Journal of Clinical Immunology

, Volume 38, Issue 6, pp 699–710 | Cite as

Novel Mutations in RASGRP1 are Associated with Immunodeficiency, Immune Dysregulation, and EBV-Induced Lymphoma

  • Ido Somekh
  • Benjamin Marquardt
  • Yanshan Liu
  • Meino Rohlfs
  • Sebastian Hollizeck
  • Musa Karakukcu
  • Ekrem Unal
  • Ebru Yilmaz
  • Turkan Patiroglu
  • Murat Cansever
  • Shirly Frizinsky
  • Vicktoria Vishnvenska-Dai
  • Erez Rechavi
  • Tali Stauber
  • Amos J. Simon
  • Atar Lev
  • Christoph Klein
  • Daniel Kotlarz
  • Raz SomechEmail author
Original Article



RAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency (PID) characterized by CD4+ T cell lymphopenia and Epstein-Barr virus (EBV)-associated B cell lymphoma. Our report of three novel patients widens the scope of RASGRP1 deficiency by providing new clinical and immunological insights on autoimmunity, immune cell development, and predisposition to lymphoproliferative disease.


One patient of Turkish origin (P1) and two Palestinian patients (P2, P3) were evaluated for immunodeficiency. To decipher the molecular cause of disease, whole exome sequencing was conducted. Identified mutations were validated by immunological and biochemical assays.


We report three patients presenting with similar clinical characteristics of immunodeficiency and EBV-associated lymphoproliferative disease. In addition, P2 and P3 exhibited overt autoimmune manifestations. Genetic screening identified two novel loss-of-function mutations in RASGRP1. Immunoblotting and active Ras pull-down assays confirmed perturbed ERK1/2 signaling and reduced Ras-GTPase activity in heterologous Jurkat cells with ectopic expression of RASGRP1 mutants. All three patients had CD4+ T cell lymphopenia. P2 and P3 showed decreased mitogen-induced lymphocyte proliferation, reduced T cell receptor excision circles, abnormal T cell receptor (TCR) Vβ repertoires, and increased frequencies of TCRγδ cells. TCR gamma repertoire diversity was significantly reduced with a remarkable clonal expansion.


RASGRP1 deficiency is associated with life-threatening immune dysregulation, severe autoimmune manifestations, and susceptibility to EBV-induced B cell malignancies. Early diagnosis is critical and hematopoietic stem cell transplantation might be considered as curative treatment.


Autoimmunity EBV lymphoproliferation PID RASGRP1 T cell development 



The authors thank the patients and their families for their collaboration and for their expert care by the interdisciplinary pediatric teams. The study would not have been possible without additional support by the German Academic Exchange Program (DAAD) and the Care-for-Rare Foundation.

Author Contributions

I.S. and B.M. analyzed and interpreted results and I.S., B.M., and D.K. drafted the manuscript. B.M. designed, performed, and analyzed experiments for the patients. A.L., A.J.S., and E.R. performed the experiments for P2, P3. Y.L. and M.R. conducted NGS. S.H. analyzed NGS results. M.K., E.U., E.Y., T.P., and M.C. followed, diagnosed, and treated P1. R.S., T.S., V.V.D., and S.F. followed, diagnosed, and treated P2, P3. C.K., D.K., and R.S. designed and supervised the experiments.

Funding Information

This work was supported by grants from the Jeffrey Modell Foundation (JMF) (Raz Somech), the German Research Foundation (DFG CRC1054, Leibniz Program) (Christoph Klein, Daniel Kotlarz), and the Else Kröner–Fresenius-Stiftung (Christoph Klein). Daniel Kotlarz has been a scholar funded by the Daimler und Benz Stiftung, Reinhard-Frank Stiftung, and Else Kröner–Fresenius-Stiftung.

