Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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 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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

BioProject

Sequence Read Archive

Referenced accessions

NCBI Reference Sequence

Swiss-Prot

References

  1. Song, W.J. et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat. Genet. 23, 166–175 (1999).

    Article  CAS  Google Scholar 

  2. Nichols, K.E. et al. Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1. Nat. Genet. 24, 266–270 (2000).

    Article  CAS  Google Scholar 

  3. Kar, A. & Gutierrez-Hartmann, A. Molecular mechanisms of ETS transcription factor–mediated tumorigenesis. Crit. Rev. Biochem. Mol. Biol. 48, 522–543 (2013).

    Article  CAS  Google Scholar 

  4. Romana, S.P. et al. Deletion of the short arm of chromosome 12 is a secondary event in acute lymphoblastic leukemia with t(12;21). Leukemia 10, 167–170 (1996).

    CAS  PubMed  Google Scholar 

  5. Patel, N. et al. Expression profile of wild-type ETV6 in childhood acute leukaemia. Br. J. Haematol. 122, 94–98 (2003).

    Article  CAS  Google Scholar 

  6. Barjesteh van Waalwijk van Doorn-Khosrovani, S. et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene 24, 4129–4137 (2005).

    Article  Google Scholar 

  7. Bejar, R. et al. Clinical effect of point mutations in myelodysplastic syndromes. N. Engl. J. Med. 364, 2496–2506 (2011).

    Article  CAS  Google Scholar 

  8. Van Vlierberghe, P. et al. ETV6 mutations in early immature human T cell leukemias. J. Exp. Med. 208, 2571–2579 (2011).

    Article  CAS  Google Scholar 

  9. Zhang, M.Y. et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nat. Genet. 47, 180–185 (2015).

    Article  CAS  Google Scholar 

  10. Rasighaemi, P., Onnebo, S.M., Liongue, C. & Ward, A.C. ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish. Haematologica 100, 23–31 (2015).

    Article  CAS  Google Scholar 

  11. Wang, L.C. et al. Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL. EMBO J. 16, 4374–4383 (1997).

    Article  CAS  Google Scholar 

  12. Wang, L.C. et al. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes Dev. 12, 2392–2402 (1998).

    Article  CAS  Google Scholar 

  13. Hock, H. et al. Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes Dev. 18, 2336–2341 (2004).

    Article  CAS  Google Scholar 

  14. Pereira, C.F. et al. Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell 13, 205–218 (2013).

    Article  CAS  Google Scholar 

  15. Orkin, S.H. et al. Abnormal RNA processing due to the exon mutation of β E-globin gene. Nature 300, 768–769 (1982).

    Article  CAS  Google Scholar 

  16. Green, S.M., Coyne, H.J. III, McIntosh, L.P. & Graves, B.J. DNA binding by the ETS protein TEL (ETV6) is regulated by autoinhibition and self-association. J. Biol. Chem. 285, 18496–18504 (2010).

    Article  CAS  Google Scholar 

  17. Kwiatkowski, B.A. et al. The ets family member Tel binds to the Fli-1 oncoprotein and inhibits its transcriptional activity. J. Biol. Chem. 273, 17525–17530 (1998).

    Article  CAS  Google Scholar 

  18. Raslova, H. et al. FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia. J. Clin. Invest. 114, 77–84 (2004).

    Article  CAS  Google Scholar 

  19. Million, R.P., Harakawa, N., Roumiantsev, S., Varticovski, L. & Van Etten, R.A. A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase. Mol. Cell. Biol. 24, 4685–4695 (2004).

    Article  CAS  Google Scholar 

  20. Roukens, M.G., Alloul-Ramdhani, M., Moghadasi, S., Op den Brouw, M. & Baker, D.A. Downregulation of vertebrate Tel (ETV6) and Drosophila Yan is facilitated by an evolutionarily conserved mechanism of F-box-mediated ubiquitination. Mol. Cell. Biol. 28, 4394–4406 (2008).

    Article  CAS  Google Scholar 

  21. Lopez, R.G. et al. TEL is a sequence-specific transcriptional repressor. J. Biol. Chem. 274, 30132–30138 (1999).

    Article  CAS  Google Scholar 

  22. Fenrick, R. et al. TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1. Mol. Cell. Biol. 20, 5828–5839 (2000).

    Article  CAS  Google Scholar 

  23. Machlus, K.R. & Italiano, J.E. Jr. The incredible journey: from megakaryocyte development to platelet formation. J. Cell Biol. 201, 785–796 (2013).

    Article  CAS  Google Scholar 

  24. Mullighan, C.G. et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 446, 758–764 (2007).

    Article  CAS  Google Scholar 

  25. Strehl, S., Konig, M., Dworzak, M.N., Kalwak, K. & Haas, O.A. PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13). Leukemia 17, 1121–1123 (2003).

    Article  CAS  Google Scholar 

  26. Wu, T.D. & Nacu, S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010).

    Article  CAS  Google Scholar 

  27. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    Article  CAS  Google Scholar 

  28. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

    Article  Google Scholar 

  29. Liu, X., Jian, X. & Boerwinkle, E. dbNSFP: a lightweight database of human nonsynonymous SNPs and their functional predictions. Hum. Mutat. 32, 894–899 (2011).

    Article  CAS  Google Scholar 

  30. Rowley, J.W. et al. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes. Blood 118, e101–e111 (2011).

    Article  CAS  Google Scholar 

  31. Love, M., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. bioRxiv. http://dx.doi.org/10.1101/002832.

  32. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  Google Scholar 

  33. Kim, D. & Salzberg, S. TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol. 12, R72 (2011).

    Article  CAS  Google Scholar 

  34. Ge, H. et al. FusionMap: detecting fusion genes from next-generation sequencing data at base-pair resolution. Bioinformatics 27, 1922–1928 (2011).

    Article  CAS  Google Scholar 

  35. Wilcox, D.A. et al. Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII. J. Thromb. Haemost. 1, 2477–2489 (2003).

    Article  CAS  Google Scholar 

  36. Kahr, W.H. et al. Abnormal megakaryocyte development and platelet function in Nbeal2−/− mice. Blood 122, 3349–3358 (2013).

    Article  CAS  Google Scholar 

  37. Urban, D. et al. The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis. Blood 120, 5032–5040 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

Authors

Contributions

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, Christopher C Porter or Jorge Di Paola.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/ng.3253

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3253

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing