Natthapol Songdej

Hematopoietic transcription factor mutations and inherited platelet dysfunction

The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is increasing evidence that mutations in hematopoietic transcription factors are major players in the pathogenesis of defective megakaryopoiesis and platelet dysfunction in patients with inherited platelet disorders. These hematopoietic transcription factors include RUNX1, FLI1, GATA-1, and GFI1B. Mutations involving these transcription factors affect diverse aspects of platelet production and function at the genetic and molecular levels, culminating in clinical manifestations of thrombocytopenia and platelet dysfunction. This review focuses on these hematopoietic transcription factors in the pathobiology of inherited platelet dysfunction.

Inherited platelet dysfunction and hematopoietic transcription factor mutations

Abstract Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet dysfunction are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for the defects in platelet production, morphology, and function. The hematopoietic TFs implicated in the patients with impaired platelet function include Runt related TF 1 (RUNX1), Fli-1 proto-oncogene, ETS TF (FLI1), GATA-binding protein 1 (GATA1), and growth factor independent 1B transcriptional repressor (GFI1B). These TFs act in a combinatorial manner to bind sequence-specific DNA within a promoter region to regulate lineage-specific gene expression, either as activators or as repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology and platelet production and function, culminating in thrombocytopenia, platelet dysfunction, and associated clinical features. Mutations in TFs may occur more frequently in the patients with inherited platelet dysfunction than generally appreciated. This review focuses on the alterations in hematopoietic TFs in the pathobiology of inherited platelet dysfunction.

Hematopoietic transcription factor mutations: important players in inherited platelet defects

Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet defects are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for defects in platelet production, morphology, and function. The hematopoietic TFs implicated in patients with impaired platelet function and number include runt-related transcription factor 1, Fli-1 proto-oncogene, E-twenty-six (ETS) transcription factor (friend leukemia integration 1), GATA-binding protein 1, growth factor independent 1B transcriptional repressor, ETS variant 6, ecotropic viral integration site 1, and homeobox A11. These TFs act in a combinatorial manner to bind sequence-specific DNA within promoter regions to regulate lineage-specific gene expression, either as activators or repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology, and platelet production and function, culminating in thrombocytopenia and platelet dysfunction. Some are associated with predisposition to hematologic malignancies. These TF variants may occur more frequently in patients with inherited platelet defects than generally appreciated. This review focuses on alterations in hematopoietic TFs in the pathobiology of inherited platelet defects.

Inherited thrombocytopenias: the beat goes on

In this issue of Blood, Bottega et al document mutations in ACTN1, which encodes the cytoskeletal protein α-actinin 1, in 10 of 239 consecutive probands with an inherited thrombocytopenia--making ACTN1 an important cause of familial thrombocytopenia.

Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events Differential Effects of RUNX1 Variants

Background: PCTP (phosphatidylcholine transfer protein) regulates the intermembrane transfer of phosphatidylcholine. Higher platelet PCTP expression is associated with increased platelet responses on activation of protease-activated receptor 4 thrombin receptors noted in black subjects compared with white subjects. Little is known about the regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses on activation. Platelet expression profiling of a patient with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of the PCTP gene compared with healthy controls. Methods: We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to the PCTP promoter using DNA-protein binding studies and human erythroleukemia cells and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction in 2 separate patient cohorts (587 total patients) with cardiovascular disease. Results: Platelet PCTP protein in the patient was reduced by ≈50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ≈1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in human erythroleukemia cells, indicating that PCTP is regulated by RUNX1. Studies in 2 cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared with white subjects and was associated with future death/myocardial infarction after adjustment for age, sex, and race (odds ratio, 2.05; 95% confidence interval 1.6–2.7; P<0.0001). RUNX1 expression is known to initiate at 2 alternative promoters, a distal P1 and a proximal P2 promoter. In patient cohorts, there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression. Conclusions: PCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.

