Platelets
ISSN: 0953-7104 (Print) 1369-1635 (Online) Journal homepage: http://www.tandfonline.com/loi/iplt20
ISTH Advanced Training Course on platelet bleeding disorders: How should they be investigated? Steve P. Watson, Martina E. Daly, Paul Harrison, Gillian C. Lowe, Andrew Paterson, Jose Rivera, Tim D. Warner & Neil V. Morgan To cite this article: Steve P. Watson, Martina E. Daly, Paul Harrison, Gillian C. Lowe, Andrew Paterson, Jose Rivera, Tim D. Warner & Neil V. Morgan (2016) ISTH Advanced Training Course on platelet bleeding disorders: How should they be investigated?, Platelets, 27:8, 719-721, DOI: 10.1080/09537104.2016.1256726 To link to this article: http://dx.doi.org/10.1080/09537104.2016.1256726
Published online: 14 Nov 2016.
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Date: 14 December 2016, At: 11:24
http://www.tandfonline.com/iplt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, 2016; 27(8): 719–721 © 2016 Taylor & Francis Group, LLC. DOI: 10.1080/09537104.2016.1256726
MEETING REPORT
ISTH Advanced Training Course on platelet bleeding disorders: How should they be investigated? St. Anne’s College, Oxford, 6–9th September 2016 Steve P. Watson 1, Martina E. Daly2, Paul Harrison3, Gillian C. Lowe1,4, Andrew Paterson5, Jose Rivera6, Tim D. Warner7, & Neil V. Morgan1 1
Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, 2Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, UK, 3Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK, 4Haemophilia Comprehensive Care Centre, University Hospital Birmingham, Edgbaston, Birmingham, UK, 5Program in Genetics & Genomic Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada, 6Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia 30003, Spain, and 7The William Harvey Research Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University of London, London, UK
Is there a need for an Advanced Training Course? The International Society of Thrombosis and Haemostasis (ISTH) currently organizes an annual Scientific Subcommittee Conference (SSC) and a biannual Congress (preceded by the SSC) which will become an annual event beginning in 2019. A standout feature of these meetings is the world-leading science that brings together basic and clinical researchers in the field of haemostasis and thrombosis. To complement these two meetings, the ISTH has introduced a series of Advanced Training Courses (which are perhaps more appropriately termed ‘Workshops’) held across the globe on focused themes such as thrombosis, bleeding, and vascular biology in a ‘Gordonstyle’ format that allows participants and presenters to interact in a relaxed and informal atmosphere. The courses are targeted at clinical and basic researchers. With the ever increasing number of meetings available, the question must be asked as to the role of these courses and whether there is space in the already over-crowded meetings calendar. The answer is an overwhelming Yes, as they allow cutting edge discussion and consideration of future developments on a focused theme by world-leading, experienced and new researchers without the need for, or emphasis on, unpublished data. In this way, the theme of the course can readily range from historical landmarks to current issues and future developments. The informal atmosphere allows discussion throughout the day and into the evening in order to stimulate interactions between experienced and new researchers alike. An Oxford College (in this case St. Anne’s) was a perfect venue for such a meeting, especially when blessed with warm late summer sun, the occasional glass of wine, and the sole use of the venue.
