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.

High-throughput RNA sequencing of the T cell receptor alpha and beta chains for simultaneous clonality and biological analyses in Sezary syndrome.

BACKGROUND: Previous investigations pointed out a role for antigen stimulation in Sezary syndrome (SS). High-throughput sequencing of the T cell receptor (TR) offers several applications beyond diagnostic purposes, including the study of T cell pathogenesis. METHODS: We performed high-throughput RNA sequencing of the TR alpha (TRA) and beta (TRB) genes focusing on the complementarity-determining region 3 (CDR3) in 11 SS and one erythrodermic mycosis fungoides (MF) patients. Five psoriasis patients were employed as controls. Peripheral blood CD4+ cells were isolated and RNA sequenced (HiSeq2500). High-resolution HLA typing was performed in neoplastic patients. RESULTS: Highly expanded predominant TRA and TRB CDR3 were only found in SS patients (median frequency: 94.4% and 93.7%). No remarkable CDR3 expansions were observed in psoriasis patients (median frequency of predominant TRA and TRB CDR3: 0.87% and 0.69%, p < 0.001 compared to SS). CDR3 almost identical to the predominant were identified within each SS patient and were exponentially correlated with frequencies of the predominant CDR3 (R2 = 0.918, p < 0.001). Forty-six different CDR3 were shared between SS patients displaying HLA similarities, including predominant TRA and TRB CDR3 in one patient that were found in other three patients. Additionally, 351 antigen matches were detected (Cytomegalovirus, Epstein-Barr, Influenza virus, and self-antigens), and the predominant CDR3 of two different SS patients matched CDR3 with specificity for Influenza and Epstein-Barr viruses. CONCLUSIONS: Besides detecting clonality, these findings shed light on the nature of SS-related antigens, pointing to RNA sequencing as a useful tool for simultaneous clonality and biological analysis in SS.