Abstract
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.