyes and wide nostrils, Large spleen, myelofibrosis Myelofibrosis Grey Platelet Syndrome Leukaemia RUNX1, ETV6, GATA1 Pulmonary cardiac Fibrosis Hermansky-Pudlak Syndrome Heart defect Jacobson/Paris-Trousseau DiGeorge/Velocardialfacial syndrome Septal defect Thrombocytopenia Absent Radius Renal Haematuria-Proteinuria MYH9-Related disease Renal tubular acidosis Arthrogryposis renal dysfunction cholestasis syndrome Congenital malformations Thrombocytopenia Absent Radius Gastrointestinal Milk-protein allergy Thrombocytopenia Absent Radius Cholestasis Arthrogryposis renal dysfunction cholestasis syndrome Granulomatosis colitis Hermansky-Pudlak Syndrome Auditory High-frequency sensory loss MYH9-Related disease DIAPH1 Eyes Cataract MYH9-Related disease Albinism Hermansky-Pudlak Syndrome Chediak-Higashi Neurological Mental retardation Paris-Trousseau Ataxia, intellectual disability Chediak-Higashi Periventricular nodular heterotopia FLNA-associated macrothrombocytopenia Epilepsy Homozygous Pelger-Huet Cognitive impairment DiGeorge/Velocardialfacial syndrome Developmental delays Homozygous Pelger-Huet Immunological Autoimmunity Wiskott-Alrich Chediak-Higashi Grey Platelet syndrome Immunodeficiency Wiskott-Alrich Chediak-Higashi Endocrine and metabolic Hyperphytosterolaemia Mediterranean macrothrombocytosis Hypercholesterolaemia Mediterranean macrothrombocytosis Parathyroid abnormalities DiGeorge/Velocardialfacial syndrome Thymus abnormalities DiGeorge/Velocardialfacial syndrome Palla et al (2015);, Lentaigne et al (2016); Bariana et al (2017);, Nurden et al (2012);, Harrison et al (2011). ª 2017 John Wiley & Sons Ltd 369

8 Table III. Differentiation between coagulation factor defects and platelet disorders based on history. Findings Coagulation factor defect not been proven. Functional assays of this protein are of little value as there is no lower limit of normal. Tests evaluating global haemostatic capacity such as the thrombin generation test and thromboelastography (TEG/ ROTEM) may provide additional insights into in vivo haemostasis. These have the advantage of testing coagulation in a more physiological whole blood setting, but abnormalities are generally not specific for a particular disorder. These may be used to predict and assess response to treatment and might better predict clinical phenotype in unusual cases as they more effectively assess the rate of and total thrombin generation, whole blood clot formation, and/or fibrin polymerization (Al Dieri et al, 2002; Van Geffen et al, 2012). While TEG/ROTEM are now widely used in clinical practice, significant steps need to be taken in standardization of preanalytical and analytical variables to make thrombin generation measurements more reproducible. Platelet function tests Von Willebrand Disease/Platelet disorder Onset of bleeding Delayed after trauma Spontaneous or immediately after trauma Mucosal bleeding Rare Common Petechiae Rare Characteristic Ecchymoses Large and solitary Small and multiple Haemarthrosis Characteristic Rare Bleeding small cut Minimal Persistent Gender 80-90% Male Equal Palla et al (2015). Light transmission aggregometry (LTA) was first utilised in the early 1960s and remains the gold standard for platelet function testing. Despite being in clinical use for five decades the test is poorly standardized. As a result, there is wide variation in laboratory practice. Guidelines for the methodology of LTA have been published (Harrison et al, 2011). LTA should be repeated on at least one occasion if there is an abnormality to show reproducibility before it can be considered diagnostic of a platelet dysfunction. Because of the technical complexity of LTA and the fact that it requires skilled biomedical scientists, the Platelet Function Analyser (PFA)-100 has been proposed as an initial screening test. This has good sensitivity for type 2 and 3 VWD and severe platelet dysfunction (Favaloro, 2008; Gresele, 2015). However, it has low sensitivity for type 1 VWD and mild platelet dysfunction. A normal test, therefore, cannot be used to exclude a bleeding disorder or the need for further tests. Similarly, other global tests of platelet-dependent haemostasis, such as the Impact-R cone and plate analyser, have insufficient negative predictive value to reduce the need for LTA (Shenkman et al, 2008). In addition, renal function and urea should be measured, as platelet function can be impaired in a high uraemic state. Moreover, the MYH9-associated platelet function disorders are associated with renal impairment. The ISTH guidelines on the diagnosis of inherited platelet function disorders recommend that in addition to LTA there should be an assessment of ATP/ADP release from platelet granules and an assay of at least one marker of granule content (Gresele, 2015). The options for nucleotide release are lumi-aggregometry, which simultaneously measures LTA and ATP secretion or high performance liquid chromatography/ luminometry. To measure granule content, the supernatant of LTA samples or activated platelets can be used to measure alpha-granule proteins by flow cytometry or enzyme-linked immunosorbent assay. This leads to a potential diagnostic yield of approximately 40% for the main platelet disorders including Glanzmann Thrombasthenia, Bernard Soulier Syndrome, Wiscott-Aldrich Syndrome, Primary Secretion Defects and Leucocyte Adhesion Deficiency III. Flow cytometry is also recommended as a first line test and can be