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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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 4

P1 lymphocytes functional analysis.

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P1 lymphocytes functional analysis. (A) Analysis of P1 PBMC spontaneous ...
(A) Analysis of P1 PBMC spontaneous and chemoattractant-induced migration in the absence (left) or presence (right) of fibronectin coating (100 mg/ml) using Transwell devices. Experiments were repeated twice in triplicates. One representative of 2 independent experiments is shown. SDF-1α (CXCL12, 1,500 ng/mL) was used as chemoattractant. Means of triplicates (with SD) are represented for patient and control cells in the graphs. At the time of the test, P1 PBMCs carried 52.5%RAP1BWT/G12E cells. (B) RAP1B activation, calculated as the ratio RAP1B-GTP/RAP1B total expression, was evaluated in P1 and control B-LCL cells by Western blotting after pull-down assay. P1 B-LCL cell bulk populations used for the experiment contained 80%, 14%, and 4% RAP1BWT/G12E cells. The blot is representative of 2 independent experiments. (C) Analysis of the relative proportion of P1 and control B-LCL cells in G1, S, and G2 cell-cycle phases after double staining with anti-BrdU monoclonal antibody and PI. P1 B-LCL cell bulk populations contained 70% and 10% RAP1BWT/G12E cells. Two different healthy B-LCL cell unrelated controls (1 age matched) were used for normalization. Experiments were performed in triplicate for 2 independent experiments. The representative graph shows the gated populations. Statistical significance was determined by Mann-Whitney U test. **P < 0.01; ***P < 0.001. (D) Percentages of RAP1BWT/G12E cells in sorted G1, S, and G2 phase populations after cell-cycle analysis. P1 B-LCL cell bulk population contained 70% RAP1BWT/G12E cells. (E) Representative histograms(left) showing cell divisions by CellTrace Violet staining of synchronized HDs (gray histograms) and P1 (blue and red histograms) B-LCL cells after 6 days of culture. P1 B-LCL cell bulk contained 0% (light blue), 2% (dark blue), 60% (light red), or 70% (dark red) RAP1BWT/G12E cells. Dot plots graph (right) showing index of proliferation of HD and P1 B-LCL cells calculated from FACS histograms shown in the left panel (with same color code) at indicated time of culture. Each symbol corresponds to 1 individual HD (black) or P1 (blue or red). Data are from 1 of 2 independent experiments. (F) Representative FACS dot plots (left) depicting annexin V and 7-AAD expressions of HD and P1 B-LCL cells containing 0% or 70% RAP1BWT/G12E cells for 0 and 24 hours. Dot plot graph (right) showing apoptotic cells (annexin V+ and 7-AAD+) of HD and P1 B-LCL cells calculated from FACS dot plots shown in the left panel with color code as in E at indicated time of culture. Each symbol corresponds to 1 individual HD (black) or P1 (blue and red). Data are from 1 of 2 independent experiments.