Compliance with Ethical Standards

All procedures were performed upon informed consent and assent from patients, first-degree relatives, and healthy donor controls in accordance with the ethical standards of the institutional and/or national research committees and with the current update of the Declaration of Helsinki.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10875_2018_533_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 16.4 kb)
10875_2018_533_MOESM2_ESM.docx (15 kb)
ESM 2 (DOCX 15.2 kb)
10875_2018_533_MOESM3_ESM.docx (16 kb)
ESM 3 (DOCX 15.5 kb)


  1. 1.
    Fischer A. Human primary immunodeficiency diseases: a perspective. Nat Immunol. 2004;5(1):23–30.CrossRefPubMedGoogle Scholar
  2. 2.
    Picard C, Bobby Gaspar H, Al-Herz W, Bousfiha A, Casanova JL, Chatila T, et al. International union of immunological societies: 2017 primary immunodeficiency diseases committee report on inborn errors of immunity. J Clin Immunol. 2018 Jan;38(1):96–128.CrossRefPubMedGoogle Scholar
  3. 3.
    Notarangelo LD. Primary immunodeficiencies. J Allergy Clin Immunol. 2010;125(2 Suppl 2):S182–94.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Seleman M, Hoyos-Bachiloglu R, Geha RS, Chou J. Uses of next-generation sequencing technologies for the diagnosis of primary immunodeficiencies. Front Immunol. 2017;8:847.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Salzer E, Cagdas D, Hons M, Mace EM, Garncarz W, Petronczki OY, et al. RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics. Nat Immunol. 2016;17(12):1352–60.CrossRefPubMedGoogle Scholar
  6. 6.
    Platt CD, Fried AJ, Hoyos-Bachiloglu R, Usmani GN, Schmidt B, Whangbo J, et al. Combined immunodeficiency with EBV positive B cell lymphoma and epidermodysplasia verruciformis due to a novel homozygous mutation in RASGRP1. Clinical immunology (Orlando, Fla.) 2017;183:142–144.Google Scholar
  7. 7.
    Mao H, Yang W, Latour S, Yang J, Winter S, Zheng J, et al. RASGRP1 mutation in autoimmune lymphoproliferative syndrome-like disease. J Allergy Clin Immunol. 2017;Google Scholar
  8. 8.
    Winter S, Martin E, Boutboul D, Lenoir C, Boudjemaa S, Petit A, et al. Loss of RASGRP1 in humans impairs T-cell expansion leading to Epstein-Barr virus susceptibility. EMBO molecular medicine. 2018 Feb;10(2):188–99.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Simanshu DK, Nissley DV, McCormick F. RAS proteins and their regulators in human disease. Cell. 2017;170(1):17–33.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Jun JE, Rubio I, Roose JP. Regulation of ras exchange factors and cellular localization of ras activation by lipid messengers in T cells. Front Immunol. 2013 Sep 04;4:239.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Stone JC. Regulation of Ras in lymphocytes: get a GRP. Biochem Soc Trans. 2006;34(Pt 5):858–61.CrossRefPubMedGoogle Scholar
  12. 12.
    Dower NA, Stang SL, Bottorff DA, Ebinu JO, Dickie P, Ostergaard HL, et al. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 2000;1(4):317–321.Google Scholar
  13. 13.
    Lev A, Simon AJ, Broides A, Levi J, Garty BZ, Rosenthal E, et al. Thymic function in MHC class II-deficient patients. J Allergy Clin Immunol. 2013;131(3):831–9.CrossRefPubMedGoogle Scholar
  14. 14.
    Lev A, Simon AJ, Bareket M, Bielorai B, Hutt D, Amariglio N, et al. The kinetics of early T and B cell immune recovery after bone marrow transplantation in RAG-2-deficient SCID patients. PLoS One. 2012;7(1):e30494.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Alamyar E, Duroux P, Lefranc MP, Giudicelli V. IMGT((R)) tools for the nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS. Methods in molecular biology (Clifton, NJ). 2012;882:569–604.CrossRefGoogle Scholar
  16. 16.
