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


Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia1,2. 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.

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Figure 1: Mutation analysis of ETV6.
Figure 2: Abnormal development of cultured megakaryocytes expressing mutant ETV6 at day 12.
Figure 3: Aberrant cytoplasmic localization of ETV6 in cultured megakaryocytes transduced with lentivirus expressing ETV6 mutants.

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NCBI Reference Sequence



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We are grateful to the families studied for their contribution to this project. We are also grateful to T. Shaikh, R. Spritz and J. Murray for their insightful comments. This work was supported by the Postle Family Chair in Pediatric Cancer and Blood Disorders (J.D.P.) and by US National Institutes of Health grants HL112311 (A.S.W.) and GM103806 (J.W.R.). W.H.A.K. was supported by operating grants from the Canadian Institutes of Health Research (CIHR; MOP-81208 and MOP-259952). P.N. and A.S. were supported by grant GGP13082 from the Telethon Foundation.

Author information




L.N., R.W.L., A.B.L.-S., A.S.W., W.H.A.K., C.C.P. and J.D.P. conceived and designed the experiments. L.N., R.W.L., A.B.L.-S., R.L., F.G.P., L. Li, L. Lu, A.S., C.G. and D.D.R. performed experiments and provided critical data. M.C., M.R., P.N., C.L.B., A.P., M.D., A.G.-H., L.X. and C.L.-M. provided patient samples and study materials, and collected and assembled data. S.H., P.H. and A.G.-H. analyzed data. K.J., K.G. and J.W.R. analyzed genomic and transcriptome data. L.N., R.W.L., A.B.L.-S., F.G.P., A.S.W., W.H.A.K., C.C.P. and J.D.P. wrote the manuscript. All authors reviewed and contributed to the final version of the manuscript. A.S.W., W.H.A.K., C.C.P. and J.D.P. jointly supervised the research.

Corresponding authors

Correspondence to Walter H A Kahr or Christopher C Porter or Jorge Di Paola.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Platelets from affected patients are comparable to controls on electron micrograph.

Thin-section transmission electron micrographs of representative platelets from a normal control (a) and an affected patient (b). The ultrastructure of patient-derived platelets is comparable to that of control cells, with occasional elongated α-granules seen in the former. Scale bars, 500 nm.

Supplementary Figure 2 The c.1252A>G change is a splice-site mutation.

RT-PCR of the transcript encoded by exons 6–8 of ETV6 for individuals I-2 and II-3 of family 3 with the c.1252A>G mutation. (a) Two transcripts are visible in the gel when this region is amplified: the expected 386-bp transcript as seen in the unaffected control (C+) and a smaller, 285-bp transcript, which by reverse sequencing, demonstrate skipping of exon 7 and a peak for a G substitution, indicating leakage of the missense mutation (b,c). (d) The sequence of the cloned product confirms the presence of the G nucleotide in exon 7.

Supplementary Figure 3 Immunoblots of ETV6 protein expression in transfected HEK293T cells.

Plasmids containing DDK-tagged cDNA encoding either WT, p.P214L, p.R418G (missense mutation acquired by c.1252A>G) or p.385_418del (deletion mutation acquired by c.1252A>G) ETV6 were transiently transfected into HEK293T cells. Whole-cell lysates were separated by 7.5% SDS-PAGE and probed with an anti-DDK antibody. Blots show equal expression of WT (lane 1), p.P214L (lane 2), p.R418G (lane 3) and p.385_418del (lane 4). A smaller protein is seen when p.385_418del is expressed.

Supplementary Figure 4 Immunoblot analysis of ETV6 protein content in patient-derived and control platelets.

Reduced whole-cell lysates from the equivalent of 107 platelets were loaded in each lane, with equal loading confirmed by probing for GAPDH. Subjects were as described in the Figure 1 pedigrees with the addition of two unrelated normal controls. ETV6 protein size and platelet levels were similar for affected individuals (family 1, II-1 and III-1; family 3, I-2 and II-3), an unaffected relative (family 1, III-2) and controls.

Supplementary Figure 5 Dimerization of mutants with WT ETV6.

