A Novel RAC2 Mutation Causing Combined Immunodeficiency

Ras-related C3 botulinum toxin substrate 2 (RAC2) acts as a molecular switch and has crucial roles in cell signaling and actin dynamics. A broad spectrum of genetic RAC2 mutations can cause various types of primary immunodeficiency, with complete penetrance. Here, we report a novel heterozygous missense mutation in RAC2 with incomplete penetrance, and the associated phenotypes, in a Chinese family. Immunological phenotype was detected by flow cytometry. T cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) were assessed by real-time quantitative PCR. Gene mutations were detected by whole-exome sequencing (WES) and confirmed by Sanger sequencing. The proband was an 11-year-old girl who presented with recurrent respiratory infections, bronchiectasis, persistent Epstein-Barr virus viremia, infectious mononucleosis, encephalitis, and cutaneous human papillomavirus infections. Laboratory analyses revealed increased serum IgG and decreased IgM levels, reduced naïve CD4+ and CD8+ T cells, an inverted CD4+/CD8+ ratio, and low TREC and KREC numbers. The mutation resulted in increased production of reactive oxygen species, while impaired actin polarization in neutrophils; diminished proliferative responses, increased cytokine production and a dysregulated phenotype in T lymphocytes; as well as accelerated apoptosis and hyperactivity of AKT in HL-60 human leukemia cells. WES identified a c.44G > A mutation in RAC2 resulting in a p.G15D substitution. Despite sharing the same mutation as the proband, her father suffered from recurrent respiratory infections and bronchiectasis, and had similar immunological defects, whereas her sister was apparently healthy, other than cutaneous human papillomavirus infections, and only mild immunological defects were detected preliminarily. Our findings broaden the clinical and genetic spectra of RAC2 mutations and underline the importance of RAC2 gain-of-function mutations with complete or incomplete penetrance.


Introduction
RAC2 is a Rho guanosine triphosphatase (GTPase) which is exclusively expressed in hematopoietic cells and executes crucial physiological roles, including regulating hematopoietic stem cells, neutrophil migration and killing, and lymphocyte development and function [1][2][3]. RAC2 can act as a molecular switch, which participates in gene transcription, cell survival, adhesion, reactive oxidant species (ROS) production, and cytoskeleton reorganization [4][5][6]. Accordingly, variable mutations in RAC2 have rarely been identified in a small number of patients, and result in different forms of primary immunodeficiency [6][7][8].
Here, we describe a novel heterozygous RAC2 missense mutation, c.44G > A (p.G15D), in an 11-year-old girl who presented with recurrent respiratory infections and bronchiectasis, cutaneous human papillomavirus infections, persistent Epstein-Barr virus (EBV) viremia, and EBV-induced disease, with low T cell receptor excision circle (TREC) and K-deleting recombination excision circles (KREC) numbers, indicating immunodeficiency. We also report that the same mutation was shared with her father, who suffered from recurrent respiratory infections and bronchiectasis, and her sister who appeared healthy, apart from cutaneous human papillomavirus infections. We found impaired proliferative responses, enhanced cytokine production and a dysregulated phenotype in T lymphocytes in the proband and her father, while all of which were less marked in her sister. We also found impaired actin polarization in neutrophils, increased ROS production, accelerated apoptosis, and AKT hyperactivity in HL-60 cells. Thus, our results broaden the clinical and genetic spectrum of known RAC2 mutations, and suggest that dominant activating monoallelic RAC2 deficiency can exhibit incomplete penetrance.

Ethics Approval and Consent
This study was performed after obtaining written informed consent from the patient's guardians, consistent with the Declaration of Helsinki, and was approved by the Medical Ethics Committee of Hunan Children's Hospital.

Analysis of T Cell Proliferation and Cytokine Production
Peripheral blood mononuclear cells were performed for T cell proliferation and cytokine production as described previously [21]. Cells were analyzed using a FACSCanto II flow cytometer.

