Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia

Abstract

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Figure 1

Mutation analysis of ETV6. (a) Pedigrees for three affected families. Solid symbols represent affected individuals with thrombocytopenia and high red cell mean corpuscular volume. Asterisks represent individuals that developed B cell leukemia. (See Supplementary Table 1 for CBC values). (b) Representative images of a bone marrow aspirate for one of the affected individuals without leukemia that shows mild dyserythropoiesis in the left panel (yellow arrows) and an immature hypolobulated megakaryocyte (yellow arrow) on the right panel. (c) Schematic of ETV6, which is composed of 8 exons encoding untranslated regions (yellow) and protein coding sequences (blue). Two mutations, c.641C>T in exon 5 and c.1252A>G in exon 7 are depicted. In the lower section of the panel a schematic of ETV6 is represented with its different domains, pointed (PNT), central and ETS (which is the DNA-binding domain) and the location of the amino acid changes. (d) Effects of ETV6 mutant alleles (p.Pro214Leu, p.Arg418Gly, and p.385_418del) in the activity of an ETV6-responsive reporter plasmid (pGL2-754TR) when expressed alone or with the wild type allele. Luciferase activity, represented here by fold repression, is shown relative to empty vector and normalized using an internal control plasmid expressing Renilla luciferase. Wild type ETV6 (WT) repressed luciferase expression by approximately 7.5 fold compared to empty vector, whereas p.P214L ETV6, p.R418G, and p.385_418del repressed transcription 4.5, 1.5, and 1.5 fold, respectively (p<0.0001, one way ANOVA) (Figure 1d). Co-expression of WT and mutant proteins caused intermediate repression of the stromelysin-1 promoter. Experiments were done in triplicates and repeated 4 times (error bars = standard deviation).

Figure 2

Abnormal development of day 12 cultured megakaryocytes expressing mutant ETV6. Control cells and those transduced with lentivirus to express myc-tagged forms of ETV6 (ETV6^WT, ETV6^P214L and ETV6^R418G) were assessed via immunofluorescence microscopy imaging; scale bars = 5 μm. (a–c) Megakaryocytes (control cells shown) were identified via expression of CD61 (violet) and VWF (red) and staged by diameter: <15 μm (a), >15μm (b), or the presence of proplatelets (c). (d) Population distributions showed no significant differences between control and ETV6^WT-transduced cells, while ETV6^P214L-transduced megakaryocytes showed a significantly higher proportion (*P<0.05, 2-tailed t-test) of cells in the earlier developmental stage (<15 μm) and fewer in the mature proplatelet-forming stage (control: n=7, WT: n=3, P214L: n=3, R418G: n=4 cultures with >300 cells for each; error bars = standard deviation). Images for control cells and those transduced with WT, P214L and R418G forms of ETV6 are shown in Supplementary Fig 6.

Figure 3

Aberrant cytoplasmic localization of ETV6 in cultured megakaryocytes transduced with ETV6 mutants. Comparison via immunofluorescence microscopy of ETV6 localization in Day 12 cultured control megakaryocytes and those transduced with lentiviral wild-type (ETV6^WT) or mutant (ETV6^P214L, ETV6^R418G) myc-tagged forms of ETV6. Confocal Z-sections of representative mature cells (>15μm) are shown stained for DNA (blue), ETV6 (both endogenous and myc-tagged; green) and myc (red). In control cells endogenous ETV6 is concentrated in the nucleus, as is endogenous and myc-tagged WT ETV6 in cells transduced with ETV6^WT. In contrast, cells transduced with mutant myc-tagged ETV6 (ETV6^P214L, ETV6^R418G) show this protein concentrated in the cytoplasm with little visible in the nucleus. Scale bars = 5 μm. See also Videos 1–4.