Functional Testing of ETV6 Variants: Is the Evaluation of Their Intracellular Localization Sufficient in Assessing Pathogenicity?

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.

Keywords: ETV6; functional studies; thrombocytopenia.

Figure 1

(A) Schematic representation of ETV6 domains. Amino acid number of the domain boundaries are reported below. (B) Structural analysis of the aminoacidic changes on ETS domain. Structural analysis of ETS bound to a specific DNA sequence (PDB ID 4MHG). The DNA cognate is reported in blue, the ETS domain is reported in green, and the residue involved in the aminoacidic change is highlighted in purple.

Figure 2

Effect of ETV6 mutation on the repressive activity on MMP3 promoter. Bars represents the P. pyralis/R. reniformis luciferase emission ratio in HEK293T cells with firefly luciferase expression controlled by the MMP3 promoter. Ratios are normalized to emission ratio of the empty vector (black bar). Mutations are reported as follows: p.Gln347Pro: Q347P; p.Ile370Met: I370M; and p.Arg378Gnl: R378Q. Error bars indicate the standard deviation from three independent experiments. Significance levels are represented as ## = p < 0.01 compared to the empty vector, and ns = p > 0.05, ** = p < 0.01, *** = p < 0.001 compared to WT-ETV6 overexpression, using Student’s t-test.

Figure 3

Functional analyses of the impact of ETV6 mutation on its intracellular localization. (A) Immunofluorescence analysis in HeLa cells transfected with Myc-tagged ETV6 constructs cloned into the pcDNA3.1 expression vector. Myc-ETV6 is shown in green, and nuclei are stained in blue (Hoechst staining). Images shown are representative of three independent experiments. Scale bar = 100 µm. (B) Quantification of ETV6 subcellular localization in cells. The histogram represents the proportion of cells with nuclear (N, black), cytoplasmic (C, light gray), or mixed nuclear/cytoplasmic (N/C, gray) staining. Data are based on results from three independent experiments counting at least 100 cells. (C) Western blot analysis of nuclear and cytoplasmic fractions from HEK293T cells 48 h post-transfection with wild type or mutant Myc-tagged ETV6. HSP90 and SP1 were used as markers for cytoplasmic and nuclear fractions, respectively. Mutations are reported as follows: p.Gln347Pro: Q347P; p.Ile370Met: I370M; and p.Arg378Gnl: R378Q.