Once considered rare, inherited platelet disorders are becoming more recognized as an important cause of bleeding in both children and adults – bleeding that ranges from mild to severe. Patients with platelet disorders may present with thrombocytopenia incidentally on a screening complete blood count or with bleeding manifestations consistent with platelet-type bleeding: mucocutanous bleeding, gastrointestinal bleeding, menorrhagia, and postsurgical or traumatic bleeding.
The diagnosis of more severe disorders is seldom in question; however, subtle platelet dysfunction may be much more difficult to diagnose, and congenital thrombocytopenia can be misdiagnosed as immune thrombocytopenia or missed entirely if the bleeding manifestations are mild. With the advent of next-generation/high-throughput DNA/RNA sequencing, the identification of novel genes clinically associated with platelet dysfunction and thrombocytopenia has become more common. Many new causes of familial thrombocytopenia and platelet dysfunction have been described in the past few years using these methods. This will only become even more common as these technologies begin to be used in clinical diagnosis. However, caution must be exercised to avoid calling variants that are not truly disease-causing ‘pathologic’ prematurely. This review will focus primarily on the most recently described platelet biology and how this has advanced our understanding of the inherited platelet disorders.
CLASSIFICATION OF INHERITED PLATELET DISORDERS
Over the years, many different authors have made attempts to categorize the inherited platelet disorders. The two main ways in which to do this are by clinical characteristics and by molecular pathways. Characterization on the basis of clinical characteristics is useful when evaluating the patients to guide diagnostic testing and allow more streamlined and consistent evaluation. Recently, guidelines were published for the evaluation of patients with suspected platelet function disorders . These guidelines provide a framework on which to build evaluations and allow consistent evaluation from center to center of all patients with suspected platelet disorders. What is important to note is that next generation sequencing (NGS) and molecular evaluations are not included early in the algorithm as polymorphisms, particularly novel polymorphisms affecting platelet function, may be difficult to sort from rare normal variants and from true clinical disease. Additionally, the most subtle platelet function disorders are likely to represent a spectrum of abnormal function and variable penetrance, with modifying genotype/phenotype characteristics resulting in a complex multifactorial picture that may present with clinical bleeding.
For understanding the mechanisms of platelet dysfunction and for fitting the newly described disorders into a framework, it is, however, easiest to categorize the platelet disorders by molecular mechanisms (Table 1), and for this reason, the present review has been organized in this manner. This combines thrombocytopenias and platelet function defects into the same framework.
Transcriptional regulation: GATA1, FLI1, RUNX1, ETV6, and GFI1B
Because the defects in transcriptional regulation were some of the first to be characterized, this review will not focus on these in detail and will mention them only in passing. Defects in GATA-binding factor-1, the X-linked transcription factor that controls both erythroid and megakaryocyte development, were the first mutations of transcriptional regulation described in humans. Patients with GATA1 mutations have X-linked macrothrombocytopenia associated with variable anemia ranging from dyserythropoiesis, anemia, β-thalassemia, or congenital erythropopoietic porphyria [2,3].
Paris–Trousseau (Jacobsen) syndrome is an autosomal dominant macrothrombocytopenia believed to result from loss of transcription factor function. In most patients, deletion of a portion of the distal portion of long arm of chromosome 11 (11q23.3–24) results in loss of the transcription factor Friend of Leukemia Integration (FLI1) locus . These patients have giant alpha granules in a proportion of their platelets (1–5%) and have a mild bleeding phenotype with mild platelet aggregation defects. Generally, patients with larger deletions have mental retardation, trigonocephaly, dysmorphic facies, congenital heart disease, and abnormalities of the urogenital and skeletal system .
