A Novel Gain‐of‐Function RAC2 Variant (p.Y64H) Associated With Lymphopenia and Leukopenia

To the Editor: Ras-related C3 botulinum toxin substrate 2 (RAC2) is a small GTPase that plays a critical role in multiple cellular processes, including NADPH oxidase activation, phagocytosis, and cytoskeletal remodeling [1]. RAC2 mutations have been implicated in immunodeficiency with defective neutrophil chemotaxis and leukocytosis, or lymphopenia or hypogammaglobulinemia. A phenotypic heterogeneity has been reported in patients with RAC2 variants with varying mutations raging from dominant-negative (loss-of-function, LOF), activating to constitutively active gain-of-function (GOF) mutations [2-4]. Several GOF mutations in RAC2 have previously been associated with severe neutrophilic inflammation and hyperactive immune responses [2, 5]. Here, we describe a novel pathogenic activation or GOF missense mutation, p.Y64H, in RAC2, which leads to a unique phenotype characterized by persistent lymphopenia and leukopenia, altered hematopoiesis, and immune dysfunction. A 7-year 6-month-old male patient was referred to the pediatric hematology outpatient clinic for persistent lymphopenia (see Case Report in the Supporting Information section). The patient had no recent infections, fever, or other systemic illnesses. Bone marrow aspiration ruled out myelodysplastic syndrome (MDS). Clinical laboratory immunophenotyping revealed a reduced CD4+/CD8+ T cell ratio (0.65 and 1.0 at different time points), reduced CD3+ T cell percentage (26.8), and a reduction in absolute CD3+, CD4+, and FOXP3+ T cell numbers (Figure 1A), despite a normal proportion of regulatory T cells. The patient also exhibited persistent neutropenia with large cytoplasmic vacuoles in neutrophils (Figure 1B), a finding consistent with previous reports of RAC2 GOF mutations [5, 6]. Genetic analysis identified a heterozygous NM_002872.5 RAC2 c.190T>C (p.Y64H) mutation (Figure 1C, D). This variant affects a highly conserved tyrosine residue, predicted to be phosphorylated, which is replaced by histidine (Figure 1E). In silico prediction tools, such as PolyPhen-2, MutationTaster, identified the mutation as probably damaging and deleterious, respectively. CADD score for the variant is 28.2. Structural modeling suggests that this substitution alters RAC2 activation dynamics, preventing normal GDP-GTP cycling and leading to constitutive activation, a hallmark of RAC2 GOF variants [2, 5]. Consistent with prior reports of activating mutations, this mutation led to increased vacuoles in patient neutrophils (Figure 1B) [5, 7]. To characterize the functional effects of the p.Y64H mutation, we performed biochemical assays to quantify the amount of active RAC2 GTP capable of binding the GST-PAK-RBD domain, which confirmed increased RAC2 activity compared to healthy donor (HD) controls (Figure 1F). Notably, previously reported RAC2 mutations, such as p.P29L and p.D57N, have been linked to exaggerated neutrophil chemotaxis and autoinflammatory syndromes [2, 5]. Additionally, with increased RAC2 activity, the patient-derived PBMCs demonstrated higher transwell migration compared to a healthy control, unlike prior reports with dominant negative mutations that lead to decreased chemotaxis. (Figure 1G) [2, 5, 7]. The patient's NK cell cytotoxicity was also comparable to controls suggesting that RAC2 is essential for immune cell function (Figure 1H). A different time-point analyses revealed slightly elevated CD56High population and increased NK percentage, suggesting that RAC2 hyperactivation does not uniformly impair killing ability that may alter NK subset composition (Figure S1a). RAC2 mutations also were shown to reduce T cells, especially, CD4 compartment [2]; similarly, p.Y64H variant decreased CD3+ T and Treg cell numbers even if the Treg frequency was high (Figure 1A). Of note, in vitro T cell proliferation responses were dramatically impaired (Figure 1I). The apoptosis of T cells was slightly higher in culture suggesting that T cell death or TCR mediated signaling defects together may account for the observed proliferation defects (Figure S1b). A bone-marrow aspirate was performed to investigate whether RAC2 hyperactivation affected hematopoiesis (see Methods section in the Supporting Information, and Table S1 for antibodies). In the mononuclear cell pool, the early hematopoietic precursors (hematopoietic stem cells [HSC], MMP, multilymphoid progenitor [MLP]), the myeloid precursors (megakaryocyte–erythroid progenitor cells [MEP], common myeloid progenitors [CMP], granulocytic myeloid progenitors [GMP]), the pre-pro B (CD19+CD34+), and immature (CD19+IgM+,IgD+) B lymphoid subsets were found in a proportion comparable to those found in HDs (Figure S2a, b) suggesting that the RAC2 mutation did not prevent HSC differentiation. In vitro assays were performed in order to measure HSPCs-CD34+ cells proliferation and differentiation capacities. In both conditions, we observed a lower proliferative index (Figure S2c) compared to the HD, especially in the presence of GM-CSF. In the methylcellulose assays, a reduced number of myeloid (colony forming unit [CFU]) and erythroid (burst forming unit [BFU]) colonies were observed in the RAC2 patient (Figure S2d), compared to healthy controls, which confirms that cell differentiation could occur, whereas cell proliferation was impaired. Altogether, our findings suggest that p.Y64H hyperactivation disrupts homeostasis of hematopoiesis, providing a novel mechanistic insight into RAC2 GOF disorders. The p.Y64H RAC2 mutation represents a novel pathogenic variant that expands the known spectrum of RAC2-associated immunodeficiencies. Similar to several reported RAC2 GOF mutations, which primarily affect neutrophil function and inflammation, this variant is characterized by leukopenia. These findings suggest that RAC2 hyperactivation disrupts homeostasis of hematopoiesis. Given that targeted inhibition of RAC2 signaling may offer therapeutic benefits, exploring Rac-specific GTPase inhibitors or PI3K pathway modulators could be a potential avenue for treatment [7]. Furthermore, the impact of RAC2 GOF mutations on thymic function and T cell development remains to be fully elucidated. T cell receptor excision circle (TREC) analysis could provide additional insights into whether the observed T cell defects arise from impaired thymopoiesis or altered peripheral homeostasis. In conclusion, we report a new RAC2 GOF mutation (p.Y64H) associated with a specific immunophenotype, highlighting the complexity of RAC2-driven immune dysregulation. These findings underscore the need for functional characterization of RAC2 variants in primary immunodeficiencies and may inform novel therapeutic strategies for RAC2-related disorders. This study was partially supported by Erciyes University BAP Unit grants TYG-2023-13110, TUBITAK GEBIP 2021, Science Academy BAGEP 2021 awards to AE. The study was conducted in accordance with the French legislation and the principles of the Helsinki declaration. Informed consent was obtained from the healthy donors, and the study protocol was approved by the regional investigational review board ethical committee and Ministry of Health (DC 2014-2272/2015/ DC-2008-329.) The patients gave their consent to participate in the current study in writing. The authors declare no conflicts of interest. Figure S1: Flow cytometry analysis of NK cells among PBMCs from the healthy donor (HD) and RAC2 patient's (Pt) and T cell apoptosis. (a) Patient PBMCs were analyzed via CD56 staining, top row shows PBMCs gated on lymphocytes, CD56 percentage. Bottom row shows high and dim fractions of CD56+ NK cells. (b) PBMCs from healthy donor (HD) and RAC2 patient were cultured overnight with 1 µg/mL (each) soluble anti-CD3/CD28 and stained for Annexin V and 7-AAD (Biolegend, cat# 640930). The left panel shows representative flow plot, whereas the right panel shows stimulated (anti-CD3/CD28) and unstimulated quantified bar graphs. Figure S2: Flow cytometry analysis was performed on mononuclear bone-marrow cells from the RAC2 patient's (Pt). Cells were stained with several antibodies and analyzed by flow cytometry. (a) in the alive (7AAD-) population (b) Comparison of the % of each subset with the one observed in several bone-marrow cells from healthy donors (HD, n = 5). (c–d) Purified CD34+-HSPCs from the Pt or an HD were cultured with a cocktail of cytokines (SCF, FLT-3L, TPO, IL-3 (CK) or SCF and GM-CSF (GM-CSF) for 8–12 days, respectively. The proliferation index was defined as the cell number obtained at the end of the culture/cell number at the beginning of the culture. Myeloid colony forming unit (CFU) and burst forming unit (BFU) were evaluated after an 8-day culture. CMP, common myeloid progenitors; GMP, granulocytic myeloid progenitors, lin- (lineage negative population); HSC, hematopoietic stem cells; MEP, megakaryocyte–erythroid progenitor cells; MLP, multilymphoid progenitor; MPP, multipotent progenitors. Table S1. Multi-labeled panel of antibodies. Table S2. Immunological studies. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.