Parsing the repertoire of GPIb-IX-V disorders

In this issue of Blood , [Sivapalaratnam et al][1][1][2] report an association between unique, rare monoallelic variants in GP1BB , which encodes the glycoprotein (GP)Ibβ subunit of the platelet GPIb-IX-V complex, and autosomal dominant macrothrombocytopenia. ![Figure][3] Schematic drawing

Platelet disorders: the next generation is in.

These studies describe elegant genomic approaches driven by HTS (also called next-generation sequencing) in unraveling the genetic abnormalities in patients with bleeding and thrombotic disorders. Each study tells an important story about the means as well as the end, establishing the disease-causing variants. They attest to the power of the HTS technology, advances in bioinformatics, large databases, and application of human phenotype ontology (HPO)–term coding,3 and of collaborative groups so essential to make rapid inroads. In the first study, Simeoni et al report the results from the ThromboGenomics Consortium encompassing investigators from across 13 countries, where a targeted panel of 63 genes was assessed in a heterogeneous group of patients with bleeding and platelet disorders (BPD) with known and unknown diagnosis. Included were patients with abnormal platelet count, volume, morphology, or function, or a tendency to bleed abnormally on a genetic basis, along with patients with an abnormal tendency for thrombosis. The HTS platform was designed to detect variants in the exonic fraction of 63 BPD genes, and many of their introns and untranslated regions. Automated variant filtering procedures and HPO coding-based prioritization of candidate variants were applied. The authors sequenced 300 samples (260 unrelated subjects) from 4 subject groups: the “known” group (n = 159) with diagnostic laboratory abnormalities and previously established pathogenic genetic variants; the “suspected” group (n = 61) with phenotypes that strongly indicated a particular disorder on the basis of laboratory abnormalities but without knowledge of causal variants; the “uncertain” group (n = 76) with phenotypes that could not be matched to any known BPD because the laboratory assays were either normal or not diagnostic of an established disorder; and 4 unaffected relatives. In the “known” group, the ThromboGenomics platform correctly called the pathogenic variants in all 159 samples (with 145 causal variants), with an impressive sensitivity of 100%. In the “suspected” group, in 56 of the 61 subjects a pathogenic or likely pathogenic variant was identified, including 5 samples where the prior Sanger sequencing was unrevealing. These patients had entities such as Glanzmann thrombasthenia (4 patients), Bernard-Soulier syndrome (1), Hermansky-Pudlak syndrome (9), May-Hegglin disorder (4), or a plasma factor deficiency (26), with clinical and laboratory findings that suggested the diagnosis and candidate gene(s). The highly “uncertain” group consisted mainly of patients with bleeding symptoms but with normal laboratory tests, storage pool disorder (presumably indicated by abnormalities on platelet function testing), or patients with a thrombotic event and low plasma protein S. In these 76 subjects (62 unrelated), pathogenic or likely pathogenic variants were detected in only 8 cases (sensitivity, 10.5%). Thus, in these patients without a clear phenotypic lead, the yield from interrogating genes previously implicated in BPD patients was low. These data indicate that there are many more causal genes involved in BPD patients than identified to date and that the unknown causal genes/variants may outnumber those known. The unknown variants may be in other genes or in the unexplored regulatory regions. Incorporating into the ThromboGenomics platform additional genes that become linked to BPD disorders may make it a stronger test. Also, the platform does not detect inversions, and these are known to occur in some BPD patients (such as hemophilia A). Whole exome sequencing, as applied in identifying the mutation in DIAPH12 and other genes,4-8 may provide a solution. Exomes account for ∼2% of the genome; whole genome sequencing may be required in some. But this imposes additional requirements, including cost. The ThromboGenomics Consortium includes patients with bleeding symptoms (platelet and coagulation defects) and thrombotic phenotypes. Eventually, the effectiveness of the platform in each group needs to be established and may be different. Overall, the studies of Simeoni et al are an impressive validation of a targeted approach using HTS and HPO coding, particularly in BPD patients with suspected etiology. Its use in the wider population of BPD patients may depend on the success rate in further studies in the “uncertain” group of patients, with additional genes incorporated into the platform. Patients with platelet function defects and a normal platelet count but without clues on evaluation to known entities are of specific interest. In such patients, the defects are largely unknown. In the second study, Stritt et al combined exome sequencing, HPO coding, and phenotype similarity regression to identify a novel heterozygous variant in DIAPH1 (p.R1213*) in 2 unrelated pedigrees with autosomal dominant MTP and early onset sensorineural hearing loss. Data were analyzed from 702 index BPD cases with unknown genetic mechanisms from the Biomedical Research Centres/Units Inherited Diseases Genetic Evaluation–BPD (BRIDGE-BPD) study3 and 3453 control subjects. A total of 1073 genes had a rare variant in at least 2 cases and predicted to impact gene translation. The DIAPH1 variant was not found within the 61 486 exomes in the Exome Aggregation Consortium database. Six affected pedigree members but not 3 asymptomatic members had the variant. DIAPH1 encodes the protein that regulates cytoskeletal processes, such as actin assembly and microtubule stability. The DIAPH1 variant predicts for truncation in the DIAPH1 diaphanous autoregulatory domain. The authors demonstrate reduced proplatelet formation from megakaryocytes and cytoskeletal alterations consistent with constitutive DIAPH1 activation, and advance a causal link between the DIAPH1 variant and MTP and hearing loss. To date, mutations in MYH9 have been associated with MTP and sensorineural hearing loss. This report extends DIAPH1 mutations to this constellation, although there were differences. Neutrophil cytoplasmic inclusions and renal failure noted in MYH9 variants were not observed. These 2 studies in this issue of Blood make important contributions to the field of platelet disorders. Since the advent of HTS, these and other studies4-8 promise exciting times ahead in the quest to decode the secrets of nature underlying human bleeding disorders.