Course summary The topics of the meeting were platelet-based bleeding disorders (focusing on clinical diagnosis), platelet function testing, next
Correspondence: Steve P. Watson, Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK. E-mail:
[email protected]
generation sequencing (NGS) and induced pluripotent stem (iPS) cell technology. The meeting was structured as a combination of lectures, case studies, meet-the-expert sessions, and poster sessions led by internationally recognized leaders from the UK, Netherlands, Canada, and Australia. The meeting began with Steve Watson (Birmingham) stating that there are three areas of uncertainty: (i) clinical diagnosis of platelet bleeding disorders; (ii) how to test for a platelet disorder; (iii) how to interpret next generation sequencing (NGS) data for platelet-based bleeding disorders. Watson went on further to say that for a single affected individual in a family (with no other affected family members), we have no current way of determining the genetic basis of their bleeding disorder with certainty due to the presence of a several inherited heterozygous, pathogenic single nucleotide variants (SNVs), possibly in combination with acquired defects. These challenges were further explored by a highly informative overview of clinical diagnosis by Mike Makris (Sheffield) in which he described two contrasting patients with Glanzmann’s thrombasthaenia; one patient required 24 units of blood at the time of first menstruation, experienced life-long excessive bleeding and bruising, and died in her early thirties. The second, considerably older patient, successfully carried three pregnancies with only one requiring a blood transfusion, and remained relatively symptom free throughout her life. Such a wide variation in phenotype of a single monogenic disorder emphasizes the need for caution in interpretation of genetic data in isolation of clinical and other phenotypic information. With this in mind, Makris reviewed the history of the bleeding assessment tools (BATs) and the important role that they now play in aiding clinical diagnosis of bleeding. The discussion on platelet function testing was led by Tim Warner (Queen Mary’s, University of London) who began with Born aggregometry, stating that this test would always be used because of its historical importance and familiarity in clinical testing laboratories. He then went on to describe a 96 well platelet aggregation assay and
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its potential for automation. The advantages of the 96 plate assay are several-fold and include cost, reduced blood volume, almost ‘bedside’ testing platform, and automation, as samples can be easily tested locally to generate standardized data suitable for both immediate and grouped analyses. Given that all of the current platelet function tests give different results on the same blood samples ([1]), such a simple, robust assay is one way to standardize results between centers, and thereby allow meaningful comparison, subject to clinical validation. Paul Harrison (Birmingham) gave an overview of flow cytometry and other techniques, with flow cytometry as the method of choice for certain conditions associated with glycoprotein receptor defects (e.g. Glanzmann’s Thrombasthenia, Bernard Soulier syndrome), or for patients with moderate to severe inherited thrombocytopenia. Martina Daly (Sheffield) later gave an overview of testing for platelet secretion disorders and the nature of such disorders, emphasising that such tests can be readily introduced into aggregometry by use of luciferase (lumiaggregometry) or 96 well plate assays. Johan Heemskerk (Maastricht) extended the discussion of functional assays to the use of a portable microfluidic assay over multiple surfaces, with the potential for automation and advantage of small volumes and whole blood analysis. While each of these assays has a place, the varying results, uncertainty over interpretation, and overlap with controls means that only severe and syndromic (i.e., presence of associated distinguishing features) disorders can be unequivocally identified on the basis of functional testing. In most cases, the functional assays should be seen as a guide to clinical diagnosis and to inform genetic testing. Martina Daly (Sheffield, UK) summarized the DNA-based approaches that have been used to investigate patients with platelet bleeding disorders, and the challenges of genetic testing in these individuals. Neil Morgan (Birmingham, UK), David Rabbolini (Sydney, Australia), Suthesh Sivapalaratnam (Barts, London, UK), and Jose Rivera (Murcia, Spain) discussed the use of Sanger and NGS in the assessment of patients with suspected platelet disorders. Morgan leads the UK Genotyping and Phenotyping of Platelets (GAPP) study which has recruited almost 1000 participants over 10 years with clinically-diagnosed platelet-based bleeding disorders of unknown aetiology, all of which have been subject to aggregometry measurements or, in the case of patients with low platelet count, platelet function testing (integrin αIIbβ3 activation and P-selectin expression) by flow cytometry [2]. The phenotypic information is used to stratify patients for genetic testing through targeted genes (Sanger sequencing) and NGS. Morgan illustrated how this approach led to the identification of a new gene, SLFN14 (encoding Schlafen-14), for inherited thrombocytopenia, with three different mutations (which were sequential) in three unrelated families [3]. Morgan also described the high number of mutations in the transcription factor RUNX1 in the GAPP cohort [4], despite the absence of a family history of leukaemia in patients enrolled. The work identifies mutations in RUNX1 as one of the major causes of mild thrombocytopenia and shows that they are not necessarily associated with leukaemia [4]. Sivapalaratnam described the BRIDGE study which uses NGS to characterise patients with all types of bleeding disorders, with the power of analysis coming from the study of large cohorts. The approach was illustrated by a single family with a novel activating mutation in SRC [5] and by the clustering of similar phenotypes in patients with a mutation in GP1BA (the GPIbα subunit of the GPIb-IX-V complex) that gives rise to Bernard Soulier syndrome. Rivera used a panel of 71 genes in the study of clinically diagnosed and undiagnosed platelet disorders. Remarkably, despite a prior clinical diagnosis of Glanzmann’s thrombasthenia, he identified four unrelated families in Spain and Portugal with function disrupting mutations in RASGRP2, which encodes the nucleotide exchange factor CalDAG-GEFI, thus adding to the only previous known family described with inherited bleeding due to loss of CalDAG function [6]. A similar approach was used by Rabbolini
