Somatic RAP1B gain-of-function variant underlies isolated thrombocytopenia and immunodeficiency
Marta Benavides-Nieto, … , Jean-Pierre de Villartay, Despina Moshous
Marta Benavides-Nieto, … , Jean-Pierre de Villartay, Despina Moshous
Published September 3, 2024
Citation Information: J Clin Invest. 2024;134(17):e169994. https://doi.org/10.1172/JCI169994.
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Research Article Hematology Immunology

Somatic RAP1B gain-of-function variant underlies isolated thrombocytopenia and immunodeficiency

Abstract

The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient’s hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.

Authors

Marta Benavides-Nieto, Frédéric Adam, Emmanuel Martin, Charlotte Boussard, Chantal Lagresle-Peyrou, Isabelle Callebaut, Alexandre Kauskot, Christelle Repérant, Miao Feng, Jean-Claude Bordet, Martin Castelle, Guillaume Morelle, Chantal Brouzes, Mohammed Zarhrate, Patricia Panikulam, Nathalie Lambert, Capucine Picard, Damien Bodet, Jérémie Rouger-Gaudichon, Patrick Revy, Jean-Pierre de Villartay, Despina Moshous

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Figure 6

RAP1B-G12E, -G12V, and -G60R variants in overexpression models.

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RAP1B-G12E, -G12V, and -G60R variants in overexpression models. (A) RAP1...
(A) RAP1B-GTP activation evaluated by Western blotting after pull-down assay. HEK293T cells were transfected with vectors containing RAP1B-WT, RAP1B variants, or equimolar RAP1B-WT and RAP1B variant combinations. Values were compared with RAP1B-WT transfected cells (set to 1). Data from 5 independent experiments. (B) RAP1B-GTP activation in RAP1B-transduced HEL cells compared with nontransduced HEL cells (set to 1). Data from 5 independent experiments. (C) Talin-1/β3 integrin association was determined by Duolink proximity ligation assay in transfected HEL cells. Graph represents the relative number of fluorescent dots per cell in comparison with nontransfected HEL cells (set to 1). At least 50 transfected HEL cells per condition; data from 8 independent experiments. (D) Integrin activation in RAP1B-transfected HEL cells evaluated by flow cytometry measuring Oregon Green 488–labeled fibrinogen binding in unstimulated conditions. Graph represents the normalized integrin activation index, calculated as the ratio between MFI of each studied variant compared with RAP1B-WT transfected HEL cells. Data from 9 independent experiments. (E) Analysis of RAP1B-transduced T cell blasts over time. Cells were cultured in medium containing 15% DC-FBS and IL-2 at 100 U/ml. (F) Activation-induced cell death or apoptosis in RAP1B-transduced T cell blasts in response to increased concentrations of anti-CD3 antibody (OKT3) after 6 hours of stimulation. (G) Proliferation of RAP1B-transduced T cell blasts in the presence of 2.5 μg/ml coated anti-CD3 antibody and 100 U/ml IL-2, performed in triplicate. Graph represents the normalized proliferation index of transduced cells at days 3 and 4 compared with nontransduced and RAP1B-WT transduced cells. (H) Representative overlaid FACS histograms showing cell divisions by CellTrace Violet staining of transduced T cell blast with lentivirus expressing empty vector (light gray), RAP1B-WT (gray), RAP1B-G12E (red), RAP1B-G12V (orange), and RAP1B-G60R (blue) variants at day 4 after stimulation. (I) Graph corresponds to the distribution of transduced T cell blasts in different cell divisions calculated from FACS histograms in H. Error bars represent SEM. Statistical significance was determined for A by Mann-Whitney U test and for B–D by 1-way ANOVA, followed by Dunnett’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.