Inherited Platelet Disorders: Diagnosis and Management

Inherited disorders of platelets are characterized by highly variable mucocutaneous bleeding manifestations and excessive hemorrhage following surgical procedures or trauma. They include patients who have decreased platelet counts, abnormal platelet function or a combination of both, in general reflecting underlying abnormalities that culminate in defective platelet production and function. Over the last two decades there has been an impressive increase in the number of genetic abnormalities identified as leading to inherited thrombocytopenias. In some of these patients platelets have impaired function as well. Most patients with inherited platelet function have mild to moderate bleeding manifestations. In the vast majority of patients suspected to have an inherited platelet function defect the molecular and genetic mechanisms are unknown. The platelet dysfunction in these patients arises by diverse mechanisms, including abnormalities in membrane glycoproteins, granules and their contents, platelet signaling and secretion mechanisms, thromboxane production pathways and in platelet procoagulant activities. The platelet dysfunction may arise due to mutation in a transcription factor (e.g. RUNX1) leading to abnormalities in multiple mechanisms involved in platelet production as well as in function. In this chapter, clinical and laboratory aspects of Inherited disorders of platelets are discussed.

Focal cranial hyperostosis from meningioma: a complication from previous radiation treatment for childhood T-cell acute lymphoblastic leukemia.

Nearly 75% of childhood cancer survivors will experience an adverse late effect from previous therapy. In patients previously treated with cranial irradiation, the late effect can manifest as secondary central nervous system tumors. Presented is a case of a 20 year man with a history of T-cell lymphoblastic leukemia diagnosed at age 22 months, treated with chemotherapy and cranial irradiation. He had developed increasing prominence of the top of his head over several months. Plain radiograph showed frontal calvarium thickening with focal “hair-on-end” periosteal reaction. Magnetic resonance imaging revealed an enhancing dural-based mass with transcalvarial extension, confirmed after resection to be meningioma (World Health Organization Grade I). This case illustrates an atypical presentation of a late effect of childhood cancer treatment and highlights the need to be informed about prior treatments received and potential attendant complications.