Immunostaining and Immunoblotting Analysis of RAC2
Immunostaining and immunoblotting analysis of RAC2 in isolated neutrophils, and immunoblotting analysis of FLAG (Proteintech), AKT (Cell Signaling Technology), and phospho-AKT (Ser473; Cell Signaling Technology) in transfected HL-60 cells were performed as described previously [13,21].

Analysis of Actin Polarization in Neutrophils
For immunofluorescence staining, isolated neutrophils were plated on slides in the presence of 500 nM PMA (Sigma-Aldrich) [22]. After fixation and blocking, cells were stained with phalloidin (Molecular Probes) at a 1:40 dilution and with 49-6-diamidino-2-phenylindole dihydrochloride at a 1:10,000 dilution, in PBS. Cover slips were imaged under a laser scanning confocal microscope (Nikon, Japan).

Analysis of Apoptosis in HL-60 Cells
Three days after transfection, cells were harvested, and apoptosis was determined by Annexin V-FITC Apoptosis Detection Kit (Beyotime), according to the manufacturer's instructions. Cells were analyzed using a FACSCanto II flow cytometer.

Clinical Description
The patient was from a nonconsanguineous kindred and had presented with recurrent respiratory infections since the age of 1 year, which occurred at a frequency of more than six times each year. She was repeatedly administered with oral and intravenous antibiotic treatment at local hospitals. She also experienced recurrent fever and petechia, along with an episode of convulsion at 1 year and 3 months old; therefore, she was transferred to our hospital. At her first admission, laboratory findings indicated leukocytosis (14,400 cells/µL; normal range 4000-10,000 cells/µL), with 48.5% lymphocytes and 28% atypical lymphocytes, alongside anemia (hemoglobin, 77 g/L), thrombocytopenia (42,000 cells/µL), and elevated liver enzymes (alanine transaminase 237.8 IU/L; aspartate transaminase 564.7 IU/L). Antiviral capsid antigen IgM to EBV was positive, and hepatosplenomegaly was detected on clinical examination and ultrasound. She was diagnosed with infectious mononucleosis, which was treated with intravenous ganciclovir, along with antibiotic treatments for respiratory infection. After a month of hospitalization, leukocytes and thrombocytes returned to the normal range. She had undergone recurrent episodes of convulsion and fever at 4 years and 2 months old. EBV DNA was detected in the serum (1.31E + 05 copies/mL) by viral DNA PCR (Table 1), along with transaminitis. Increased atypical lymphocytes and large vacuoles were observed by light microscopy (Fig. 1A). Cerebrospinal fluid (CSF) was colorless and not turbid, with normal cell counts and protein content, although glucose levels were elevated. Magnetic resonance imaging scan was performed and showed extensive and cytotoxic edematous changes in the left brain and thalamus (Fig. 1B, left). Based on these findings, viral encephalitis attributable to EBV was diagnosed. She received intravenous ganciclovir treatment. She has not had a convulsion episode since discharge; however, encephalomalacia, brain atrophy, and gliosis were detected in the left temporoparietal occipital lobe on reexamination 16 months later (Fig. 1B, right). Furthermore, she experienced recurrent pneumonia along with episodes of asthma, and bronchiectasis was determined by chest computer tomography scan from 5 years old (Fig. 1C). Etiological examination verified repeated isolations of Haemophilus influenzae and Streptococcus pneumoniae from sputum samples. She has undergone repeated and prolonged hospitalizations, as well as treatment with higher grade intravenous antibiotics, approximately ten times annually; furthermore, dyscalculia was found when she started school. These health issues forced her to drop out of school. No obvious cognitive or motor deficiencies were discovered by physical examination, and she was good at handicrafts. Recently, primary immunodeficiency was suspected in the proband and she was admitted to our department at 11 years old. She was given interval intravenous immunoglobulin infusion, which significantly ameliorated her condition, and she has since required fewer and shorter hospitalizations.
The father of the proband is a 39-year-old man with a history of recurrent sinopulmonary infections and bronchiectasis, and has received traditional Chinese medicine treatment locally, while her sister is 9 years old and has cutaneous human papillomavirus infections, while appearing otherwise healthy, without recurrent infections.

