Patient Clinical Characteristics
The patient was from a nonconsanguineous kindred with no significant family history. She first presented with fever and cough at 4 months and was diagnosed with pneumonia, and since had recurrent pneumonia at a frequency of three times each year, which was treated locally with repeated oral and intravenous antibiotics. She also experienced recurrent episodes of hemoptysis from 8 years old, with documented bronchiectasis. Due to the recurrent fever and pneumonia, requiring increasingly longer hospitalization and higher grade antibiotic treatment, our hospital was consulted when she was 10 years old; she weighed 20 kg and her height was 122 cm on admission. On clinical examination, clubbing of her fingers was detected, along with moderate moist rales and hepatosplenomegaly. Laboratory investigations showed constitutive severe lymphopenia and fluctuating granulocytopenia, which was particularly prominent during infection, with large vacuoles visible by light microscopy (Fig. 1a–d), and decreased serum immunoglobulin G (IgG) (Table 1), with no commonly positive autoantibodies, such as anti-nuclear, anti-neutrophil cytoplasmic, or anti-thyroid peroxidase antibodies. Haemophilus influenzae was repeatedly isolated from sputum on etiological examination, while fungal, cytomegalovirus (CMV), and Epstein-Barr virus (EBV) infections were absent. Chest computer tomography scan revealed extensive bronchiectasis (Fig. 1e). From the beginning of 2019, she began to receive regular intravenous immunoglobulin treatment each month; however, pulmonary infections continued to occur more than five times per year and required hospitalization for combined antibiotic treatment.
The patient had severe T, B, and NK cell lymphopenia, with high relative percentages of effector/memory T cells, while a markedly reduced CD4:CD8 ratio, as well as recent thymic and bone marrow emigrants (Table 2). She also showed diminished proliferative responses to stimulation with mitogens or T-cell receptor (TCR), while cytokine production was elevated (Fig. S1 and Fig. S2). Further work-up revealed extended active cTfh, as well as increased percentages of Th1 and Th1/17-like subsets among non-cTfh and cTfh cells (Fig. 2a, b). Additionally, the patient had a significatively increased percentage of double-negative B cells, along with CD21low B cells (Fig. 2c). Collectively, these results indicated combined immunodeficiency with a dysregulated phenotype.
A Novel Heterozygous RAC2 Mutation Identified by Gene Sequencing
Sequencing analysis using an NGS-based immunodeficiency panel revealed a novel de novo heterozygous variant in RAC2 (c.86C>G), which was confirmed by Sanger sequencing, and causes a proline to arginine substitution at position 29 (p.P29R) of the RAC2 protein (Fig. 3a, b). Pathogenicity analysis of this novel mutation predicted that it is deleterious (Table 3). The mutated P29 residue resides within a highly-conserved Switch I domain (Fig. 3c), which is vitally important for interactions with GEFs and downstream effectors ; however, 3D-modeling structural analysis of the mutant protein using Swiss PdbViewer did not show any obvious destruction of hydrogen bonds or influence on interactions with GDP (data not shown). Nevertheless, data on RAC2 and RAC1 mutations in cancer cells support the hypothesis that this novel mutation is disease causing; RAC1 p.P29S substitutions have been identified in sun-exposed melanomas, head and neck squamous cell carcinomas, as well as analogous RAC2 p.P29L and p.P29Q mutations [37-39], supporting the importance of this proline residue for normal Switch I region function. This variant meets the American College of Medical Genetics (ACMG) criteria for pathogenesis , as follows: pathogenic strong (PS1) evidence, as it is a de novo mutation; 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; and pathogenic supporting 3 (PP3), since multiple lines of computational evidence support a deleterious effect. Taken together, these findings indicate that the variant is plausibly a novel pathogenic heterozygous RAC2 mutation.
