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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.

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Figure 1: New ETV6 germline variants encoding p.Pro214Leu, p.Arg369Gln and p.Arg399Cys in association with thrombocytopenia and hematologic malignancy.
Figure 2: Missense alterations in the ETS domain abrogate ETV6 DNA binding.
Figure 3: ETV6 mutation reduces nuclear localization.
Figure 4: ETV6 mutants are deficient in transcriptional repression and act in a dominant-negative manner.
Figure 5: ETV6 mutants impair hematopoietic stem cell proliferation and alter the ETV6 transcriptome.

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NCBI Reference Sequence

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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).

    CAS  PubMed  Google Scholar 

  2. Smith, M.L., Cavenagh, J.D., Lister, T.A. & Fitzgibbon, J. Mutation of CEBPA in familial acute myeloid leukemia. N. Engl. J. Med. 351, 2403–2407 (2004).

    CAS  PubMed  Google Scholar 

  3. Hahn, C.N. et al. Heritable GATA2 mutations associated with familial myelodysplastic syndrome and acute myeloid leukemia. Nat. Genet. 43, 1012–1017 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Kazenwadel, J. et al. Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature. Blood 119, 1283–1291 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pippucci, T. et al. Mutations in the 5′ UTR of ANKRD26, the ankirin repeat domain 26 gene, cause an autosomal-dominant form of inherited thrombocytopenia, THC2. Am. J. Hum. Genet. 88, 115–120 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Noris, P. et al. Mutations in ANKRD26 are responsible for a frequent form of inherited thrombocytopenia: analysis of 78 patients from 21 families. Blood 117, 6673–6680 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Kirwan, M. et al. Exome sequencing identifies autosomal-dominant SRP72 mutations associated with familial aplasia and myelodysplasia. Am. J. Hum. Genet. 90, 888–892 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shah, S. et al. A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia. Nat. Genet. 45, 1226–1231 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Auer, F. et al. Inherited susceptibility to pre B-ALL caused by germline transmission of PAX5 c.547G>A. Leukemia 28, 1136–1138 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Holmfeldt, L. et al. The genomic landscape of hypodiploid acute lymphoblastic leukemia. Nat. Genet. 45, 242–252 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Powell, B.C. et al. Identification of TP53 as an acute lymphocytic leukemia susceptibility gene through exome sequencing. Pediatr. Blood Cancer 60, E1–E3 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Zhang, M.Y. et al. Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity. Haematologica 10.3324/haematol.2014.113456 (19 September 2014).

  13. De, S. et al. Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6. J. Mol. Biol. 426, 1390–1406 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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  PubMed  PubMed Central  Google Scholar 

  16. Coyne, H.J. et al. Autoinhibition of ETV6 (TEL) DNA binding: appended helices sterically block the ETS domain. J. Mol. Biol. 421, 67–84 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chakrabarti, S.R. & Nucifora, G. The leukemia-associated gene TEL encodes a transcription repressor which associates with SMRT and mSin3A. Biochem. Biophys. Res. Commun. 264, 871–877 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Park, H., Seo, Y., Kim, J.I., Kim, W. & Choe, S.Y. Identification of the nuclear localization motif in the ETV6 (TEL) protein. Cancer Genet. Cytogenet. 167, 117–121 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Fenrick, R. et al. Both TEL and AML-1 contribute repression domains to the t(12;21) fusion protein. Mol. Cell. Biol. 19, 6566–6574 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  22. 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  PubMed  Google Scholar 

  23. Kim, C.A. et al. Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression. EMBO J. 20, 4173–4182 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 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  PubMed  PubMed Central  Google Scholar 

  25. 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  PubMed  PubMed Central  Google Scholar 

  26. Pritchard, C.C. et al. Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens. J. Mol. Diagn. 16, 56–67 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Marquez, R. et al. A new family with a germline ANKRD26 mutation and predisposition to myeloid malignancies. Leuk. Lymphoma 10.3109/10428194.2014.903476 (22 April 2014).

  28. Novershtern, N. et al. Densely interconnected transcriptional circuits control cell states in human hematopoiesis. Cell 144, 296–309 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bluteau, D. et al. Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation. J. Clin. Invest. 124, 580–591 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330–337 (2012).