Immunological Assessment
It was retrospectively noted that the proband's laboratory examinations showed an inverted CD4:CD8 ratio (0.43) and a reduced B cell percentage (6%) at her first admission, as well as fluctuating neutropenia, progressive lymphopenia, and increased serum IgG levels, while IgM was decreased, except during her first episode of infectious mononucleosis ( Fig. 1D and Table 2). Further investigations revealed that were detected by light microscopy in neutrophils isolated from bone marrow when the patient was aged 3 years and 4 months. B Magnetic resonance imaging (MRI) scans of the patient at age 4 years and 2 months (left) and at age 5 years and 6 months (right). C Computed tomography (CT) scans of the patient at age 5 years. D Absolute counts of white blood cells, neutrophils, and lymphocytes over time.
E Oxidative burst in dihydrorhodamine 123-loaded neutrophils from patients P1 (the proband's father), P2 (the proband), P3 (younger sister), and the proband's mother after stimulation with fMLP. F Representative of superoxide production from HL-60 cells transfected with either wide type or G15D mutated RAC2 expression constructs. ****P < .0001  (Table 3). Neutrophils from the patient, her father, and her sister demonstrated increased oxidative burst capacity, relative to healthy control samples, after fMLP stimulation (Fig. 1E); baseline ROS production by HL-60 cells transfected with the following identified RAC2 G15D mutant was higher than that by cells transfected with the RAC2 wild-type (Fig. 1F), consistent with the characteristic features of gain-of-function RAC2 mutations.

Gene Sequencing Identified a Novel Heterozygous RAC2 Mutation
In an attempt to define a genetic cause of the immunodeficiency, WES was performed and a previously unreported heterozygous variant, c.44G > A, in RAC2, was detected and subsequently confirmed by Sanger sequencing. This mutation was also detected in her father and younger sister by Sanger sequencing, while it was not found in her mother, uncle, or grandparents ( Fig. 2A, B). This missense mutation results in a glycine to aspartic acid substitution at position 15 (p.G15D) of RAC2, which is predicted to be deleterious/ pathogenic ( Table 4). The missense mutated p.G15 residue is located within the G1 box (Fig. 2C), which is a highly conserved guanine nucleotide binding region [23]; therefore, the crystal structure of RAC2 was used as a template and the structural impact of the Gly15Asp mutant analyzed using Swiss PdbViewer. Structural analysis by 3D modeling showed that this mutation leads to destruction of hydrogen bonds and prevents interaction with GDP (Fig. 2D). According to the American College of Medical Genetics criteria [24], the p.G15D variant meets the pathogenic criteria: pathogenic moderate 1 (PM1), due to its location in a well-established functional domain; pathogenic moderate 2 (PM2), as the mutation is not reported in population databases, such as the 1000 Genomes Project; pathogenic supporting 1 (PP1), because the mutation cosegregates with disease in family members; and pathogenic supporting 3 (PP3), since multiple lines of computational evidence support a deleterious effect.

Analysis of T Cell Proliferation and Cytokine Production
Recently, we reported that a dominant activating RAC2 mutation led to diminished T cell proliferation in response to stimulation of the T cell receptor (TCR), in addition to increased cytokine production and skewing of the CD4 T cell subsets toward the Th1-like phenotype [21]. Impaired proliferative responses, enhanced cytokine production, and a dysregulated phenotype in T lymphocytes were recapitulated in the proband and her father ( Fig. 3 and 4). Interestingly, only mildly impaired proliferative responses were detected in her sister, in whom cytokine production and subsets phenotype seemed more closely resembled the control (Fig. 3  and 4).