Enhanced Neutrophil ROS Production and Activation
RAC2 has non-redundant and crucial roles in multiple biological functions of neutrophils, including in oxidase activity . Neutrophils from RAC2-/- mice and patients with the RAC2 p.D57N mutation exhibit impaired ROS production in response to fMLP, whereas patients with activating gain-of-function mutations, such as RAC2 p.E62K and p.P34H, show enhanced ROS generation [19, 20]. Samples from our patient also showed increased ROS production in response to fMLP and WKYMVM, while levels were normal following PMA stimulation (Fig. 4a), since the latter is independent of RAC2 . These findings were reminiscent of the enhanced ROS production and stimulation-induced apoptosis in BTK-deficient human neutrophils, resulting from increased RAC2 activity in the plasma membrane in a primed state, which has been proposed as responsible for neutropenia and tissue damage . Further, neutrophil activation was quantified by multicolor flow cytometry to detect cell-surface CD11b and CD62L expression, and degranulation was assessed by analysis of cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules). Samples from our patient showed increased neutrophil activation, but no obvious degranulation abnormalities in response to different stimuli (Fig. S3).
Impaired Actin Polarization in T Cells and Neutrophils
The activated state of the RAC2 GTPase is tightly regulated, and appropriate inactivation of RAC2 is required for F-actin disassembly and phagocytic cup closure . Elevated F-actin content was detected in neutrophils from our patient (Fig. 4b). Decreased cycling between F-actin and G-actin is expected to result in impaired actin polarization, which was observed in patient T cells and neutrophils (Fig. 5a, b), along with impaired RAC2 polarization in neutrophils (Fig. 5c). The apparent macropinosomes detected in neutrophils from our patient were consistent with the abnormal macropinocytosis reported in patients with RAC2 p.E62K mutations . These abnormalities are established as associated with numerous functions, including cellular migration, intracellular trafficking, and phagocytosis, among others. These results demonstrate that tight regulation of RAC2 activity is pivotal for the control of actin dynamics and actin cytoskeleton reorganization required for physiological cell polarization and macropinocytosis.
Increased Apoptosis in T/B Cells and Neutrophils
The RAC2 p.G12R mutation is proven to abrogate the survival and differentiation capacities of hematopoietic stem and progenitor cells, because of consequent defects in cellular and mitochondrial networks . Analogously, transgenic mice expressing the constitutively active RAC2 p.G12V or p.Q61L mutations exhibit increased thymocyte and T lymphocyte apoptosis . Further, the RAC2 p.P34H mutation results in increased apoptosis of unstimulated and TCR-stimulated T cells in vitro and EBV-transformed B cells after serum starvation . Mechanistically, these elegant studies have established preliminary and causative roles for RAC2 in lymphopenia. Similarly, significantly increased apoptosis/necrosis of unstimulated, TCR-stimulated, or FASL-induced T cells, and unstimulated or CPG/F(ab)2-stimulated B cells, respectively, were observed in vitro in samples from our patient. In addition, we detected increased apoptosis/necrosis of unstimulated and FASL-induced neutrophils, which could not be rescued by LPS stimulation (Fig. 6a). Furthermore, intracellular active caspase 3 expression was markedly increased in leukocytes from the patient (Fig. 6b). Collectively, our data suggest that RAC2 p.P29R leads to increased apoptosis, partly through influencing caspase 3, which may help to dissect the signaling mechanisms underlying her observed severe peripheral lymphopenia and neutropenia.
Strengthened Expression of RAC2 in Neutrophils and Lymphocytes
The RAC2 mutants, p.G12R, p.P34H, p.Q61R, p.E62K, and p.N92T, along with the RAC1 p.P29S and p.N92I alterations, have been shown to exist preferentially in a GTP-bound state, as a result of rapid transition from the GDP-bound state, ultimately resulting in hyperactivity and combined immunodeficiency [19, 20, 22, 23, 37-39]. By contrast, the p.D57N mutation acts in a dominant negative fashion to interfere with RAC1 and RAC2 function, and displayed 10% GTP-binding ability, relative to the wild-type protein [12-14]. Intriguingly, expression of the homozygous null p.W56X RAC2 mutation was completely absent in cells transfected with the mutant . As no changes in hydrogen bonds or interactions with GDP were predicted to result from the novel mutation reported here, we investigated its influence on RAC2 expression in our patient, and detected increased mRNA and protein levels in neutrophils and lymphocytes (Fig. 7a–d). Hence, we identified altered expression of RAC2 protein arising from this gain-of-function mutation.