used to investigate abnormalities in the fibrinogen (Glycoprotein [GP]IIb/IIIa), VWF (GPIb/IX/V), collagen (GPVI and GPIa/ IIa) and thrombin (protease activated receptor 1, PAR-1) receptors. In the remaining 60% of suspected inherited platelet function defects, first line testing will not reach a definitive diagnosis and further investigation is required. This includes extended LTA using an additional panel of platelet agonists, extended flow cytometry and assays of granule content, such as direct measurement of platelet nucleotides in a platelet lysis assay or ATP release following agonist stimulation. The latter has the advantage of also assessing signalling pathways. In addition, impaired clot retraction aids the diagnosis of Glanzmann Thrombasthenia or Stormorken Syndrome. The supernatant of non-anticoagulated whole blood can be used for thromboxane B2 assays to detect abnormalities of arachidonic acid release or metabolism. Although transmission electron microscopy has, for many years, been the gold standard for counting platelet alpha or dense granule defects, it has limited availability. Recent advances in super high resolution light microscopy suggest that this may provide greater diagnostic potential and may be a more clinically useful imaging solution for structural platelet abnormalities (Westmoreland, 2016). Finally, if enhanced ristocetin-induced platelet aggregation is detected, mixing tests, using a combination of LTA and flow cytometry, should be performed to differentiate the plasmatic (VWD type 2B) from the platelet (platelet type VWD) causes of ristocetin hyperreactivity (Gresele, 2015). These second line tests have the potential to reach a definitive diagnosis in a further 7% of cases, and will suggest a probable diagnosis of a number of other platelet disorders, which need to be confirmed by third line tests. 370 ª 2017 John Wiley & Sons Ltd

9 For the remaining unexplained platelet disorders, where a more definitive diagnosis is still required, the recommended approaches to third line testing are a range of biochemical studies and receptor binding assays. These include Western blot analysis for surface glycoproteins and spreading assays, MYH10 detection and protein phosphorylation (Gresele, 2015). Flow cytometry may also be used to assess protein phosphorylation. Adhesion and thrombus formation should be assessed under flow conditions. Additional studies may be warranted to evaluate second messengers (Ca 2+, camp, inositol trisphosphate) and receptor-binding studies may also help reach a diagnosis. This broad range of tests will usually require the coordination of multiple specialist laboratories. It is at the stage of third line testing for inherited platelet disorders that molecular genetic testing has previously been recommended. Genetic platforms and relevance for clinical medicine Three major genetic platforms have emerged in the last two decades: whole genome expression arrays, single nucleotide variant (SNV) chips for genome wide association studies (GWAS) and next generation sequencing (NGS). Through whole genome expression arrays, the transcriptomes have been generated, mainly facilitating gene expression mapping (Watkins et al, 2009). However, investigators turned quickly to association studies with clinical or intermediate outcomes because it has proven to be quite laborious to obtain platelets and megakaryocytes for gene expression studies. The rationale behind GWAS is the common disease, common variant hypothesis in which a limited number of genetic variants with a high frequency (typically above 5%) in the general population contribute to susceptibility for disease (Manolio et al, 2009). In general, these high frequency variants have a small effect size on the phenotype. The small effect sizes mean that large sample sizes are needed to identify novel variants. The most recent GWAS has discovered 2796 variants contributing to blood phenotypes in 36 blood cell indices of participants (Astle et al, 2016). These are scientifically relevant but not for clinical purposes because they primarily associate regions with platelet counts and volume. Platelet count is not always a predictor of bleeding, although there seems to be a relationship between volume and function suggesting that the total platelet mass may be more useful. Even though increasingly high effect variants are found, the majority still have small effects. Studies of the regions identified will quite possibly lead to the discovery of new mechanisms in platelet biology. It is the third of the listed genetic platforms, NGS, which has the most relevance for clinical diagnosis. Due to technical advancement it is now possible to sequence the coding regions of the genome (the exome) and/or pre-selected regions of the genome (targeted sequencing) at a low cost. The price of whole genome sequencing (WGS) has also dropped drastically to per genome. The application of NGS in the investigation of bleeding disorders has led to multiple gene discoveries such as NBEAL2, RBM8A, ACTN1, SRC and DIAPH1 (Gunay-Aygun et al, 2011; Lentaigne et al, 2016; Stritt et al, 2016; Turro et al, 2016; Bariana et al, 2017) and expansion of the distinct genes responsible for previously characterised disorders, such as Hermansky-Pudlak syndrome. Genes discovered through application of NGS are marked by an asterisk in Table I. Benefits of (next generation) genetic testing Reaching a definitive molecular diagnosis is