Analysis of RAC2 Expression in Neutrophils and HL-60 Cells
Structural analysis by 3D modeling revealed that this p.G15D mutation leads to destruction of hydrogen bonds and prevents interaction with GDP, a finding consistent with  the delineated RAC2 mutants, such as p.G12R, p.P34H, and p.E62K. These mutations occur preferentially in a GTPbound state due to the rapid transition from the GDP-bound state, which results ultimately in hyperactivity and combined immunodeficiency [13,14,17]. Previously, we showed that the p.P29R mutation does not alter hydrogen bonds, and that it strengthens expression of RAC2 in neutrophils [21]. Due to limitations regarding biochemical analysis in our group, we only examined expression of RAC2 in isolated neutrophils by immunostaining and immunoblotting analysis, neither of which revealed significant differences in RAC2 expression (Fig. 5A, B). We also performed Flagtagged WT and mutant RAC2 in HL-60 cells, and found comparable expression between WT and mutant forms of RAC2 (Fig. 5C). As it has been reported that RAC2 hyperactivation leads to increased PI3K activity and functionally affect mainly the CD56 dim NK cell subset [25], we tried to perform the expression of phospho-AKT (p-AKT) in transfected HL-60 cells; the results revealed significant heyperactivation of PI3K in the presence of the mutant RAC2 (Fig. 5C).

Analysis of Actin Polarization and Apoptosis
As tight regulation of RAC2 activity is pivotal for the control of actin dynamics and actin cytoskeleton reorganization, logically, impaired actin polarization has been documented in patients with dominant activating mutations [13,14,21]. Impaired actin polarization was also detected in neutrophils from the proband (Fig. 6).
As the dominant activating monoallelic RAC2 mutations, p.G12R, p.P34H, and p.P29R abrogate the survival capacities of hematopoietic stem and progenitor cells, unstimulated or stimulated T cells and B cells in vitro, and neutrophils, partially through increased apoptosis mechanistically [13,17,21]; therefore, we attempted to examine apoptosis of HL-60 cells. Analogously, increased apoptosis was found in HL-60 cells transfected with the mutant RAC2 form (Fig. 7).

