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