important for a variety of reasons. Without genetic testing it can be challenging to establish the exact cause of a bleeding or platelet disorder because multiple different genetic variants can present with a very similar phenotype. It enables the clinician to tailor clinical management and discuss the prognosis with the patient. An understanding of the inheritance enables screening of family members, including those who are unchallenged and, therefore currently unaffected, and can help inform patients in family planning discussions. It can also inform us which patients need non-haematological investigations. For example, monitoring for lung disease is required in type 1 Hermansky-Pudlak syndrome due to abnormalities in HPS1 and HPS4, but not in the milder forms of this disorder. Similarly, the MYH9-related platelet function disorders carry a risk of subsequent nephritis even if the initial presentation seems to be limited to platelet abnormalities. Another example is RUNX1 variants, which not only cause mild platelet function disorders, but also carry a lifelong increased risk for acute myeloid leukaemia (Lentaigne et al, 2016). In the past obtaining a genetic diagnosis was often challenging. It was not always clear which genetic tests were available and testing was based on Sanger sequencing, which is costly and time consuming. As a result of this, genes were tested sequentially rather than in parallel, meaning that it could take several years to reach a diagnosis. These issues have been overcome with the advent of targeted sequencing platforms. The relative ease of these platforms means one could argue that after the initial work up with FBC, blood film, coagulation screen, coagulation factor levels and initial platelet function testing, the next diagnostic step should be genetic testing. The traditional stepwise testing approach requires multiple hospital visits to discuss test results and additional investigations. The cost of these interventions, which may require the patient to attend multiple specialist laboratories, and the consequent delay in diagnosis are compelling reasons for introducing genetic testing much earlier in the diagnostic process. To our knowledge there are currently five next generation sequencing platforms designed for use with IBDs described in the literature. Two are platelet-specific, one is for coagulation factors only, one specifically for F8 and one platform ª 2017 John Wiley & Sons Ltd 371

10 covers genes associated with platelet disorders, coagulation factor deficiencies and thrombotic disorders. The UK-Genotyping And Phenotyping of Platelets project (UK-GAPP) has established a targeted panel of 216 genes with an established or hypothetical role in platelet disorders (Jones et al, 2012). This was initially tested in 10 participants with different platelet function disorders yielding 4500 potential SNVs in these candidate genes. Subsequently, a systematic filtering strategy, with the following prior assumptions that the pathogenic SNV in the study subject was: (i) within a candidate gene coding region or splice site, (ii) not a population variant identified in dbsnp 132 (the Single Nucleotide Polymorphism Database, a free public archive for genetic variation within and across different species), and (iii) identified with two different bioinformatics tools, made it possible to refine the initial yield to a shortlist of 10 potentially pathogenic SNVs. Leo et al (2015) took a different approach to the initial targeted sequencing approach and whole exome sequencing was performed on 18 unrelated cases. Subsequently, they focused their analysis on 329 genes regulating platelet function, number and size in order to identify candidate gene defects in patients with inherited platelet disorders. They also assessed the potential pathogenicity of candidate gene defects using computational predictive algorithms. Analysis of the 329 candidate platelet dysfunction genes identified 63 candidate defects, affecting 40 genes, among index cases with Gi-coupled protein signalling abnormalities, while 53 defects, within 49 genes, were identified among patients with secretion abnormalities. The advantage of a whole exome approach is that all genes expressed in human cells can be interrogated. The disadvantage is that deeper intronic regions and regulatory non-coding regions are not tested. A Spanish team generated a platform with 71 genes implicated in platelet disorders (Bastida et al, 2016a; Lozano et al, 2016). They applied this platform and a bioinformatics pipeline, to identify a novel variant in a case of Wiskott-Aldrich syndrome. They found a novel hemizygous 1 bp deletion in the WAS gene, c.802delc, leading to a frameshift and stop codon at amino acid 308 (p.arg268glyfs*40). Lozano et al (2016) applied the same panel of 71 genes, to identify a homozygous change (c.1142c>t) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin. This variant led to a p.ser381phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In another pedigree, whole exome sequencing identified a nonsense homozygous variation (c.337c>t) (p.arg113x) in exon 5 of RASGRP2. This group have also generated a 23-gene NGS panel for coagulation factors (Bastida et al, 2016b). A custom target enrichment library captured 31 coagulation genes. For all exons and flanking regions of these genes, probes were generated for 296 targets to cover 863 kb. A total of 20 patients with an inherited