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A.T. Nurden and P. Nurden
features; not all patients develop hematologic abnormali- ties while macrothrombocytopenia can also be isolated. Studies in mice show knock-on effects with sterol interca- lation within the demarcation membrane system of megakaryocytes leading to release of hyper-reactive enlarged platelets with deregulation of multiple signaling pathways including GPIba shedding.114
Diagnosis
The continuing difficulties in diagnosis of inherited thrombocytopenias should not be underestimated. While classic disorders such as BSS, MYH9-related disease and WAS are now relatively straightforward to identify, the majority are not and in the absence of a well-defined fam- ily history of bleeding, new cases can still be confused with immune thrombocytopenic purpura. Diagnostic algorithms with steps based on clinical data and laborato- ry testing aid the work-up and management of patients.115- 118 The widespread use of whole blood electronic counters to evaluate platelet count and size has helped, while assessing platelet function, distinguishing between syn- dromic or non-syndromic forms, identifying associated hematologic or marrow defects are important parts of any strategy. A successful diagnosis avoids unnecessary splenectomy and/or the use of drugs given for immune thrombocytopenic purpura. It helps to predict disease evo- lution, and is indispensable for selecting patients for HSC transplantation or gene therapy. Noteworthy advances have been the application of Human Phenotype Ontology terms and the identification of phenotype clusters of asso- ciated pathologies as an aid to establishing phenotype/genotype relationships.119 Nonetheless prepar- ing an extensive Human Phenotype Ontology listing is not always compatible with day-to-day hospital practice. Classic tests for evaluating platelet function, such as aggre- gometry (Born platelet aggregometer, impedance aggre- gometry or other methods), secretion assays, flow cytom- etry, western blotting and electron microscopy, are time consuming, expensive to perform and require trained per- sonnel. Critically, many such tests are difficult or impossi- ble to perform when the platelet count is low. Cytochemical or immunofluorescence staining of blood smears is a recognized first step for detecting enlarged platelets, and is particularly useful in third-world coun- tries.32,117 The evaluation of platelet spreading on single protein substrates provides more information.120 The use of computer-based high-throughput testing of platelet function and thrombus formation under flow on extracel- lular matrix proteins on microchips is full of promise but has been little tested for thrombocytopenias.121
As highlighted throughout our review, applying NGS to inherited thrombocytopenias is greatly expanding the list of causative genes as well as increasing the number of variants implicated in the classic diseases. The initial suc- cess of WES in genotyping gray platelet syndrome and the TAR syndromes led to the BRIDGE-Bleeding and Platelet Disorders (BRIDGE-BPD) project, orchestrated by Professor W. Ouwehand, which combines the unique sequencing and bioinformatics resources of the Sanger Institute in Cambridge (UK).122 The Genotyping and Platelet Phenotyping (GAPP) consortium led by Professor S. Watson in Birmingham, UK has been another major player.123 As the number of causal genes for all forms of
inherited platelet disorders increased, BRIDGE-BPD and
GAPP both put together platforms to test patients for
potentially pathogenic variants against previously identi-
fied target genes concentrating on exomes, untranslated
regions and selected intronic regions.124-127 Indeed, GAPP
designed a gene panel specific for inherited thrombocy-
topenias.127 In contrast, the ThromboGenomics
Consortium (Department of Haematology, University of
Cambridge, UK) included a limited number of genes
causal for other blood and thrombotic disorders; the panel
was regularly updated and a large cohort of 2,396 patients
screened.125,126 The use of NGS and high-throughput proce-
dures for diagnosing platelet disorders including thrombo-
cytopenias has quickly expanded worldwide as is illustrat-
ed by reports from Italy, Japan, Spain, France, Holland and Scandinavia as well as North America.46,60,77,78,80,87,108,113,128, 129
The question now is not whether to apply NGS proce- dures in the mainstream of diagnosis but when and how.128 Certainly, a strong argument can now be made to use them upfront: early identification of a causal mutation in a known gene will avoid much unnecessary biological characterization, as we have stated recently in this journal.130 It is certainly necessary if prenatal diagnosis or HSC transplantation is on the agenda. Nonetheless, upwards of 200 potential gene variants can be located for each patient, so prioritizing and filtering the variants is key.131 Variant selection includes the exclusion of synony- mous variants and those with a minor allele frequency >0.01 in the normal population. Therefore, in the absence of a previously validated mutation in a known causal gene for inherited thrombocytopenia much care must be taken. Only for a limited number of cases can linkage in large families be performed. Data evaluation in terms of quality control, depth of coverage of each gene, the conservation of affected nucleotides or amino acids, the choice of in sil- ico pathogenicity prediction software, and the classifica- tion of variants according to appropriate genetic guide- lines have been nicely reviewed elsewhere.125,131
In taking account of these advances, the fundamentals for a systematic approach to the diagnosis and manage- ment of inherited thrombocytopenia remain largely those expertly outlined in a recent European Hematology Association consensus report on mild and moderate bleeding disorders, to which the reader is directed.132 In the context of immune thrombocytopenia, a systematic approach includes careful primary tier assessment of the basic clinic-pathological information for each new patient prior to next stage investigations and, if needed, referral to a specialist center. The recommendations in this European Hematology Association report are very useful in emer- gency situations and for prevention or control of sponta- neous bleeding, such as epistaxis or gum bleeding and at- risk situations, including dental and surgical procedures or traumatic events when immediate practical decisions must be made.
Treatment
As with all inherited platelet disorders, spontaneous bleeding includes epistaxis, gum bleeding, easy bruising and petechiae while gastrointestinal hemorrhage occurs in some severe cases; bleeding may be prolonged after cuts, trauma, or surgery, while for women, menorrhagia and childbirth are added risks.133,134 For example, in a European
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haematologica | 2020; 105(8)