Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

Abstract

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Figure 1

New ETV6 germline variants encoding p.Pro214Leu, p.Arg369Gln and p.Arg399Cys in association with thrombocytopenia and hematologic malignancy. Families A, B and C have ETV6 p.Arg399Cys, p.Arg369Gln and p.Pro214Leu variants, respectively, that segregate with thrombocytopenia and hematologic malignancy in each family. WT indicates genotyped subjects with only wild-type ETV6 alleles; R399C, R369Q, and P214L indicate subjects heterozygous for the variant allele. Arrows indicate the proband in each family.

Figure 2

Missense alterations in the ETS domain abrogate ETV6 DNA binding. (a) Positions of germline and somatic alterations in ETV6 relative to the PNT oligomerization and ETS DNA-binding domains. The germline alterations reported in this study are highlighted in red. Somatic alterations affecting the same amino acids as the germline alterations are boxed. Somatic alterations reported in the literature include ones associated with MDS^,,, AML^,,–, CMML, immature T cell ALL, mature T cell ALL, B cell precursor ALL^,, hypodiploid ALL, multiple myeloma, colorectal adenocarcinoma^, and melanoma. Truncating alterations, including nonsense, frameshift and splice-site changes, are shown in bold. (b) Hydrogen bonding (dotted lines) of Arg399 (orange) with guanine (magenta) in the ETS binding element. The protein structure of the mouse ETV6 ETS domain (Protein Data Bank (PDB), 4MHG) is shown. The ETS domains of the mouse and human ETV6 proteins have 100% protein sequence identity. (c) Molecular modeling of the Arg399Cys (orange) variant using SWISS-MODEL predicts loss of hydrogen bonding to DNA. (d) Coomassie-stained SDS-PAGE gel with recombinant histidine affinity (HAT)-tagged ETS domains from wild-type (WT), Arg369Gln or Arg399Cys ETV6. (e) EMSA of the ETS domains for wild-type, Arg369Gln or Arg399Cys ETV6. Biotinylated EBS DNA probe was incubated with the indicated concentrations of purified recombinant HAT-tagged ETS domain from wild-type or mutant ETV6. Open and closed triangles indicate the positions of the protein-bound and unbound probes, respectively.

Figure 3

ETV6 mutation reduces nuclear localization. (a) Fluorescence images of HeLa cells transiently expressing EGFP-tagged wild-type, Pro214Leu, Arg369Gln or Arg399Cys ETV6. Scale bar, 25 μm. (b) Percentage of cells exhibiting predominantly nuclear (Nuc > Cyto), predominantly cytoplasmic (Cyto > Nuc) or equivalent nuclear and cytoplasmic (Nuc = Cyto) EGFP signal. Three individual experiments were performed, and at least 300 total cells were counted for each condition. Pairwise comparisons between wild-type protein and each mutant were performed using the χ² test (*P < 1 × 10⁻⁴⁸).

Figure 4

ETV6 mutants are deficient in transcriptional repression and act in a dominant-negative manner. (a) Schematic of the pGL3 reporter constructs harboring the MMP3 and PF4 promoters upstream of the firefly luciferase gene. Black rectangles represent core ETS DNA-binding motifs. (b) Protein blot analysis of ETV6 expression in HeLa whole-cell lysates. (c) HeLa cells were cotransfected with the pGL3-MMP3 reporter construct, a pHAGE expression vector (empty vector, wild-type ETV6 or mutant ETV6) and pCS2 Renilla luciferase. Firefly to Renilla luciferase ratios (Fluc/Rluc) were calculated to control for transfection efficiency. Bars show the mean (+ s.e.m.) fold change in the Fluc/Rluc ratio relative to empty vector. Data represent at least two individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type (**P < 0.0005). (d) Experiments are as in c except that the pGL3-PF4 reporter construct was used. Data represent at least three individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type (**P < 0.0005). (e) HeLa cells were cotransfected with 50 ng of wild-type ETV6 expression vector with increasing amounts (50, 150 and 250 ng) of ETV6 expression vector encoding the Pro214Leu, Arg369Gln, Arg399Cys or monomeric Arg399Cys mutant, pGL3-PF4 reporter construct and pCS2 Renilla luciferase. Bars show the mean (+ s.e.m.) fold change in the Fluc/Rluc ratio relative to empty vector. Data represent at least three individual experiments for each condition with duplicate measurements. Pairwise Student’s t tests were performed comparing each condition to wild type alone (*P < 0.005, **P < 0.0005; NS, not significant).

Figure 5

ETV6 mutants impair hematopoietic stem cell proliferation and alter the ETV6 transcriptome. (a) Proliferation of human CD34⁺ cells expressing wild-type, Pro214Leu, Arg369Gln or Arg399Cys ETV6 cultured under non-differentiating conditions. Viable cells were counted in triplicate every 2 d. Plotted points represent means ± s.d. Pairwise Student’s t tests were performed comparing each mutant to wild-type ETV6 on day 6 (*P < 0.01). (b) Genome-wide mRNA expression profiling with wild-type or mutant ETV6. PCA plot of the first two principal components representing 68% of the total variance in the transcriptome data set from K562 cells expressing wild-type protein or the indicated mutant ETV6 species. The data from three independent experiments are shown. (c) Heat map showing the log₂-transformed and mean-centered transcript levels for differentially expressed genes in K562 cells expressing wild-type ETV6 or the indicated mutant ETV6 species. Differentially expressed genes were partitioned into seven distinct clusters by k-means clustering using Euclidian distance. Yellow and blue indicate higher and lower expression, respectively.