coagulation disorder were studied using NGS technology. In all of these patients, a causative genetic variant was identified. Twenty-one pathogenic variants were found, all of which were single nucleotide variations. Six novel variants affecting F8, F9, F10, F11 and VWF, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant (Bastida et al, 2016b). A team in Germany has developed a platform specifically to target the entire F8 gene to detect deep intronic variants that are typically missed by WES (Bach et al, 2016). Conventional techniques fail to identify a genetic cause in 4% of cases with haemophilia, suggesting that some may be due to deep intronic variants. In this study, 15 cases with proven haemophilia A without a known coding variant were sequenced. This revealed 23 deep intronic candidate variants in several F8 introns, including six recurrent variants and three variants that have been described before. One patient additionally showed a deletion of 92 kb in intron 1. Prediction of effect was done in silico. In each of these 15 individuals a likely pathogenic variant was found. These pathogenic variants co-segregated with the phenotype in pedigree studies. Finally, ThromboGenomics (TG) is a targeted NGS panel, which has been developed in Cambridge, UK (Simeoni et al, 2016; Version 3.0 of the platform contains 78 genes with a confirmed role in bleeding and thrombotic disorders including coagulation pathways and platelet disorders due to defects important for platelet formation, morphology or function. By selecting only genes with an established role in inherited coagulation, platelet and thrombotic disorders, clinical utility is enhanced. The technology allows the rapid addition of new genes, once they have been approved by the ISTH Scientific and Standardization Committee (SSC) for Genomics, as described below. To date, more than 1450 samples have been processed by the TG pipeline. A variant that would explain the phenotype has been reported in 60% of cases of suspected coagulation disorders and 52% of suspected platelet disorders. An initial clinical assessment and laboratory testing are still required to identify which patients are likely to have an inheritable disorder and to optimize application of Human Phenotype Ontology terms to enable correlation of variants with clearly defined phenotypes. A limitation of NGS platforms that must be taken into consideration, especially on receipt of a negative report, is that they are less suited to the detection of gross genetic abnormalities, such as large inversions. These abnormalities are not detected by Sanger sequencing either, and conventionally require tailored methods for the detection of specific abnormalities, such as the intron 1 and 22 inversions in F8. Novel private inversions remain a challenge and require techniques, such as Southern Blotting, which is becoming increasingly unavailable in clinical laboratories. Fortunately, these are extremely rare and gross abnormalities are mostly due to copy number variants (CNVs) resulting in deletion or duplication of one or more exons. Conventionally, these 372 ª 2017 John Wiley & Sons Ltd

11 have also required a separate technique, such as Multiplex Ligation-dependent Probe Amplification, but they can now be detected on the TG platform with equivalent efficacy. The genes in which pathogenic CNVs are known are marked with dagger symbol in Table I. CNVs are relatively common in the VWF gene. An example of this is deletion of exons 4 and 5 (c _ del [p.asp75_gly178del]), identified in patients with Type 1 and Type 3 VWD, in whom direct sequencing of the VWF gene had previously not identified causative variants (Sutherland et al, 2009). The TG platform compares relative read depth to identify large deletions and duplications and has, to date, identified 26 pathogenic CNVs, 14 of which are in VWF, including 6 cases with VWF exon 4 and 5 deletions, with additional cases of whole gene deletions, single exon deletions and one duplication. Another example is two suspected cases of Quebec platelet disorder from independent pedigrees that were both found on TG to have duplication of PLAU. At present, work is being done to optimise the TG pipeline for detection of smaller CNVs that are too small for the coverage depth calling methods but too large for the variant callers. In cases of suspected IBDs, where a variant is not identified on the TG platform, WGS, for example through the Genomes Project, is the appropriate and recommended next step to look for novel genetic causes. Dealing with incidental genetic findings and variants of uncertain significance The large increase in variant data produced by NGS platforms means that low frequency variants at loci not directly relevant to the phenotype under investigation will be uncovered. If these variants are not known to be connected with any phenotype then they are of no further concern. However, if they are associated with a clinical phenotype, particularly a severe one, then these incidental findings become pertinent and should be reported. Genomics England ( have determined which phenotypes are severe enough to require reporting as an incidental finding and the only bleeding disorder on this list is severe haemophilia. This is because the finding of such a variant in a female would effectively make her a carrier regardless of her own clinical phenotype and this has implications for any future male progeny. For NGS gene panels focusing