Chiara Gnan

Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell–precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

ANKRD26 -related thrombocytopenia and myeloid malignancies

To the editor: Since the discovery that mutations in the 5′ untranslated region (UTR) of ANKRD26 are responsible for an autosomal-dominant form of thrombocytopenia ( ANKRD26 -RT),[1][1] 21 affected families were reported.[2][2] A study analyzing this series of patients suggested that ANKRD26 -RT

Clinical and pathogenic features of ETV6-related thrombocytopenia with predisposition to acute lymphoblastic leukemia

ETV6-related thrombocytopenia is an autosomal dominant thrombocytopenia that has been recently identified in a few families and has been suspected to predispose to hematologic malignancies. To gain further information on this disorder, we searched for ETV6 mutations in the 130 families with inherited thrombocytopenia of unknown origin from our cohort of 274 consecutive pedigrees with familial thrombocytopenia. We identified 20 patients with ETV6-related thrombocytopenia from seven pedigrees. They have five different ETV6 variants, including three novel mutations affecting the highly conserved E26 transformation-specific domain. The relative frequency of ETV6-related thrombocytopenia was 2.6% in the whole case series and 4.6% among the families with known forms of inherited thrombocytopenia. The degree of thrombocytopenia and bleeding tendency of the patients with ETV6-related thrombocytopenia were mild, but four subjects developed B-cell acute lymphoblastic leukemia during childhood, resulting in a significantly higher incidence of this condition compared to that in the general population. Clinical and laboratory findings did not identify any particular defects that could lead to the suspicion of this disorder from the routine diagnostic workup. However, at variance with most inherited thrombocytopenias, platelets were not enlarged. In vitro studies revealed that the maturation of the patients’ megakaryocytes was defective and that the patients have impaired proplatelet formation. Moreover, platelets from patients with ETV6-related thrombocytopenia have reduced ability to spread on fibrinogen. Since the dominant thrombocytopenias due to mutations in RUNX1 and ANKRD26 are also characterized by normal platelet size and predispose to hematologic malignancies, we suggest that screening for ETV6, RUNX1 and ANKRD26 mutations should be performed in all subjects with autosomal dominant thrombocytopenia and normal platelet size.

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Summary.  Background:  Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource‐limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina.

Mutations of RUNX1 in families with inherited thrombocytopenia

To the Editor: Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is a rare autosomal dominant disease characterized by thrombocytopenia and platelet functional defects. Although bleeding tendency is usually mild to moderate, an important hallmark of FPD/AML is the increased risk of myeloid neoplasms, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). FPD/AML is caused by different, usually private, mutations of RUNX1, a gene encoding the DNA binding subunit (also known as CBF-alpha) of the core binding factor (CBF) transcription complex. The N-terminal domain of RUNX1 (runt-homologous domain) mediates DNA binding and heterodimerization to CBF-beta, the other subunit of the CBF complex. At the C-terminus, RUNX1 is constituted of domains involved in transcription activation and repression. We report three families with pathogenic variants of RUNX1 identified in our cohort of 274 consecutive unrelated probands with inherited thrombocytopenia (IT) (Supporting Information Materials and Methods). They carry three different heterozygous mutations, all segregating with thrombocytopenia in the respective families: one missense (c.578T>A/p.Ile193Asn) variant affecting a well conserved residue of the runt-homologous domain, and two nucleotide substitutions of the canonical “gt” dinucleotide in the donor splice sites of intron 4 (c.35111G>A) and intron 8 (c.96712_5del) (Supporting Information Figures S1-S3). The effect of c.35111G>A on splicing mechanisms was not determined because RNA from members of family 2 was not available. In family 3, RT-PCR analysis revealed two alternative spliced products, sp1 and sp2 that led to frameshift/truncation and in-frame deletion alterations at the transactivation domain of RUNX1, respectively. The sp1 RNA was expressed at higher level than the sp2 form. The 13 individuals from the three families carrying the RUNX1 mutations had mild thrombocytopenia (platelet count ranging from 70 to 130 3 10/L) and mild bleeding tendency (Table 1). The platelet size was normal in all the six cases analyzed. Consistent with the mild reduction in platelet count, patients’ serum thrombopoietin levels were normal or moderately increased (Table 1). No specific morphological alteration of platelets was detected at the May–Grunwald–Giemsa staining, except for moderate reduction in the alpha-granule content in family 1, as confirmed by immunofluorescence analysis (data not shown). Flow cytometry of platelets showed normal expression of the major surface glycoprotein (GP) complexes GPIIb-IIIa and GPIb-IX-V (Table 1). In family 1, we found moderate reduction (25-45%) of GPIa-IIa, whose alteration was independent of genotypes at the ITGA2 locus. In this family, platelet aggregation was defective after stimulation with low doses of collagen (4 lg/mL), a defect that could not be explained by the reduction of GPIa-IIa, as the specific reagent used for the platelet aggregation assay does not bind this receptor. No second wave aggregation was observed in response to low doses of ADP (2 lM) in the five individuals investigated, suggesting alterations of the platelet delta-granules (Table 1). Although we did not support this hypothesis through specific assays, deficiency, or defective release of the dense-granules is one prevalent defect in individuals with FPD/ AML. Increasing the concentration of ADP (5 lM), we found defective response only in family 1. Normal responses were observed using higher concentrations of both the agonists (collagen 20 lg/mL and ADP 20 lM) and with ristocetin (1.5 mg/mL), suggesting that the platelet dysfunction was mild. Consistent with data from literature, our findings indicate that multiple aspects of platelet structure and function are compromised in FPD/AML, which is expected considering that RUNX1 regulates expression of multiple genes and that their expression is modulated by many genetic factors. Individuals with heterozygous mutations of RUNX1 are at risk of hematological malignancies, which have been reported in almost 40% of patients with a median age of onset in the early 30s. In our cohort of 14 carriers (individual II-3 of family 3 is an obligate carrier), only three individuals (one from family 2 and two from family 3) had developed AML (Supporting Information Materials and Methods). This low prevalence (20%) could depend on the relatively young age of some of the individuals studied, as well as on the fact that the probands were not all selected on the basis of their personal or family history of hematological malignancies. Indeed, families 1 and 2 were identified by diagnostic work-up of an IT of unknown origin, which was not associated with MDS or AML. Therefore, only systematic molecular genetic testing will provide the actual risk of malignancies in individuals with RUNX1 mutations. Although AML remains the most prevalent neoplasm, we cannot exclude that mutations of RUNX1 predispose also to solid tumors, as those (lung and uterine cancer) diagnosed in two members of family 3. Although mutations of RUNX1 are relatively rare (1% in our cohort), their identification in families with IT is important because of the associated risk of malignancies. However, it is difficult to discriminate FPD/AML from other forms of IT due to lack of pathognomonic signs. The most promising features could be the defects of the alpha-