Abstract

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

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Acknowledgements

We thank the patient and his family for participating in this study; B. Fleckenstein and M. Schmidt for generating and providing patient-derived T cell lines; and G. Superti-Furga, J. Bigenzahn for providing the inducible protein expression system used for Jurkat T cells and together with N. Serwas, C.D. Conde, A. Kalinichenko, K. Ackerman and R. Martins for critically reviewing the manuscript and providing comments. The research leading to these results was funded by the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement 310857 (K.B.), the Vienna Science and Technology Fund (WWTF) through project LS14-031 (J.B.H. and K.B.), an unrestricted research grant from Celgene Austria (U.J.), the National Institutes of Health (R01AI067946 to J.S.O.), Boehringer Ingelheim Fonds (R.P.), the Austrian Science Fund (FWF): Project M1809-B19 (K.L.W.), and the French Agence Nationale de la Recherche (ANR-13-BSV1-0031 to L.D.).

Author information

Author notes

    • Deniz Cagdas
    • , Miroslav Hons
    • , Emily M Mace
    • , Michael Sixt
    • , Ilhan Tezcan
    •  & Jordan S Orange

    These authors contributed equally to this work.

Affiliations

  1. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

    • Elisabeth Salzer
    • , Wojciech Garncarz
    • , Özlem Yüce Petronczki
    • , Laurène Pfajfer
    • , Ivan Bilic
    • , Sol A Ban
    • , Katharina L Willmann
    • , Keiryn L Bennett
    • , Loïc Dupré
    •  & Kaan Boztug
  2. Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria.

    • Elisabeth Salzer
    • , Wojciech Garncarz
    • , Özlem Yüce Petronczki
    • , Laurène Pfajfer
    • , Loïc Dupré
    •  & Kaan Boztug
  3. Section of Pediatric Immunology, Hacettepe University, Ihsan Dogramaci Children's Hospital, Ankara, Turkey.

    • Deniz Cagdas
    • , Özden Sanal
    •  & Ilhan Tezcan
  4. Institute of Science and Technology Austria, Klosterneuburg, Austria.

    • Miroslav Hons
    •  & Michael Sixt
  5. Center for Human Immunobiology, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas, USA.

    • Emily M Mace
    • , Malini Mukherjee
    • , Hsiang Ting Hsu
    • , Pinaki P Banerjee
    • , Papiya Sinha
    •  & Jordan S Orange
  6. Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

    • René Platzer
    • , Verena Supper
    • , Hannes Stockinger
    •  & Johannes B Huppa
  7. Centre for Haemato-Oncology, Barts Cancer Institute - a CR-UK Centre of Excellence, Queen Mary University of London, London, UK.

    • Fabienne McClanahan
    •  & John G Gribben
  8. Institute of Immunology, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

    • Gerhard J Zlabinger
  9. Christian Doppler Laboratory for Immunomodulation and Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

    • Winfried F Pickl
  10. Centre de Physiopathologie de Toulouse Purpan (CPTP), INSERM, UMR1043, Toulouse Purpan University Hospital, Toulouse, France.

    • Loïc Dupré
  11. Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

    • Ulrich Jäger
  12. Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria.

    • Kaan Boztug
  13. St. Anna Kinderspital and Children's Cancer Research Institute, Department of Pediatrics, Medical University of Vienna, Vienna, Austria.

    • Kaan Boztug

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Contributions

E.S. performed most of the experiments, analyzed data, interpreted results, and, together with K.B., wrote the initial draft and revised version of the manuscript. D.C., I.T. and Ö.S. cared for the patient, and provided and interpreted clinical and immunological data. M.H. and E.S. performed migration and Lifeact experiments. M.S. provided critical input to the content of the manuscript. E.M.M., P.S., M.M., P.P.B., H.T.H. and J.S.O. performed and interpreted NK-cell immunological synapse experiments and detailed flow-cytometry-based NK-cell immunophenotyping. S.A.B. and E.S. identified the RASGRP1 mutation and performed initial experiments. R.P. and J.B.H. performed lipid bilayer calcium-flux experiments, and analyzed and interpreted data. L.P. generated an untransformed CD8+ T cell line from the patient and performed the CD8+ T cell cytotoxic assays. H.S. and V.S. performed proliferation analyses together with E.S. and provided critical input. Ö.Y.P. and L.D. performed the immunofluorescence experiments to quantify RhoA activation. K.L.W., W.G. and I.B. performed experiments and provided critical input. F.M., J.G.G. and U.J. helped with migration analyses. K.L.B. performed mass spectrometry analyses. W.F.P. and G.J.Z. performed thymidine incorporation assays, chromium release assays and analyses of autoantibody titers. K.B. conceived of and coordinated the study, provided laboratory resources, interpreted data, supervised E.S., W.G., Ö.Y.P., I.B., S.B. and K.W., wrote the manuscript together with E.S. and took overall responsibility for the study. All of the authors provided critical input and agreed to this publication.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kaan Boztug.

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https://doi.org/10.1038/ni.3575

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