  31. Seshagiri, S. et al. Recurrent R-spondin fusions in colon cancer. Nature 488, 660–664 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Welch, J.S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Walter, M.J. et al. Clonal diversity of recurrently mutated genes in myelodysplastic syndromes. Leukemia 27, 1275–1282 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang, J. et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 481, 157–163 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Xu, L. et al. Genomic landscape of CD34+ hematopoietic cells in myelodysplastic syndrome and gene mutation profiles as prognostic markers. Proc. Natl. Acad. Sci. USA 111, 8589–8594 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ding, L. et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature 481, 506–510 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yoshida, K. et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature 478, 64–69 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Dolnik, A. et al. Commonly altered genomic regions in acute myeloid leukemia are enriched for somatic mutations involved in chromatin remodeling and splicing. Blood 120, e83–e92 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Padron, E. et al. ETV6 and signaling gene mutations are associated with secondary transformation of myelodysplastic syndromes to chronic myelomonocytic leukemia. Blood 123, 3675–3677 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Griesinger, F., Janke, A., Podleschny, M. & Bohlander, S.K. Identification of an ETV6-ABL2 fusion transcript in combination with an ETV6 point mutation in a T-cell acute lymphoblastic leukaemia cell line. Br. J. Haematol. 119, 454–458 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Zhang, J. et al. Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group. Blood 118, 3080–3087 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, Q. et al. ETV6 mutation in a cohort of 970 patients with hematologic malignancies. Haematologica 99, e176–e178 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lohr, J.G. et al. Widespread genetic heterogeneity in multiple myeloma: implications for targeted therapy. Cancer Cell 25, 91–101 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hodis, E. et al. A landscape of driver mutations in melanoma. Cell 150, 251–263 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Walsh, T. et al. Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of non-syndromic hearing loss DFNB82. Am. J. Hum. Genet. 87, 90–94 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Gulsuner, S. et al. Spatial and temporal mapping of de novo mutations in schizophrenia to a fetal prefrontal cortical network. Cell 154, 518–529 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Walsh, T. et al. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc. Natl. Acad. Sci. USA 108, 18032–18037 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mostoslavsky, G., Fabian, A.J., Rooney, S., Alt, F.W. & Mulligan, R.C. Complete correction of murine Artemis immunodeficiency by lentiviral vector–mediated gene transfer. Proc. Natl. Acad. Sci. USA 103, 16406–16411 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Delaney, C., Varnum-Finney, B., Aoyama, K., Brashem-Stein, C. & Bernstein, I.D. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 106, 2693–2699 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Anders, S., Pyl, P.T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics doi:10.1093/bioinformatics/btu638 (25 September 2014).

  53. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  Google Scholar 

  54. Reiner, A., Yekutieli, D. & Benjamini, Y. Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19, 368–375 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Yeung, K.Y., Haynor, D.R. & Ruzzo, W.L. Validating clustering for gene expression data. Bioinformatics 17, 309–318 (2001).

    Article  CAS  PubMed  Google Scholar 

  56. Saeed, A.I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003).

    Article  CAS  PubMed  Google Scholar 

  57. Young, M.D., Wakefield, M.J., Smyth, G.K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Burwick, N., Coats, S.A., Nakamura, T. & Shimamura, A. Impaired ribosomal subunit association in Shwachman-Diamond syndrome. Blood 120, 5143–5152 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all patients and their families for participation in this research study. We thank M. Chin (University of Washington), B. Turok-Storb (Fred Hutchinson Cancer Research Center) and S. Tapscott (Fred Hutchinson Cancer Research Center) for luciferase plasmids and reagents. We thank H. Hock, B. Stoddard, S. Meshinchi, G. Smith, A. Kumar, C. Toledo, S. Yu, A. Fong and K. MacQuarrie for helpful discussions. We thank S. Castro for clinical sample processing. This work was supported by US National Institutes of Health grants R24DK093425 and R24DK099808-01 to A.S., M.-C.K. and J.L.A.; by the Ghiglione Aplastic Anemia Fund and Julian's Dinosaur Guild from Seattle Children's Hospital to A.S.; by Medical Scientist Training Program Training grant T32GM007266 and Genetic Approaches to Aging Training grant T32AG000057 to M.Y.Z.; and by grants from the US National Institutes of Health (K12CA139160) and the Cancer Research Foundation to J.E.C. M.-C.K. is an American Cancer Society professor.

