Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease (original) (raw)

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References

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Acknowledgements

We thank J. Chilton, A. Damhuis, R. Raman and B. Yang for technical assistance. This work was supported by the UK National Institute for Health Research (NIHR) Exeter Clinical Research Facility through funding for S.E. and A.T.H. A.T.H. is an NIHR Senior Investigator. S.E. and A.T.H. are supported by Wellcome Trust Senior Investigator awards. Further funding was provided by Diabetes UK and the Finnish Medical Foundation.

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Author notes

  1. Sarah E Flanagan, Emma Haapaniemi and Mark A Russell: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, UK
    Sarah E Flanagan, Mark A Russell, Richard Caswell, Hana Lango Allen, Elisa De Franco, Timothy J McDonald, Noel G Morgan, Sian Ellard & Andrew T Hattersley
  2. Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland
    Emma Haapaniemi & Juha Kere
  3. Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland
    Emma Haapaniemi, Kaarina Heiskanen, Timo Otonkoski & Juha Kere
  4. Department of Hematology, Hematology Research Unit Helsinki, University of Helsinki, Helsinki, Finland
    Hanna Rajala & Satu Mustjoki
  5. Helsinki University Central Hospital Cancer Center, Helsinki, Finland
    Hanna Rajala & Satu Mustjoki
  6. Department of Clinical Sciences, Lund University, Lund, Sweden
    Anita Ramelius & Åke Lernmark
  7. Clinical Research Centre, Skåne University Hospital, Malmö, Sweden
    Anita Ramelius & Åke Lernmark
  8. Bristol Royal Hospital for Children, Bristol, UK
    John Barton
  9. Children's Hospital, Helsinki University Central Hospital, Helsinki, Finland
    Kaarina Heiskanen, Merja Kajosaari & Timo Otonkoski
  10. Department of Pediatrics, Kuopio University Hospital, Helsinki, Finland
    Tarja Heiskanen-Kosma
  11. Department of Diabetes and Endocrinology, Children's University Hospital, Dublin, Ireland
    Nuala P Murphy
  12. Department of Endocrinology, Institute for Mother and Child Health Care of Serbia 'Dr Vukan Cupic', Belgrade, Serbia
    Tatjana Milenkovic
  13. Division of Infectious Diseases, Immunodeficiency Unit, Helsinki University Central Hospital, Helsinki, Finland
    Mikko Seppänen
  14. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden
    Juha Kere
  15. Center for Innovative Medicine, Karolinska Institutet, Huddinge, Sweden
    Juha Kere

Authors

  1. Sarah E Flanagan
  2. Emma Haapaniemi
  3. Mark A Russell
  4. Richard Caswell
  5. Hana Lango Allen
  6. Elisa De Franco
  7. Timothy J McDonald
  8. Hanna Rajala
  9. Anita Ramelius
  10. John Barton
  11. Kaarina Heiskanen
  12. Tarja Heiskanen-Kosma
  13. Merja Kajosaari
  14. Nuala P Murphy
  15. Tatjana Milenkovic
  16. Mikko Seppänen
  17. Åke Lernmark
  18. Satu Mustjoki
  19. Timo Otonkoski
  20. Juha Kere
  21. Noel G Morgan
  22. Sian Ellard
  23. Andrew T Hattersley

Contributions

S.E.F., S.E. and A.T.H. designed the study. N.P.M., T.M., T.O., E.H., K.H., T.H.-K., M.K., A.R., A.L. and J.B. recruited subjects to the study. R.C. and E.D.F. performed the exome sequencing and targeted next-generation sequence analysis. H.L.A. performed the bioinformatics analysis. S.E.F. and E.H. performed the Sanger sequencing analysis and the interpretation of the resulting data. S.E.F., T.J.M., E.H., M.S., J.K. and A.T.H. analyzed the clinical data. M.A.R. and N.G.M. designed and performed the functional studies. H.R. and S.M. performed the T cell assays. S.E.F., M.A.R. and A.T.H. prepared the draft manuscript. All authors contributed to discussion of the results and to manuscript preparation.

Corresponding authors

Correspondence toJuha Kere, Noel G Morgan or Andrew T Hattersley.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 STAT3 expression in HEK293 cells

Western blot showing the expression of STAT3 protein in HEK293 cells transfected with STAT3 constructs. HEK293 cells were lysed, and protein extracts were probed with anti-STAT3 antibody. β-actin was used as a loading control. The experiment was repeated twice with similar results.

Supplementary Figure 2 Genotype-phenotype relationship in STAT3 alterations.

The predicted effects of the STAT3 alterations were modeled in PDB structure 1BG1 (mouse STAT3/DNA complex) using SWISS-MODEL and visualized in the Swiss-PdbViewer. (a) Overview of the STAT3 dimer bound to DNA; STAT3 chains are shown in ribbon form, with residues N646 (red) and N647 (green) shown as space-filling residues on the left chain only; DNA strands are shown as blue and turquoise ribbons. (b) As in a, but expanded to show the proximity of residues N646 and N647 to both the DNA-binding and dimerization surfaces. (c) Predicted molecular surfaces of wild-type STAT3 (wt) and mutants N646K, N647D and N647I; surfaces are colored for positive charge (blue; top row), negative charge (red; middle row) and hydrophobicity (brown (most polar) to blue (most hydrophobic); bottom row); structures have been rotated compared to in a and b to show relevant groups more clearly. The N646K alteration reported here results in increased positive charge (circled, N646K column, upper row) at the DNA-binding surface; this is likely to result in higher DNA binding affinity due to electrostatic interaction with the DNA backbone and, hence, increased STAT3 activity. Conversely, the N647D substitution, previously reported as a loss-of-function alteration in HIES, leads to increased negative surface charge in this region (circled, N647D column, middle row) and is likely to inhibit DNA binding and/or dimerization. By comparison, a different substitution at this position, N647I, has been previously reported as an activating alteration in LGLL; it has been postulated that STAT3 mutations in LGLL promote STAT3 dimerization and, hence, biological activity, as a result of increased hydrophobicity at the dimerization surface. This is consistent with protein modeling in silico, which predicts increased hydrophobicity in this region (circled, N646I column, bottom row) compared to wild-type STAT3 or other variants.

Supplementary Figure 3 Increased basal STAT3 activity in vitro.

The intracellular expression of IFN-γ and TNF-α was measured from unstimulated and stimulated (anti-CD3, anti-CD28, anti-CD49d) CD4+ and CD8+ T cells after 6-h incubation using flow cytometry. Samples were available from six healthy controls and two patients (patient 3 (p.K392R) and patient 5 (p.K658N)). In all analyzed cases, the expression of IFN-γ/TNF-α was under 1% in unstimulated cells. The median percentage of stimulated cytokine-producing CD4+ and CD8+ cells was 8.0% and 12%, respectively, among healthy controls (data of one control shown). CD4+ cells from patient 2 showed increased cytokine production when stimulated. IFN, interferon; TCR, T cell receptor; TNF, tumor necrosis factor.

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Flanagan, S., Haapaniemi, E., Russell, M. et al. Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.Nat Genet 46, 812–814 (2014). https://doi.org/10.1038/ng.3040

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