Autosomal dominant mutations in Runt related transcription factor-1 (RUNX1) result in familial platelet disorder with predisposition to acute myeloid leukemia. In these families, there is an increased incidence of myeloid leukemia (up to 35%), as well as thrombocytopenia with normal platelet size, and platelet dysfunction with abnormal aggregation responses and abnormal secretion . Most patients present with mucocutaneous bleeding symptoms: easy bruising, epistaxis, and bleeding after minor surgery or dental challenges, but the penetrance is variable and unaffected family members carrying mutations in involved families are reported. The UK Genotyping and Phenotyping of Platelets (UK-GAPP) study – an ongoing study in the United Kingdom that is genotyping and phenotyping families with inherited platelet dysfunction – showed that heterozygous mutations in FLI1 and RUNX1 were more prevalent in patients with dense granule secretion defects and mild thrombocytopenia than previously suspected . In this study, Stockley et al. reported mutations in either RUNX1 or FLI1 in 13 patients from six families selected from a group of 56 patients with secretion defects and an affected relative. While this is a relatively high prevalence of transcription factor mutations in patients with relatively nonspecific findings of mild platelet dysfunction, 5/6 of the families also had mild thrombocytopenia, which may have increased the likelihood of finding a single molecular defect .
The most recently described transcription factor gene associated with platelet disorders is Ets variant 6 (ETV6). Mutations in ETV6 have been associated with autosomal dominant thrombocytopenia of varying degrees (67–132 000 ×109 platelets/l), erythrocyte macrocytosis, and various hematologic malignancies including B-cell acute lymphocytic leukemia, multiple myeloma, chronic myelomonocytic leukemia, T-cell/myeloid mixed phenotype leukemia, refractory anemia with excess blasts, and myelodysplastic syndrome [8,9]. Several family members were also reported to have skin cancer and colorectal cancer. Interestingly, two of the family members in one study (Zhang et al.) were initially diagnosed as immune thrombocytopenia (ITP), emphasizing the importance of suspecting alternate diagnoses in cases of ITP that do not respond typically to ITP therapy or have significantly more bleeding than is expected. In both studies of ETV6 mutations, the combination of NGS sequencing and good family studies were able to identify the causative mutations.
Finally, the combined use of NGS and candidate gene sequencing led to the description of autosomal dominant mutations in growth factor independent 1B (GFI1B), resulting in mild clinical bleeding, red blood cell anisopoikilocytosis, and macrothrombocytopenia, with absent alpha granules and abnormal aggregation responses . GFI1B is a transcription factor that had previously thought to be important in murine erythroid and platelet development, but before this had not been linked to human disease. Since then, a second, dominant-negative mutation in GFI1B was reported in autosomal dominant gray platelet syndrome [11▪].
TPO signaling: c-MPL, ANKRD26, TAR
Defects in thrombopoietin (TPO) signaling underlie congenital amegakaryocytic thrombocytopenia (CAMT), which is the prototypical disease in this category. Mutations in the thrombopoietin receptor (MPL) are primarily responsible for the disease in these patients, who present with profound thrombocytopenia from birth and classically progress to bone marrow failure generally within the first few years of life (see  for review). The degree of dysregulation of TPO signaling correlates with time to bone marrow failure, and patients progress to bone marrow failure by 4 years of age, but the course may be variable, with some patients recovering platelet counts initially . A recent study described bone marrow failure in a 1-month-old infant with CAMT and documented compound heterozygosity in MPL.
Thromocytopenia absent radii (TAR) syndrome also presents at birth with thrombocytopenia, but with associated skeletal abnormalities generally not seen in CAMT. In additional contrast, most of these children have improvement in the platelet count sometime in the first 5 years of life and generally do not develop additional cytopenias. In 2007, proximal microdeletions in 1q21.1 were identified in patients with TAR syndrome, but these were shown also to be present in some unaffected parents of children with TAR syndrome . Albers et al. identified low-frequency noncoding SNPs in the 5’UTR of RBM8A as the second ‘mutation’ required in the autosomal recessive disorder. Since then, it has become clear that TAR syndrome results from reduced expression of Y14 (the protein encoded by RBM8A), either through heterozygous mutations resulting in decreased expression or the complex genetics initially described by Albers et al. Patients with TAR syndrome have very high thrombopoietin levels and impaired phosphorylation of Jak2 kinase that appears to improve with age corresponding to improved platelet counts .
In contrast, recent evidence suggests that the mechanism of thrombocytopenia in families harboring mutations in the 5’UTR of Ankyrin repeat domain 26 (ANKRD26) resulting in familial thrombocytopenia 2 (THC2) and predisposition to leukemia is loss of appropriate ANKRD26 silencing and persistent TPO/MPL pathway signaling leading to impaired platelet release by megakaryocytes [18▪]. Normally, RUNX1 and FLI1 bind to the 5’UTR region of ANKRD26 to repress expression in late megakaryocytes. In patients with the 5’UTR changes of THC2, RUNX1 and FLI1 are unable to bind and the expression of ANDRD26 remains high, leading to a defect in proplatelet formation as a result of increased signaling through the TPO/MPL pathway [18▪].