Discussion
In this study, we identified a novel heterozygous RAC2 mutation, c.44G > A (p.G15D), in a Chinese family. Our findings broaden the clinical and genetic spectrum of RAC2 gainof-function mutations and underlines that such mutations can cause immunodeficiency with either complete or incomplete penetrance. Meanwhile, our investigations emphasize the consensus that the activity of RAC2 GTPase should be tightly regulated as a molecular switch to execute normal immunological functions, which is completely in accord with its essential attribute.
Recent investigations of RAC2 mutations have expanded our understanding of the range of accompanying clinical presentations, which can manifest from the newborn period into adulthood. To date, RAC2 mutations have been reported in 19 patients with different forms of primary (younger sister), and the proband's mother, in response to CD3/CD28 antibodies determined by carboxyfluorescein succinimidyl ester (CFSE) dilution and analyzed by flow cytometry immunodeficiency. Recurrent respiratory infections leading to bronchiectasis development are shared phenotypes of patients with autosomal recessive loss-of-function RAC2 mutations and those with dominant gain-of-function mutations; these were also the main symptoms noted in our patient. Viral infections, including varicella zoster, herpes stomatitis, herpetic skin, shingles after varicella vaccination, and cutaneous human papillomavirus infections, have predominately been recorded in patients with RAC2 gain-of-function mutations [13][14][15]. Intriguingly,  Fig. 4 Skewed peripheral T subsets and cytokine release after stimulation. A Intracellular IFN-γ (upper) and TNF-α (lower) expression in CD4 and CD8 T cells from patients P1 (the proband's father), P2 (the proband), P3 (younger sister), and the proband's mother, after stimulation with PMA/ionomycin; and B skewed differentiation of CD4 T cells toward the Th1-like phenotype by flow cytometry the proband in this study presented with persistent EBV viremia, infectious mononucleosis, and viral encephalitis, as well as cutaneous human papillomavirus infections.
EBV, also known as human herpesvirus 4, can cause severe disease in immunodeficient patients. EBV viremia and/or EBV-induced diseases, including fulminant infectious mononucleosis, hemophagocytic lymphohistiocytosis, lymphoproliferation, and lymphoma, are common clinical features of individuals affected with defined congenital primary immunodeficiencies. These immunodeficiencies primarily affect CD8 + T and NK cell functions, resulting in expansion or effector defects [26]. Examples of such  immunodeficiencies include activated PI3Kδ syndrome (APDS) [26,27]. APDS is caused by gain-of-function mutations in PI3KCD [28] and consequent direct binding and cross-linking between RAC and PI3K [13,29]; therefore, it has been assumed that many specific features and pathophysiological mechanisms are likely shared between patients with APDS patients and those with monoallelic activating RAC2 mutations [13,25]. In this context, lymphopenia, with accumulation of senescent CD8 + CD57 + T cells and terminal effector CD8 + T cells, impaired T cell receptor signaling, disturbed homing, and defective NK cell maturation or function, may theoretically lead to viral infections analogous to those characteristic of APDS, including with EBV, in patients with RAC2 mutations [13,25]. This hypothesis is supported by evidence of increased AKT phosphorylation levels and modified degranulation in vitro following selective inhibition of p110δ in patients with the RAC2 p.P34H mutation [14,25].
It should be noted that, in our patient, neurological symptoms were accompanied by active EBV infection, characterized by elevated liver enzymes, atypical lymphocytes, and a particularly high serum EBV replication load. Despite the lack of serological confirmation and no detection of EBV DNA in CSF, EBV encephalitis was presumed. There was a previous case report of EBV-associated neurological involvement in primary immunodeficiency [30]. The amount of EBV DNA detected in serum correlated with the severity of her clinical symptoms. Furthermore, cytotoxic edema can result in persistent disability, with irreversible brain tissue damage, which may indicate necrosis and unfavorable prognosis [31]. Our patient had cytotoxic edema, followed by atrophy, consistent with this scenario. Hence, we cannot rule out an alternative explanation that the lesions observed in our patient were caused primarily or only by EBV infection.
To our knowledge, the majority of patients with RAC2 mutations are characterized by reduced total IgG levels. Our patient was initially described as having clearly high IgG levels, resulting in reasonable suspicions of primary immunodeficiency disease being ignored. The mechanisms underlying this phenomenon remain unclear, although, possibly analogously, approximately 4% of patients with APDS also exhibit increased IgG levels [32]. Intriguingly, our patient only exhibited normal IgM levels at the onset of progressive infectious mononucleosis, then presented with constitutively decreased levels of this immunoglobulin, along with lymphopenia and undetectable serum EBV replication load. Further investigations into the unique role of RAC2 in humoral immunity and its distinct mutation spectrum are warranted.
Papilloma viral infections have been described in patients with the RAC2 p.P34H mutation [13], and were also observed in our patients with RAC2 p.G15D. This is reminiscent of homozygous nonsense RhoH mutations resulting in loss of RhoH protein, which led to T cell defects causing susceptibility to papilloma virus infections in two adult siblings [33]. Mechanistically, RhoH is physiologically constitutively activated and plays regulatory roles in competitive inhibition of Rac GTPases, coordinating as an antagonist at the downstream effectors of Rac [34]. Impairment of T cell receptor function and reduced numbers of tissue homing integrin β7-positive T cells likely contribute to susceptibility to β-papilloma viral infections in human RHOH deficiency [33]. Whether an analogical scenario underlies the effects of activating RAC2 mutations requires further study.
Finally, incomplete penetrance is frequently encountered in some forms of definitive primary immunodeficiency, predominantly those which are typically inherited in an autosomal dominant manner [35]. Hence, it is plausible that activating mutations in RAC2 can present with incomplete penetrance, similar to reports of APDS [32]. This hypothesis is further supported by the fact that the father of the proband in this study predominantly suffered from recurrent respiratory infections, and harbored similar immunological defects; by contrast, her younger sister appeared to be healthy, with no history of respiratory infections, and only mild immunological defects were detected preliminarily. The pathogenesis remains poorly characterized, and further detailed research is warranted.

Conclusion
In conclusion, we report a Chinese patient with a novel heterozygous activating mutation in RAC2 who presented with combined immunodeficiency. Our findings broaden the clinical and genetic spectra of RAC2 mutations. Importantly, our results suggest that RAC2 gain-of-function mutations can result in incompletely penetrant phenotypes.