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.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

    & Molecular mechanisms of ETS transcription factor–mediated tumorigenesis. Crit. Rev. Biochem. Mol. Biol. 48, 522–543 (2013).

  4. 4.

    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).

  5. 5.

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

  6. 6.

    et al. Somatic heterozygous mutations in ETV6 (TEL) and frequent absence of ETV6 protein in acute myeloid leukemia. Oncogene 24, 4129–4137 (2005).

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    , , & ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish. Haematologica 100, 23–31 (2015).

  11. 11.

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

  12. 12.

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

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

    , , & DNA binding by the ETS protein TEL (ETV6) is regulated by autoinhibition and self-association. J. Biol. Chem. 285, 18496–18504 (2010).

  17. 17.

    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).

  18. 18.

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

  19. 19.

    , , , & 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).

  20. 20.

    , , , & 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).

  21. 21.

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

  22. 22.

    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).

  23. 23.

    & The incredible journey: from megakaryocyte development to platelet formation. J. Cell Biol. 201, 785–796 (2013).

  24. 24.

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

  25. 25.

    , , , & PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13). Leukemia 17, 1121–1123 (2003).

  26. 26.

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

  27. 27.

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

  28. 28.

    , & ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).

  29. 29.

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

  30. 30.

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

  31. 31.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. bioRxiv. .

  32. 32.

    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).

  33. 33.

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

  34. 34.

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

  35. 35.

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

  36. 36.

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

  37. 37.

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

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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

Author notes

    • Leila Noetzli
    • , Richard W Lo
    • , Walter H A Kahr
    • , Christopher C Porter
    •  & Jorge Di Paola

    These authors contributed equally to this work.

    • Walter H A Kahr
    • , Christopher C Porter
    •  & Jorge Di Paola

    These authors jointly supervised this work.

Affiliations

  1. Department of Pediatrics, University of Colorado Anschutz Medical Campus (AMC), Aurora, Colorado, USA.

    • Leila Noetzli
    • , Alisa B Lee-Sherick
    • , Stephen Hunger
    • , Christopher C Porter
    •  & Jorge Di Paola
  2. Human Medical Genetics and Genomics Program, University of Colorado AMC, Aurora, Colorado, USA.

    • Leila Noetzli
    •  & Jorge Di Paola
  3. Program in Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.

    • Richard W Lo
    • , Ling Li
    • , Lily Lu
    • , Richard Leung
    • , Fred G Pluthero
    •  & Walter H A Kahr
  4. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

    • Richard W Lo
    •  & Walter H A Kahr
  5. Children's Hospital of Michigan, Department of Pediatrics, Wayne State University, Detroit, Michigan, USA.

    • Michael Callaghan
    •  & Madhvi Rajpurkar
  6. Department of Internal Medicine, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico San Matteo Foundation, University of Pavia, Pavia, Italy.

    • Patrizia Noris
    • , Carlo L Balduini
    •  & Alessandro Pecci
  7. Department of Medical Sciences, University of Trieste, Trieste, Italy.

    • Anna Savoia
    • , Chiara Gnan
    •  & Daniela De Rocco
  8. Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.

    • Anna Savoia
    • , Chiara Gnan
    •  & Daniela De Rocco
  9. Department of Biochemistry and Molecular Genetics, University of Colorado AMC, Aurora, Colorado, USA.

    • Kenneth Jones
    • , Katherine Gowan
    •  & Arthur Gutierrez-Hartmann
  10. Department of Internal Medicine, Haematology/Oncology, University Hospital Brno, Brno, Czech Republic.

    • Michael Doubek
  11. Department of Medicine, Division of Rheumatology, University of Toronto, University Health Network, Toronto, Ontario, Canada.

    • Carolina Landolt-Marticorena
  12. Instituto de Investigaciones Médicas Alfredo Lanari, Universidad de Buenos Aires, Buenos Aires, Argentina.

    • Paula Heller
  13. Department of Medicine, University of Colorado AMC, Aurora, Colorado, USA.

    • Arthur Gutierrez-Hartmann
  14. Department of Pathology, University of Colorado AMC, Aurora, Colorado, USA.

    • Liang Xiayuan
  15. Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA.

    • Jesse W Rowley
    •  & Andrew S Weyrich
  16. Molecular Medicine Program, University of Utah, Salt Lake City, Utah, USA.

    • Jesse W Rowley
    •  & Andrew S Weyrich
  17. Department of Paediatrics, Division of Haematology/Oncology, University of Toronto and The Hospital for Sick Children, Toronto, Ontario, Canada.

    • Walter H A Kahr

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1 and 2, and Supplementary Note.

Videos

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

Excel files

  1. 1.

    Supplementary Data Set

    List of 351 platelet-specific transcripts.

About this article

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DOI

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

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