Summary: MECOM‐associated syndrome (MECOM‐AS) is a rare disease characterized by amegakaryocytic thrombocytopenia, progressive bone marrow failure, pancytopenia and radioulnar synostosis with high penetrance. The clinical phenotype may also include finger malformations, cardiac and renal alterations, hearing loss, B‐cell deficiency and predisposition to infections. The syndrome, usually diagnosed in the neonatal period because of severe thrombocytopenia, is caused by mutations in the MECOM gene, encoding for the transcription factor EVI1. The mechanism linking the alteration of EVI1 function and thrombocytopenia is poorly understood. In a paediatric patient affected by severe thrombocytopenia, we identified a novel variant of the MECOM gene (p.P634L), whose effect was tested on pAP‐1 enhancer element and promoters of targeted genes showing that the mutation impairs the repressive activity of the transcription factor. Moreover, we demonstrated that EVI1 controls the transcriptional regulation of MPL, a gene whose mutations are responsible for congenital amegakaryocytic thrombocytopenia (CAMT), potentially explaining the partial overlap between MECOM‐AS and CAMT.

Background/Objectives: ETV6-related thrombocytopenia (ETV6-RT) is a rare autosomal dominant disorder characterized by mild thrombocytopenia since birth and an increased predisposition to hematologic malignancies. ETV6 functions as a transcriptional repressor, and its pathogenic variants, predominantly within the ETS domain, disrupt nuclear localization and transcriptional activity. In individuals with congenital thrombocytopenia, we identified two missense variants: c.1110A>G, p.Ile370Met, a novel variant, and c.1133G>A, p.Arg378Gln, a known variant with conflicting pathogenicity interpretations, for which functional characterization is necessary to provide an accurate molecular diagnosis. Methods: In silico bioinformatic tools and structural modeling were used to predict the impact of the variants. Functional assays included a dual-luciferase reporter assay to measure transcriptional activity and immunofluorescence/immunoblotting to assess intracellular localization in transfected HEK293T and HeLa cells. Results: Bioinformatic predictions and structural analyses suggested that the two variants might play a role in altering the folding or function of the ETS domain. Functional analysis revealed that p.Ile370Met abolished the ETV6 transcriptional repression activity, confirming its pathogenicity. p.Arg378Gln had no effect on the reporter gene levels, and, as expected, it localized in the nucleus. Interestingly, unlike the known mutations, which fail to enter the nucleus, p.Ile370Met retained partial nuclear localization. Conclusions: Since we described the first ETV6 mutation localized in the ETS domain that causes a loss of transcriptional activity, although it maintains the ability to enter the nucleus, we suggest that both transcriptional activity and intracellular localization assays are important for the accurate classification of ETV6 variants by functional studies.

Mutations in the genes ANKRD26, RUNX1, and ETV6 cause three clinically overlapping thrombocytopenias characterized by a predisposition to hematological neoplasms. The ANKRD26 gene, which encodes a protein involved in protein-protein interactions, is downregulated by RUNX1 during megakaryopoiesis. Mutations in 5′UTR of ANKRD26, leading to ANKRD26-RT, disrupt this regulation, resulting in the persistent expression of ANKRD26, which leads to impaired platelet biogenesis and an increased risk of leukemia. Although ANKRD26 and ETV6 exhibit inverse expression during megakaryopoiesis, ETV6 does not regulate the ANKRD26 expression. Hypothesizing an interplay between ETV6 and ANKRD26 through in vitro studies, we explored the interactions between the two proteins. In this study, we found that ANKRD26 interacts with ETV6 and retains it in the cytoplasm, phenocopying ETV6-RT-related mutants. We found that GPS2, a component of the NCoR complex, binds both ANKRD26 and ETV6, mediating this interaction. Furthermore, ANKRD26 overexpression deregulates ETV6 transcriptional repression, supporting a common pathogenic mechanism underlying ANKRD26-RT, FPD/AML, and ETV6-RT. Our results unveil a novel ANKRD26-ETV6-GPS2 axis, providing new insights to investigate the molecular mechanism underlying thrombocytopenias with a predisposition to myeloid neoplasms that need to be further characterized.