Cell lysates of HEK293T cells transfected with WT ETV6-Myc/DDK or WT ETV6-His alone or cotransfected with WT ETV6-His in addition to Myc/DDK-tagged WT ETV6, c.641C>T (p.P214L) ETV6, c.1252A>G (p.R418G) ETV6 or c.1153_1253del (p.385_418del) ETV6 were incubated with anti-Myc antibody–conjugated beads, and protein complexes were isolated. Eluate was probed for DDK (top) and His (bottom). Lanes 1 and 2 show that WT ETV6-Myc/DDK was pulled down by the beads, but WT ETV6-His was not. Cotransfection experiments (lanes 3–6) show that both the indicated Myc/DDK-tagged protein and the His-tagged WT ETV6 protein were pulled down, indicating that the WT ETV6-His protein was complexed with the corresponding Myc/DDK-tagged protein and that dimerization occurred.

Supplementary Figure 6 Subcellular distribution of protein produced by transduced wild-type and mutant ETV6 alleles in maturing (>15-μm) day 12 cultured megakaryocytes.

Protein produced by transduced alleles was detected via staining for Myc tag (green), while size and stage were determined from nuclear morphology (DNA, blue) and staining for megakaryocyte-specific Von Willebrand factor (VWF, red) and CD61 (magenta). Transduced cells showed differing subcellular distribution patterns of Myc-tagged ETV6, with ETV6P214L and ETV6R418G showing a largely cytoplasmic distribution, in contrast to the nuclear localization observed for ETV6WT. Confocal z sections; scale bars, 5 μm.

Supplementary Figure 7 Transcriptional changes induced by mutant ETV6.

(a) Relationship of RNA-seq transcript profiles in platelets between two affected individuals (P214L mutation), two unaffected relatives and three unrelated controls. Shown is the sample-to-sample distance matrix, with hierarchical clustering using rlog-transformed read counts, of 15,865 detected transcripts. (b) Hierarchical clustering and heat map analysis of the relative expression of 351 transcripts involved in platelet biogenesis or function. Expression values are DESeq2 normalized and rlog transformed.

Supplementary Figure 8 Heat map analysis of the relative expression of 351 transcripts involved in platelet biogenesis or function.

Heat map showing higher-resolution hierarchical clustering of all transcripts associated with platelet function or platelet biogenesis. Expression values are DESeq2 normalized and rlog transformed. This list of transcripts was curated from the Reactome, and Gene Ontology databases and from transcripts enriched in platelets compared to all other tissues in Illumina’s Human Body Map 2.0.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 630 kb)

Supplementary Video 1

The nuclear concentration of ETV6 observed for endogenously expressed protein (control, non-transduced megakaryocyte). Three-dimensional volume renders (maximum intensity) prepared from confocal laser fluorescence microscopy z sections of representative day 12 cultured megakaryocytes stained for ETV6 (green), DNA (blue) and tubulin (magenta) via Imaris 7.6. (MOV 5669 kb)

Supplementary Video 2

Nuclear concentration of ETV6 is also seen in a cell transduced with wild-type ETV6. Three-dimensional volume renders (maximum intensity) prepared from confocal laser fluorescence microscopy z sections of representative day 12 cultured megakaryocytes stained for ETV6 (green), DNA (blue) and tubulin (magenta) via Imaris 7.6. (MOV 3554 kb)

Supplementary Video 3

Cells expressing ETV6 P214L show extensive cytoplasmic ETV6 staining. Three-dimensional volume renders (maximum intensity) prepared from confocal laser fluorescence microscopy z sections of representative day 12 cultured megakaryocytes stained for ETV6 (green), DNA (blue) and tubulin (magenta) via Imaris 7.6. (MOV 7364 kb)

Supplementary Video 4

Cells expressing ETV6 R418G show extensive cytoplasmic ETV6 staining. Three-dimensional volume renders (maximum intensity) prepared from confocal laser fluorescence microscopy z sections of representative day 12 cultured megakaryocytes stained for ETV6 (green), DNA (blue) and tubulin (magenta) via Imaris 7.6. (MOV 4899 kb)

Supplementary Data Set

List of 351 platelet-specific transcripts. (XLSX 79 kb)

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Noetzli, L., Lo, R., Lee-Sherick, A. et al. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nat Genet 47, 535–538 (2015).

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