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using a smaller gene panel leading to identification of additional mutations in the transcription factor GFI1B, as well as a novel homozygous variant of FLI1 [7] (whereas all other previously reported patients had heterozygous FLI1 mutations). While all four of these talks illustrate the power of genetic sequencing, the above examples are largely in known genes or in families with clear patterns of dominant inheritance, although in the case of SLFN14 (Schlafen-14), this led to the identification of the genetic cause in a single individual without knowledge of other affected family members [3]. The value of family studies in mapping the cause of bleeding was beautifully illustrated by Andrew Paterson (Toronto, Canada) who provided a highly informative and entertaining insight into the strengths and limitations of genetics, and gave us all food for thought by proposing the investigation of ‘superfamilies’ who share significant regions of genetic information due to cryptic relatedness. The significance of genetic testing was illustrated in a series of case studies presented by Gillian Lowe (Birmingham) including a postnatal identification of a mutation in RUNX1 following a bereavement and the significance of these findings to the family. Anne Goodeve (Sheffield) illustrated the clinical laboratory approach to the identification of platelet function disrupting genes and the use of NGS for a targeted gene panel in disorders implicated in haemostasis. Will Lester (Birmingham) discussed the clinical diagnosis of von Willebrand’s disease (VWD) which shares many of the features of a platelet disorder. This is particularly true for type 1 VWD which is characterised by reduced levels of otherwise normal VWF, and which shows large variation in levels between family members, and overlap with levels in controls. While the frequency of VWD is reported to be as high as 1:100 in some populations, the discussion indicated that the true frequency is probably closer to that of a platelet disorder, namely in the order of 1:10,000. The importance of correct diagnosis was illustrated by a large family in which several members had been diagnosed with VWD, but were subsequently found to have a heterozygous defect in the P2Y12 ADP receptor (P2RY12) that disrupted function and segregated with a bleeding tendency independent of the VWD-associated VWF defect [8]. Indeed, it would seem that the bleeding in this family is largely due to the dominant inheritance of the P2Y12 defect. While P2Y12 deficiency is usually recessively inherited, an ever increasing number of cases indicate that it can also be inherited as a dominant trait, and an association with bleeding, possibly by exerting a dominant-negative effect over the wild-type receptor or by combining with other function-disrupting mutations to increase overall bleeding risk. This further illustrates the challenge of linking genotype to phenotype without additional information and investigation. The need to confirm that mutations are indeed functionally disrupting by expressing in cell lines or in mouse platelets was discussed, although as illustrated by Watson, there are significant quantitative and qualitative differences between the function of protein families between mouse and human platelets, as well as significant sequence differences. The clinical challenge in treating women with platelet disorders and patients with VWD was eloquently illustrated by Paula James (Kingston, Canada) through description of several challenging cases. This was followed by a thoughtful presentation from Julie Vogt, a consultant clinical geneticist, on the issues raised by genetic testing in patients and in extended family members. The importance of talks such as these to the non-clinical members of the audience is in placing the work into perspective while at the same time such talks are highly appreciated by the clinical trainees. The meeting ended with discussion of iPS cell technology from two world leaders, Cedric Ghevaert (Cambridge, UK) and David Rabbolini. Ghevaert has significantly increased the yield of platelets in vitro from iPS cell-derived megakaryocytes and, although we are not there yet, the production of high yields of in vitro-generated platelets would seem to be around the corner. Rabbolini illustrated the use of patients’ iPS cells to explore the
DOI: 10.1080/09537104.2016.1256726
ISTH Advanced Training Course 2016 Meeting Report
mechanisms underlying thrombocytopenia and platelet dysfunction, focussing his talk on GFI1b. This approach speaks to a future when further insight into platelet disorders can be gleamed through studies on autologous iPS cell-derived megakaryocytes. The meeting ended with very positive summaries from Paterson, Sivapalaratnam and Rabbolini on the role of genetics in the assessment of clinically diagnosed patients but with the need for caution in relying solely on sequence information. Susie Shapiro (Oxford) brought the discussion to a close by illustrating the importance of a correct diagnosis to the patient in a manner that was understated but recognized by all.
