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Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

Nature Genetics volume 47, pages 180–185 (2015)Cite this article

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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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Sequence Read Archive

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NCBI Reference Sequence

Protein Data Bank

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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.

Author information

Authors and Affiliations

  1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Michael Y Zhang, Keith R Loeb, Marilyn Sanchez-Bonilla, Boglarka Gyurkocza, Scott A Coats & Akiko Shimamura

  2. Section of Hematology/Oncology, Center for Clinical Cancer Genetics, University of Chicago, Chicago, Illinois, USA

    Jane E Churpek, Barbara Neistadt, Rafael Marquez, Christopher J Mariani & Lucy A Godley

  3. Comprehensive Cancer Center, University of Chicago, Chicago, Illinois, USA

    Jane E Churpek, Barbara Neistadt, Rafael Marquez, Christopher J Mariani & Lucy A Godley

  4. Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington, USA

    Siobán B Keel & Janis L Abkowitz

  5. Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

    Tom Walsh, Ming K Lee, Suleyman Gulsuner & Mary-Claire King

  6. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Tom Walsh, Ming K Lee, Suleyman Gulsuner & Mary-Claire King

  7. Department of Pathology, University of Washington, Seattle, Washington, USA

    Keith R Loeb & Marshall S Horwitz

  8. Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA

    Colin C Pritchard

  9. Genomics and Bioinformatics Shared Resources, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    Jeffrey J Delrow & Ryan S Basom

  10. Pediatric Hematology Oncology, Madigan Army Medical Center, Tacoma, Washington, USA

    Melissa Forouhar

  11. Morgridge Institute for Research, University of Wisconsin, Madison, Wisconsin, USA

    Bradford S Schwartz

  12. Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA

    Bradford S Schwartz

  13. Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Inga Hofmann & David A Williams

  14. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA

    Inga Hofmann & David A Williams

  15. Harvard Stem Cell Institute, Boston, Massachusetts, USA

    Inga Hofmann & David A Williams

  16. Division of Hematology, Brigham and Women's Hospital, Boston, Massachusetts, USA

    R Coleman Lindsley

  17. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    R Coleman Lindsley

  18. Pediatric Hematology/Oncology, Seattle Children's Hospital, Seattle, Washington, USA

    Akiko Shimamura

  19. Department of Pediatrics, University of Washington, Seattle, Washington, USA

    Akiko Shimamura

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  1. Michael Y Zhang
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  2. Jane E Churpek
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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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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).

Source data

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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