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 1

RAP1B variants and RAP1B protein structure.

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RAP1B variants and RAP1B protein structure. (A) May-Grünwald-Giemsa sta...
(A) May-Grünwald-Giemsa staining of P1 BM smear at age of M18 showing reduced richness for the patient’s age, elements at all stages of maturation, predominance of the granular lineage, absence of atypical cells, and presence of rare hypolobed megakaryocytes (black arrowheads). Original magnification, ×500 (left); ×1,000 (center and right). (B) P1 pedigree and familial segregation. Sanger sequencing of RAP1B in whole peripheral blood from P1 and his parents shows the heterozygous RAP1B c.35G>A (p.G12E) variant in P1 (red arrow), but not in P1’s parents, confirming its de novo nature. (C) Ribbon representation of the 3D structure of rat Rap1B bound to a nonhydrolyzable GTP analog (GppNHp, pdb 3X1X) (42). The sequences of rat Rap1B and human RAP1B are identical, except for C139 in the human sequence, which is replaced by serine in the rat sequence. This surface residue is far from the nucleotide-binding site. P-loop, switch I, and switch II regions are shown in pink. Magnesium ion is shown in green, water molecules in red, and the residues G12, A59, and G60, which have been found mutated in patients (Table 3), are in blue. (D) CADD score and amino acid position of all human RAP1B missense variants listed in gnomAD (11) as of February 6, 2023. RAP1B variants reported in patients are shown in red: G12E (P1) and G12V, A59G, and G60R (P2, P3, and P4) (5, 6). (E) Schematic representation of the secondary structure of human RAP1B with G domain–containing P-loop (G1), switch I and switch II (G2 and G3), G4, and G5 functional domains (33, 61), and hypervariable region. (F) Multiple sequence alignment of RAP1B G1–G3 functional domains from different species (62). (G) Multiple sequence alignment of G1–G3 functional domains of human small GTPases: RAP1B, RAP1A, HRAS, NRAS, and KRAS (62).