Granule biogenesis: NBEAL2, HPS genes, VSP33B/VIPAS39, LYST
Perhaps the best known success of modern sequencing in the inherited platelet disorders is the identification of Neurobeachin-like 2 (NBEAL2) in gray platelet syndrome. In 2011, three groups simultaneously described mutations in NBEAL2 as a cause of this autosomal recessive macrothrombocytopenia with absent α-granules and moderate bleeding diathesis [19–21]. Many of these patients also developed myelofibrosis. NBEAL2 deficiency resulted in defective α-granule biogenesis and impaired inflammatory response in a murine model [22▪].
Granule biogenesis clearly plays an important role in both platelet number and function as multiple platelet syndromes exist in which granule biogenesis is affected. However, in many of these other syndromes, the platelet defect is a part of a larger syndromic disease such as Hermansky–Pudlak syndrome (mutations in HPS genes) , arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome with platelet dysfunction and a paucity of alpha granules [mutations in vacuolar protein sorting gene 33B (VPS33B) or VPS33B interacting protein 39 (VIPAS39)] , and Chediak–Higashi syndrome due to mutations in LYST with deficiency of platelet-dense granules, but also a severe immunologic defect resulting in significant infection and progressive neurological dysfunction .
Cytoskeletal defects: MYH9, FLNA, TUBB1, ACTN1
The MYH9 gene (nonmuscle myosin heavy chain II-A, NMMHC-IIA) is now well established as the causative gene in the clinical continuum of diseases now called the MYH9-related disorders. This is an autosomal dominant macrothrombocytopenia with giant platelets, leukocyte inclusions, and variable association of nephropathy, hearing loss, and cataracts. Platelet counts range from 10 to 136 × 109/l with giant platelets. A study from a large French cohort of patients with MYH9-related disease suggested that bleeding occurred more commonly in patients with mutations in the motor domain versus the tail domain, and 40% of patients had at least one extrahematological manifestation (renal disease, hearing loss, or cataracts) [26▪].
The description of mutations in filamin A (FLNA) presenting with an isolated platelet phenotype expanded the reach of platelet cytoskeletal affected genes . FLNA – which tethers GP1bα and integrin αIIb/βIII to the underlying cytoskeleton in platelets – results in disruption of the platelet plasma membrane under high shear. This gene, coded on the X-chromosome, has been associated with pleotrophic presentations loosely termed the filaminopathies, with bleeding and low platelet counts previously occasionally described in some patients. Only recently have mutations in FLNA been associated with an isolated platelet phenotype , but since this association, the role of FLNA in platelet and megakaryocyte biology has become more apparent [28▪].
Another cytoskeletal protein, tubulin B1, plays a role in an autosomal dominant macrothrombocytopenia. A missense mutation in TUBB1 was found in a Japanese family [29▪]. Subsequently, a polymorphism in TUBB1 has been associated with lack of response to ITP therapy , and now an abstract has also presented that in a large cohort of patients (480) with bleeding phenotypes, TUBB1 mutations are present in 11 probands with macrothrombocytopenia; one patient shared the mutation previously reported, four patients shared the phenotype with round platelets on electron microscopy with heterogeneous distribution of granules and characteristic elongated abnormal large channels formed from the internal membrane but had different mutations; six of the patients required further characterization, but had similar phenotypes and had suspicious mutations and consistent clinical presentations . Meanwhile, a genome-wide association study of more than 65 000 individuals of European ancestry demonstrated that the TUBB1 locus was a major determinant of mean platelet volume , demonstrating again that the genetics of platelet number and size are complex, and differentiating disease states from normal may become very complex as we begin to explore the most mild phenotypes.