With thanks to the ISTH
Feedback
Steve P. Watson
The feedback on the meeting was overwhelmingly positive with strong support for both repeating and extending the format to further topics. Everyone left the meeting with a much greater understanding of the challenges in the field and its future direction. The meeting emphasised a number of take home messages that will govern future work and serve to emphasise that a pathogenic single nucleotide variant (SNV) should be judged only as a risk factor for bleeding, and that its true significance can only be made based on clinical history and platelet function testing. Points to take away include:
References
● a monogenic disorder and its presenting phenotype can vary
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widely between families and individuals even in cases such as Glanzmann’s Thrombasthenia some platelet bleeding disorders (e.g. P2Y12 deficiency) can be inherited as either recessive or dominant traits there will never be a universal platelet function test, and resources should be put towards a near bedside test (e.g. 96 well plate) that can be authenticated, standardised and analysed by test centres extending families as much as possible, and linking cryptically related famileis are needed to gain a full picture but these studies will clearly have important ethical governance issues a genetic defect in isolation should be reviewed only as a risk factor for bleeding there will be a day when iPS cell technology will replace the generation of gene-mutated animals to study in vitro function and perhaps will pave the way for a new approach in the study of platelet disorders using autologous material the paucity of cases of platelet function disorders outside of those that give rise to very severe bleeding e.g. Glanzmann’s thrombasthenia justifies regular reporting. With this in mind, the journal Platelets will shortly introduce a new series of articles dedicated to the genetics of the more common causes of platelet disorders.
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The meeting was superbly organised through the ISTH. The involvement of the ISTH in these Advanced Training Courses adds to their ever increasing contribution to understanding, research and disease management in thrombosis and haemostasis.
Declaration of Interest The authors report no conflicting interests.
ORCID http://orcid.org/0000-0002-7846-7423
1. Lordkipanidze M, Pharand C, Schampaert E, Turgeon J, Palisaitis DA, Diodati JG. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur. Heart J. 2007;28:1702–1708. 2. Watson SP, Lowe GC, Lordkipanidze M, Morgan NV. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 2013;11:351–363. 3. Fletcher SJ, Johnson B, Lowe GC, Bem D, Drake S, Lordkipanidzé M, Sánchez Guiú I, Dawood B, Rivera J, Simpson MA, et al. SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects. J Clin Invest 2015;125:3600–3605. 4. Stockley J, Morgan NV, Bem D, Lowe GC, Lordkipanidze M, Dawood B, Simpson MA, Macfarlane K, Horner K, Leo VC, et al. Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects. Blood 2013;122:4090–4093. 5. Turro E, Greene D, Wijgaerts A, Thys C, Lentaigne C, Bariana TK, Westbury SK, Kelly AM, Selleslag D, Stephens JC, et al. A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. Sci Transl Med 2016;8:328ra30. 6. Lozano ML, Cook A, Bastida JM, Paul DS, Iruin G, Cid AR, AdanPedroso R, Ramon Gonzalez-Porras J, Hernandez-Rivas JM, Fletcher SJ, et al. Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction. Blood 2016;128:1282–1289. 7. Stevenson WS, Rabbolini DJ, Beutler L, Chen Q, Gabrielli S, Mackay JP, Brighton TA, Ward CM, Morel-Kopp MC. ParisTrousseau thrombocytopenia is phenocopied by the autosomal recessive inheritance of a DNA-binding domain mutation in FLI1. Blood 2015;126:2027–2030. 8. Daly ME, Dawood BB, Lester WA, Peake IR, Rodeghiero F, Goodeve AC, Makris M, Wilde JT, Mumford AD, Watson SP, 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;113:4110–4113.
