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Inherited thrombocytopenias
other predicted pathological variants have little effect on platelet count or size. Interestingly, TUBB1 dysfunction may lead to genome instability; co-segregation with myeloid malignancy has been reported, but this tendency needs confirmation as TUBB1 mutations are relatively common. Thyroid dysgenesis has also been linked to TUBB1 mutations in some patients. Variants identified by WES in exons 5 and 6 of ACTB encoding b-cytoplasmic actin cause syndromic thrombocytopenia by compromis- ing microtubule organization during the final stages of megakaryocyte maturation, but bleeding has not been reported.77 The defect has been located in five families and is accompanied by platelet size variability including large forms; associated and highly variable clinical features are given in Online Supplementary Table S1. The defect inter- venes in the final stages of platelet biogenesis and pre- platelet (a purported intermediate platelet precursor) frag- mentation but no defects were seen in megakaryocyte proplatelet formation in culture.77 Interestingly, variants in exons 2 to 4 give rise to a severe developmental disorder, Baraitser-Winter cerebrofrontofacial syndrome, but no thrombocytopenia.77
stituents. Glanzmann thrombasthenia is the classic disor- der of platelet function; with AR inheritance; bleeding occurs because thrombus formation and platelet aggrega- tion fail in the absence or non-functioning of integrin aIIbb3. Caused by AR mutations in ITGA2B and ITGB3, the platelet count is normal in the absence of major bleed- ing.82 However, rare gain-of-function mutations affecting the structure of aIIb or b3 can give rise to macrothrombo- cytopenia. Our group made the initial report in 1998, describing a single allele p.R995Q mutation in the aIIb cytoplasmic domain; this was later shown to be associated with a null allele.83 Platelet aggregation was much decreased with surface aIIbb3 at 18%, the macrothrombo- cytopenia was modest and bleeding infrequent. The affect- ed residue forms an intracellular salt bridge with D723 of b3, itself mutated in a variant of Glanzmann thrombasthe- nia with macrothrombocytopenia (detailed in Nurden et al.82). Breaking this bond leads to conformational changes that are transmitted to the extracellular domains with a “partial” activation of aIIbb3. Since these reports, macrothrombocytopenia with AD inheritance has been shown for other intracytoplasmic or membrane proximal gain-of-function variants affecting aIIbb3.84,85 One hypoth- esis is that the partially activated integrin on megakary- ocytes modifies megakaryocyte contact with extracellular matrix proteins in the marrow. This may promote cytoskeletal re-organization with proplatelets having fewer branches and enlarged tips. Interestingly, a gain-of- function mutation affecting aIIbb3 had a dominant pheno- typic effect with respect to a loss-of-function variant.86
Next-generation sequencing is expanding the landscape of inherited thrombocytopenias
Careful phenotyping and the use of NGS have led to the identification of a number of new genes responsible for inherited thrombocytopenia with or without large platelets or accompanying platelet function defects. Some have been discussed in preceding sections, others, confined to a limited number of families or of unusual phenotype and with links to signaling pathways, are presented below. All of the upwards of 40 genes now recognized as causative of inherited thrombocytopenia are included in Online Supplementary Table S1.
Microthrombocytopenia
The classic disorder with a low platelet count and small platelets is WAS, but microthrombocytopenia and a simi- lar phenotype including eczema, leukocytoclastic vasculi- tis, eosinophilia and elevated IgA and IgE levels was recently linked in two unrelated patients by WES to AR mutations in ARPC1B, a gene that encodes a subunit of the human actin-related protein 2/3 complex (Arp2/3).87 In fact, Arp2/3 acts with WAS protein in regulating the branching of actin filaments and plays a central role in cell migration, vesicular trafficking and cytokinesis. Platelets from the patients showed defective spreading. Loss of Arp2/3 function is syndromic in that it predisposed to inflammatory disease althoug many of the immunodefi- ciencies characteristic of WAS have so far not been observed. Microthrombocytopenia was also linked by NGS in two consanguineous families to a loss-of-function homozygous AR mutation in FYB, encoding the cytosolic
Single allele missense mutations in ACTN1 encoding a- actinin were first identified in Japanese patients with moderate thrombocytopenia and in a large French pedi- gree by WES and by genome-wide linkage analysis.78,79 Their presence was later revealed as quite common in a non-syndromic form of thrombocytopenia with mild or no bleeding although blood loss can be a problem after surgery. a-Actinin is a dimeric protein that cross-links actin filaments into bundles; mutations affecting function- al domains act in a dominant-negative manner to disrupt actin filament organization and proplatelet structure in megakaryocytes. Platelet size changes are variable between patients. It is a quite common form of mild inher- ited thrombocytopenia.78-80
A final cytoskeletal gene identified by WES in two human pedigrees with macrothrombocytopenia is TPM4 encoding tropomyosin 4, another actin-binding protein.81 A premature stop codon on a single allele segregated with platelet size increases and a low platelet count. Gene iden- tification was helped by phenotype comparison with a mouse conditional knockout model and confirmed by short hairpin RNA knockdown of TPM4 in normal human megakaryocytes. Platelet function was affected although bleeding was mild. Megakaryocytes in culture produced abundant proplatelets but with fewer branches and enlarged tips, the latter being a standard finding in patients with defects of genes encoding for cytoskeletal proteins, which results in a dynamic re-organization of the cytoskeleton.
Quite remarkably, all of the eight genes encoding cytoskeletal proteins and which give rise to macrothrom- bocytopenia do so with AD inheritance (Online Supplementary Table S1).
Abnormal integrin interactions with extracellular matrix proteins
The final steps of megakaryocyte maturation and the migration of these cells towards the vascular sinus depend not only on cytoskeletal proteins, but also on interactions between surface receptors and extracellular matrix con-
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