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Authors and Affiliations

Authors

Contributions

M.Y.Z., J.E.C., S.B.K., T.W., J.L.A., M.-C.K., L.A.G. and A.S. conceived and designed the experiments. M.Y.Z., S.B.K., T.W., C.C.P., M.S.-B., C.J.M. and S.A.C. performed the experiments. M.Y.Z., S.B.K., T.W., M.K.L., K.R.L., S.G., C.C.P., J.J.D., R.S.B., R.C.L., M.-C.K. and A.S. analyzed the data. J.E.C., S.B.K., M.F., B.G., B.S.S., B.N., R.M., I.H., D.A.W., M.S.H., L.A.G. and A.S. identified study subjects, performed clinical phenotyping and contributed biological samples. M.Y.Z., J.E.C., T.W., J.J.D., L.A.G., M.-C.K. and A.S. wrote the manuscript. A.S. and M.-C.K. jointly supervised the research.

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Correspondence to Akiko Shimamura.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Bone marrow morphology in III-2 of family A.

(a) Pre-transplant peripheral blood smears from patient III-2 illustrating hypogranulated neutrophils (left) and hypolobated neutrophils (pseudo Pelger-Huët cell) (right). (b) Platelets from a Wright-Giemsa stained peripheral blood smear. (c) Upper left, Wright-Giemsa stained particle preparation depicting small, hypolobated megakaryocytes (triangles). Lower left panel: Bone marrow biopsy depicting hypolobated micro-megakaryocytes. Right, megakaryocyte dysplasia.

Supplementary Figure 2 Sanger sequencing of the ETV6 c.1195C>T mutation in family A.

(a) Electropherograms from Sanger sequencing of ETV6 c.1195C>T in peripheral blood genomic DNA from family A. (b) Sanger sequencing of ETV6 c.1195C>T in genomic DNA and cDNA derived from an RAEB-1 MDS bone marrow sample of family A III-2 shows no loss of heterozygosity.

Supplementary Figure 3 The ETV6 p.Arg369Gln mutation disrupts internal hydrogen bonding.

(a) Hydrogen bonding (dotted lines) between the guanidinium nitrogen of Arg369 (orange) in β sheet 2 with the backbone carbonyl oxygen of Arg414 (magenta) in the wing of the ETS domain. Protein structure of the murine Etv6 ETS domain (PDB ID: 4MHG) is shown. The ETS domains of mouse and human ETV6 have 100% amino acid sequence identity. (b) Molecular modeling of the Arg369Gln (orange) variant using SWISS-MODEL predicts loss of this hydrogen bonding interaction.

Source data

Supplementary Figure 4 Cell fractionation shows decreased nuclear localization of mutant ETV6.

HeLa cells were transfected with empty vector, cDNA for wild-type ETV6 or ETV6 cDNA encoding p.Arg399Cys. Lysates of nuclear versus cell fractions as well as whole-cell lysates were analyzed by western blot for ETV6, GAPDH (cytoplasmic marker) and NPM1 (nuclear marker).

Source data

Supplementary Figure 5 Clonal evolution of MDS in the context of germline ETV6 mutation.

Analysis of germline and serial bone marrow samples in family A III-2. Electropherograms from Sanger sequencing of BCOR, RUNX1, and KRAS mutations in genomic DNA derived from (a) marrow fibroblasts, (b) bone marrow mononuclear cells at age 17 when the patient had refractory cytopenias with multilineage dysplasia (RCMD) and (c) bone marrow mononuclear cells at age 21 when the patient’s disease progressed to refractory anemia with excess blasts type 1 (RAEB-1).

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–3 and 8–10, and Supplementary Note. (PDF 2630 kb)

Supplementary Table 4

Genes downregulated with mutant ETV6 compared to WT ETV6. (XLSX 70 kb)

Supplementary Table 5

Genes upregulated with mutant ETV6 compared to WT ETV6. (XLSX 76 kb)

Supplementary Table 6

GO-seq categories for genes downregulated with mutant ETV6 compared to WT ETV6. (XLSX 77 kb)

Supplementary Table 7

GO-seq categories for genes upregulated with mutant ETV6 compared to WT ETV6. (XLSX 46 kb)

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Zhang, M., Churpek, J., Keel, S. et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nat Genet 47, 180–185 (2015). https://doi.org/10.1038/ng.3177

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