    Keylock CJ. Simpson diversity and the Shannon–Wiener index as special cases of a generalized entropy. Oikos. 2005;109:203–7.CrossRefGoogle Scholar
  17. 17.
    Kotlarz D, Marquardt B, Baroy T, Lee WS, Konnikova L, Hollizeck S, et al. Human TGF-beta1 deficiency causes severe inflammatory bowel disease and encephalopathy. Nat Genet. 2018;50(3):344–8.CrossRefPubMedGoogle Scholar
  18. 18.
    Seidemann K, Tiemann M, Schrappe M, Yakisan E, Simonitsch I, Janka-Schaub G, et al. Short-pulse B-non-Hodgkin lymphoma-type chemotherapy is efficacious treatment for pediatric anaplastic large cell lymphoma: a report of the Berlin-Frankfurt-Munster Group Trial NHL-BFM 90. Blood. 2001;97(12):3699–706.CrossRefPubMedGoogle Scholar
  19. 19.
    Minard-Colin V, Brugières L, Reiter A, Cairo MS, Gross TG, Woessmann W, et al. Non-Hodgkin lymphoma in children and adolescents: progress through effective collaboration, current knowledge, and challenges ahead. J Clin Oncol. 2015;33(27):2963–74.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    McCarthy MI, MacArthur DG. Human disease genomics: from variants to biology. Genome Biol. 2017;18(1):20.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Current protocols in human genetics/editorial board, Jonathan L Haines [et al]. 2013 Jan;0 7:Unit7 20.Google Scholar
  22. 22.
    Chen Y, Ci X, Gorentla B, Sullivan SA, Stone JC, Zhang W, et al. Differential requirement of RasGRP1 for gammadelta T cell development and activation. Journal of immunology (Baltimore, Md : 1950). 2012;189(1):61–71.Google Scholar
  23. 23.
    Ebinu JO, Stang SL, Teixeira C, Bottorff DA, Hooton J, Blumberg PM, et al. RasGRP links T-cell receptor signaling to Ras. Blood 2000;95(10):3199–3203.Google Scholar
  24. 24.
    Priatel JJ, Teh SJ, Dower NA, Stone JC, Teh HS. RasGRP1 transduces low-grade TCR signals which are critical for T cell development, homeostasis, and differentiation. Immunity. 2002;17(5):617–27.CrossRefPubMedGoogle Scholar
  25. 25.
    Lee SH, Yun S, Lee J, Kim MJ, Piao ZH, Jeong M, et al. RasGRP1 is required for human NK cell function. Journal of immunology (Baltimore, Md : 1950). 2009;183(12):7931–7938.Google Scholar
  26. 26.
    Coughlin JJ, Stang SL, Dower NA, Stone JC. RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling. Journal of immunology (Baltimore, Md : 1950). 2005;175(11):7179–7184.Google Scholar
  27. 27.
    Fuller DM, Zhu M, Song X, Ou-Yang CW, Sullivan SA, Stone JC, et al. Regulation of RasGRP1 function in T cell development and activation by its unique tail domain. PLoS One 2012;7(6):e38796.Google Scholar
  28. 28.
    Sun C, Molineros JE, Looger LL, Zhou XJ, Kim K, Okada Y, et al. High-density genotyping of immune-related loci identifies new SLE risk variants in individuals with Asian ancestry. Nat Genet 2016;48(3):323–330.Google Scholar
  29. 29.
    Ferretti A, Fortwendel JR, Gebb SA, Barrington RA. Autoantibody-mediated pulmonary alveolar proteinosis in Rasgrp1-deficient mice. Journal of immunology (Baltimore, Md : 1950). 2016;197(2):470–479.Google Scholar
  30. 30.
    Yasuda S, Stevens RL, Terada T, Takeda M, Hashimoto T, Fukae J, et al. Defective expression of Ras guanyl nucleotide-releasing protein 1 in a subset of patients with systemic lupus erythematosus. Journal of immunology (Baltimore, Md : 1950). 2007;179(7):4890–4900.Google Scholar
  31. 31.
    Rapoport MJ, Bloch O, Amit-Vasina M, Yona E, Molad Y. Constitutive abnormal expression of RasGRP-1 isoforms and low expression of PARP-1 in patients with systemic lupus erythematosus. Lupus. 2011;20(14):1501–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Golinski ML, Vandhuick T, Derambure C, Freret M, Lecuyer M, Guillou C, et al. Dysregulation of RasGRP1 in rheumatoid arthritis and modulation of RasGRP3 as a biomarker of TNFalpha inhibitors. Arthritis research & therapy 2015;17:382.Google Scholar