Finally, the most recent addition to the cytoskeletal mutations causing macrothrombocytopenia is ACTN1 (Actin-1), mutations which were described in six Japanese pedigrees with congenital macrothrombocytopenia after whole exome sequencing (WES) . These families all had autosomal dominant macrothrombocytopenia with platelet counts ranging from 54 to 132 × 109/l and a mean increase in platelet size of 30% with platelet anisocytosis. There was only a mild bleeding diathesis associated and no apparent platelet functional defects noted, but there was increased expression of GPIb/IX and GPIIb/IIIa, thought to be due to increased platelet size. A recent study using WES added more families with ACTN1 mutations in a large cohort of patients with thrombocytopenia, again demonstrating that carefully phenotyped patients, in appropriate research settings, can be studied by WES and that phenotype clustering tools may also be useful to help identify patients .
Thrombocytopenia 4 is an autosomal dominant thrombocytopenia caused by mutations in cytochrome c (CYCS) that has so far been described in two kindreds: a single family in New Zealand  and a second family in Italy with a different mutation in the same region [36▪]. Y48H and C41S are the two mutations described, both of which are in highly conserved amino acids. Platelet counts ranged from 75 to 135 × 109/l with a normal platelet size. These mutations in cytochrome c did not seem to cause alterations in respiratory chain or oxidative stress response, but did seem to cause a defect in apoptosis: transfected cells and yeast cells showed increased apoptosis. This is manifest in megakaryocytes as abnormal platelet budding and therefore decreased platelet count [36▪].
Signaling and secretion: G-protein coupled signaling, TXA2R, P2Y12
Using WES, investigators recently described three siblings from consanguineous parents who presented with a severe bleeding phenotype due to mutations in the RASGRP2 gene. This codes for a guanine nucleotide exchange factor critical for outside in signaling through αIIb/βIII (CalDAG-GEFI) through Rac1 and Rap1. These patients presented with a significant bleeding diathesis with normal platelet counts and abnormal platelet responses to low dose-agonists, except ristocetin, which could be overcome with higher doses; normal expression of surface platelet receptors, normal clot retraction and normal total thrombin generation, although a delayed time to peak thrombin generation suggested a defect in platelet signaling seen mainly at low doses [37▪▪]. Patient platelets had abnormal expression levels of protein, and failed to form filopoidia on activation. An intermediate defect in platelet spreading was seen in platelets from family members who were heterozygous for the mutation suggesting biological plausibility.
In several patients, exome sequencing or targeted sequencing that has followed careful phenotyping has allowed the identification of specific mutations in the thromboxane receptor [38,39], P2Y12[40,41], and Gi [42▪▪] and Gs  that lead to platelet dysfunction and bleeding diatheses. These mutations in known genes associated with platelet function demonstrate that as we are getting better at understanding platelet function, we will get better at identifying molecular causes of human disease.
Abnormal cAMP levels: PRKACG
A final novel mechanism of platelet dysfunction was recently described by investigators who reported on a family with autosomal recessive thrombocytopenia with defects in proplatelet formation and platelet activation due to mutations in PRKACG, the gene encoding the γ subunit of cyclic-adenosine monophosphate (c-AMP)-dependent kinase A (PKA). These patients have severe macrothrombocytopenia (5–8 × 109/l) and significant bleeding [44▪▪] Sequencing revealed the above mutation and biochemical studies revealed that family members who were homozygous for the mutation had decreased FLNA in megs and platelets, and increased levels of platelet cAMP, whereas patients who were heterozygous had intermediate levels compared to controls. The increased cAMP in platelets leading to platelet dysfunction is a novel mechanism of dysfunction, not previously reported in human disease, although animal models have suggested that this would be a mechanism of platelet dysfunction.
In summary, our knowledge of platelet and megakaryocyte biology has significantly increased over the past several years due in large part to increased understanding of the pathobiology of the inherited platelet disorders. Rational use of next-generation or targeted sequencing after careful phenotyping has led to significant advances in our understanding of biology and surprises and novel avenues of investigation. We will need to continue to carefully apply insights gained from whole exome/genome studies to be sure we do not erroneously assign disease to normal variants as all of these studies have shown careful biological follow-up of variants to demonstrate biological plausibility.
Financial support and sponsorship
The author acknowledges the NIH-NIAID and NHLBI, as well as HRSA, for their financial support.
Conflicts of interest
Dr Lambert has no relevant conflicts of interest to disclose.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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