on haemostasis genes this is currently the only pertinent incidental finding that needs reporting. All other bleeding disorders of similar severity have an autosomal recessive mode of inheritance and, therefore, the current status is that these do not need to be reported. As we move to WGS, the list of pertinent incidental findings that needs to be reported increases greatly, but would still only include variants that would be expected to result in an as yet unrecognised severe phenotype in the patient or offspring. For genes such as F8, F9, VWF and F7, multi-national, locus-specific databases have been publicly available online for several years ( Rallapalli et al, 2013). These, and some of the other coagulation factor genes, have been extensively studied and so most variants will have been previously described. For example, the F8 database ( contains 2015 unique variants garnered from 5,480 separate cases. These are examples of tier 1 genes in which variants have been proven to cause human disease, for example through biochemical studies or segregation with the phenotype in at least 4 independent pedigrees. Tier 2 contains genes where an association with a disorder has been hypothesized but further study is required to establish pathogenicity. Table I shows the ISTH SSC approved tier 1 coagulation and platelet disorder genes on the TG platform and a further seven tier 2 genes, which may be moved by the ISTH SSC to tier 1 after consideration of the available evidence (Matthijs et al, 2016). Unlike F8, the majority of coagulation factor genes have fewer than 50 unique variants described, and so many variants discovered through NGS will be novel. If there is good understanding of the structurefunction relationship in a gene, a variant might be assigned class 4 status, but caution should prevail in assigning pathogenicity and many will be class 3 (variant of uncertain significance or VUS). Feedback of a VUS to patients and clinicians without expertise in the specific genes is challenging. Greater certainty regarding a variant s pathogenicity may be achieved through testing of both phenotypically affected and unaffected relatives, allowing co-segregation based upon understanding of the mode of inheritance. Tools that pool available knowledge regarding the structure-function relationship of the variant and the gene in question can be sought through online resources such as ClinVar (Landrum et al, 2016), GeneCards ( and OMIM ( In some situations, this might enable predictions about the phenotypic consequences but otherwise there needs to be recognition that the relationship between the variant and phenotype is unclear and that further information might be available as reports of the variant are deposited in databases. Outlook for the future Although stepwise platelet function testing is costly, laborious and restricted to specialized laboratories, for the moment it remains essential for defining laboratory phenotypes that correlate with genetic variants. Strategies for reducing the requirement for specialized testing, such as using a BAT to screen out individuals with a low probability of having an IBD or pre-testing with the PFA-100, have not been particularly successful as they lack sensitivity for the more common mild IBDs. As the database of variants associated with specific phenotypic traits increases, the value of poorly standardized laboratory tests may diminish. This requires engagement of the clinical community in identifying the genetic cause of all ª 2017 John Wiley & Sons Ltd 373

12 cases of inheritable bleeding disorders, including those in which the diagnosis is clear from other laboratory tests. Moreover, the turnaround time for sequencing and most importantly analysis needs to be reduced to compete with more traditional tests. However, one should bear in mind that to complete all steps of a traditional investigation can take several years. NGS can provide us with a wealth of information on variants either in targeted genes or spread throughout the genome in a timeframe of 2 weeks to 4 months. Despite the benefits of targeted NGS platforms in the investigation of IBDs, we expect that with the cost of WGS dropping significantly, the role of targeted sequencing or exome sequencing will reduce in the next 5 years. In parallel, ongoing bio-banking efforts of peripheral blood samples and analysis of genome-wide markers will allow linkage of genetic loci to specific phenotypic traits. When a clinical question, such as the cause of abnormal bleeding, arises, it is expected that a bioinformatics filter will analyse the genes related to IBDs for pathological variants. In order to make sense of the vast amount of data generated by NGS, and particularly WGS, in a manner that is clinically useful, a multi-disciplinary approach is essential, involving clinicians, bioinformaticians and genetic scientists. The need for clinicians to develop a deeper understanding of the genetic basis of bleeding disorders will be paramount for incorporating genetic testing into the routine management of patients. Acknowledgement JC is funded by the British Heart Foundation at the time of writing. Author contributions All authors reviewed the relevant literature on this topic. SS and JC drafted the paper. KG critically reviewed the paper. Conflict of interest disclosure SS is a part of the ThromboGenomics Clinical Care and Curating team. 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