  33. 33.
    Qu HQ, Grant SF, Bradfield JP, Kim C, Frackelton E, Hakonarson H, et al. Association of RASGRP1 with type 1 diabetes is revealed by combined follow-up of two genome-wide studies. J Med Genet 2009;46(8):553–554.Google Scholar
  34. 34.
    Zhou XJ, Nath SK, Qi YY, Sun C, Hou P, Zhang YM, et al. Novel identified associations of RGS1 and RASGRP1 variants in IgA Nephropathy. Sci Rep 2016;6:35781.Google Scholar
  35. 35.
    Somech R. T-cell receptor excision circles in primary immunodeficiencies and other T-cell immune disorders. Curr Opin Allergy Clin Immunol. 2011;11(6):517–24.CrossRefPubMedGoogle Scholar
  36. 36.
    Germain RN. T-cell development and the CD4-CD8 lineage decision. Nat Rev Immunol. 2002;2(5):309–22.CrossRefPubMedGoogle Scholar
  37. 37.
    Sharp LL, Schwarz DA, Bott CM, Marshall CJ, Hedrick SM. The influence of the MAPK pathway on T cell lineage commitment. Immunity. 1997;7(5):609–18.CrossRefPubMedGoogle Scholar
  38. 38.
    Bommhardt U, Basson MA, Krummrei U, Zamoyska R. Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus. Journal of immunology (Baltimore, Md : 1950). 1999;163(2):715–722.Google Scholar
  39. 39.
    Guilbault B, Kay RJ. RasGRP1 sensitizes an immature B cell line to antigen receptor-induced apoptosis. J Biol Chem. 2004;279(19):19523–30.CrossRefPubMedGoogle Scholar
  40. 40.
    Bartlett A, Buhlmann JE, Stone J, Lim B, Barrington RA. Multiple checkpoint breach of B cell tolerance in Rasgrp1-deficient mice. Journal of immunology (Baltimore, Md : 1950). 2013;191(7):3605–3613.Google Scholar
  41. 41.
    Priatel JJ, Chen X, Zenewicz LA, Shen H, Harder KW, Horwitz MS, et al. Chronic immunodeficiency in mice lacking RasGRP1 results in CD4 T cell immune activation and exhaustion. Journal of immunology (Baltimore, Md : 1950.) 2007;179(4):2143–2152.Google Scholar
  42. 42.
    Palendira U, Rickinson AB. Primary immunodeficiencies and the control of Epstein-Barr virus infection. Ann N Y Acad Sci. 2015 Nov;1356:22–44.CrossRefPubMedGoogle Scholar
  43. 43.
    Menasche G, Feldmann J, Fischer A, de Saint Basile G. Primary hemophagocytic syndromes point to a direct link between lymphocyte cytotoxicity and homeostasis. Immunol Rev. 2005 Feb;203:165–79.CrossRefPubMedGoogle Scholar
  44. 44.
    Parvaneh N, Filipovich AH, Borkhardt A. Primary immunodeficiencies predisposed to Epstein-Barr virus-driven haematological diseases. Br J Haematol. 2013 Sep;162(5):573–86.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ido Somekh
    • 1
  • Benjamin Marquardt
    • 1
  • Yanshan Liu
    • 1
  • Meino Rohlfs
    • 1
  • Sebastian Hollizeck
    • 1
  • Musa Karakukcu
    • 2
  • Ekrem Unal
    • 2
  • Ebru Yilmaz
    • 2
  • Turkan Patiroglu
    • 2
  • Murat Cansever
    • 2
  • Shirly Frizinsky
    • 3
  • Vicktoria Vishnvenska-Dai
    • 4
  • Erez Rechavi
    • 3
  • Tali Stauber
    • 3
  • Amos J. Simon
    • 3
  • Atar Lev
    • 3
  • Christoph Klein
    • 1
  • Daniel Kotlarz
    • 1
  • Raz Somech
    • 3
    Email author
  1. 1.Dr. von Hauner Children’s HospitalUniversity Hospital, Ludwig Maximilian UniversityMunichGermany
  2. 2.Department of Pediatrics, Division of Pediatric Hematology & OncologyErciyes UniversityKayseriTurkey
  3. 3.Pediatric Department A and the Immunology Service, Jeffrey Modell Foundation Center; Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, 52621, affiliated to the Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael
  4. 4.Ocular Oncology and Autoimmune center, The Goldschleger Eye Institute; Sheba Medical Center, affiliated to the Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael

Personalised recommendations