Mutations of <i>RUNX1</i> in families with inherited thrombocytopenia
Authors: Daniela De Rocco, Federica Melazzini, Caterina Marconi, Alessandro Pecci, Roberta Bottega, Chiara Gnan, Flavia Palombo, Paola Giordano, Maria Susanna Coccioli, Ana C. Glembotsky, Paula G. Heller, Marco Seri, Anna Savoia, Patrizia Noris
Journal: American Journal of Hematology (2017)

Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is a rare autosomal dominant disease characterized by thrombocytopenia and platelet functional defects.1 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.2 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.351 + 1G > A) and intron 8 (c.967 + 2_5del) (Supporting Information Figures S1-S3). The effect of c.351 + 1G > 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 × 109/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.3 In this family, platelet aggregation was defective after stimulation with low doses of collagen (4 μg/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 μM) 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.4 Increasing the concentration of ADP (5 μM), we found defective response only in family 1. Normal responses were observed using higher concentrations of both the agonists (collagen 20 μg/mL and ADP 20 μM) and with ristocetin (1.5 mg/mL), suggesting that the platelet dysfunction was mild. Consistent with data from literature,4 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.1 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- and delta-granules, or persistence of MYH10 protein expression in platelets.4 However, these investigations cannot be performed on a routine basis, as they require specialized laboratories. Since mutations not only of RUNX1 but also of the ANKRD26 and ETV6 genes are responsible for thrombocytopenia (ANKRD26-related thrombocytopenia, ANKRD26-RT; ETV6-related thrombocytopenia, ETV6-RT) and predisposition to hematological malignancies,5 the recognition of these disorders would provide patients with appropriate genetic counseling, clinical follow-up, and treatment, especially in case of selection of donors for hematopoietic stem cell transplantation in patients who develop AML or MDS. Like FPD/AML, ANKRD26-RT and ETV6-RT are rare autosomal dominant forms of IT that are hard to diagnose because they lack specific clinical or laboratory features. However, they are characterized by normal platelet size, which is a relatively uncommon finding in IT.6 Therefore, we suggest that all the individuals with autosomal dominant thrombocytopenia and normal platelet size should be tested for mutations in RUNX1, ANKRD26, and ETV6. This study was supported by the ERA-Net for Research Program on Rare Diseases (EUPLANE), Telethon Foundation (Grant GGP13082), Cariplo Foundation (2012-0529), Italian Ministry of Health (RF-2010-2309222). C.G. receives a fellowship (RF13679 2013-2014 and RF16440 2015) from AIRC (Italian Association of Cancer Research). All the authors report no potential no conflicts of interest. DDR, CG, CM, FP performed whole exome, Sanger sequencing and bioinformatics analysis. RB carried out RT-PCR analysis. FM, AP, PG, MSC, ACG, PH, MS, PN enrolled patients for the study, collected patients' clinical and laboratory data. DDR, FM, MS and AS were responsible for writing the manuscript; DDR, AS, PN participated in the study design. All authors revised and approved the manuscript. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.