Clinical and functional spectrum of RAC2-related immunodeficiency

Abstract

Mutations in the small Rho-family guanosine triphosphate hydrolase RAC2, critical for actin cytoskeleton remodeling and intracellular signal transduction, are associated with neonatal severe combined immunodeficiency (SCID), infantile neutrophilic disorder resembling leukocyte adhesion deficiency (LAD), and later-onset combined immune deficiency (CID). We investigated 54 patients (23 previously reported) from 37 families yielding 15 novel RAC2 missense mutations, including one present only in homozygosity. Data were collected from referring physicians and literature reports with updated clinical information. Patients were grouped by presentation: neonatal SCID (n = 5), infantile LAD-like disease (n = 5), or CID (n = 44). Disease correlated to RAC2 activity: constitutively active RAS-like mutations caused neonatal SCID, dominant-negative mutations caused LAD-like disease, whereas dominant-activating mutations caused CID. Significant T- and B-lymphopenia with low immunoglobulins were seen in most patients; myeloid abnormalities included neutropenia, altered oxidative burst, impaired neutrophil migration, and visible neutrophil macropinosomes. Among 42 patients with CID with clinical data, upper and lower respiratory infections and viral infections were common. Twenty-three distinct RAC2 mutations, including 15 novel variants, were identified. Using heterologous expression systems, we assessed downstream effector functions including superoxide production, p21-activated kinase 1 binding, AKT activation, and protein stability. Confocal microscopy showed altered actin assembly evidenced by membrane ruffling and macropinosomes. Altered protein localization and aggregation were observed. All tested RAC2 mutant proteins exhibited aberrant function; no single assay was sufficient to determine functional consequence. Most mutants produced elevated superoxide; mutations unable to support superoxide formation were associated with bacterial infections. RAC2 mutations cause a spectrum of immune dysfunction, ranging from early onset SCID to later-onset combined immunodeficiencies depending on RAC2 activity. This trial was registered at www.clinicaltrials.gov as #NCT00001355 and #NCT00001467.

Graphical abstract

Figure 1.

Clinical findings in patient with RAC2 mutation. (A) Age at clinical presentation across groups: CA, reticular dysgenesis without deafness caused by constitutively active mutations; DN, dominant-negative, D57N; N, homozygous null; A, CID caused by activating mutations; NBS, detected by TREC NBS; AR, autosomal recessive. (B) Lymphocyte counts by age for each patient with data. Circles colored by presentation group, gray shading indicates normal range for age. (C) Patient cohort sorted by presentation group including sex (M, male; F, female), mutations, and presence of clinical findings. “H” denotes homozygosity for listed mutation. Gray shading of RAC2 function indicates identification by NBS. Clinical manifestations denoted by filled box. % CID, percent patients with CID-manifesting specified phenotype; LRTI, lower respiratory tract infection; nd, no data; URTI, upper respiratory tract infection; X, deceased. ∗Forty three of 44 patients with CID had available clinical data, where a indicates streptococcal abscesses; b, necrotizing pneumonia and pulmonary abscesses; c, Escherichia coli skin; d, Serratia marcescens; e, bacterial skin; f, Staphylococcus aureus and Streptococcus pyogenes.

Figure 2.

Functional assessment of patient variants. (A) Cumulative luminescence of cells cotransfected with NADPH oxidase components (gp91^phox, p67^phox, p47^phox) and specific RAC2 mutants (30-minute time course). Unstimulated cells (basal) or after addition of 1 μM PMA (PMA). Bottom row of lower set shows expanded y-axis to detect minor activation levels. (B) Summary bar graphs from integrated kinetics of unstimulated (left) or PMA-stimulated (right) superoxide production normalized to WT basal. Dashed and solid lines correspond to WT basal and WT PMA-stimulated levels, respectively. Bars shows average ± standard of the mean (SEM), n = 3-5 independent experiments. (C) Summary of RAC2 protein stability quantified by Western blot densitometry. Graph shows RAC2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels, normalized to WT, and expressed as mean ± SEM, n = 3 to 5. (D) Summary of RAC2-PAK1 binding expressed as bound/total mutant RAC2 normalized to WT bound/total, determined by densitometry of Western blots, displayed as mean ± SEM, n = 3-5 independent experiments. (E) Summary of pAKT levels determined by densitometry of Western blots. pAKT levels were normalized to RAC2 expression to control for protein stability and GAPDH for cell loading. Values are mean ± SEM, n = 3 to 4 independent experiments. In all plots, significance determined by Kruskal-Wallis test using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli comparing RAC2 variants with WT RAC2; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001.

Figure 3.

Effects of RAC2 mutations on cellular localization and morphology. COS-7 cells cotransfected with GFP (green) and various RAC2 mutants were stained for F-actin (red) and RAC2 (white), nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Representative merged images are shown. Scale bar, 10 μm. Yellow arrows, PM ruffling; yellow arrowheads, macropinosomes.

Figure 4.

Summary of mutation functional assays and location on RAC2 structure. (A) Summary of each mutant tested across functional assays. Functional groups: A, activating; CA, constitutively active; and N, null. Values derived from functional studies were row normalized and colored. Red and blue correspond to increased or decreased activity, respectively, relative to WT RAC2; gray indicates variant not tested in specified assay. (B) Identified patient mutations shown on 3-dimensional structure of RAC1 amino acids 2-177 (3TH5) with switch regions (blue) and P-loop (orange) highlighted. Constitutively active and D57N mutations are noted in red; CID mutations in pink.