Simple diagnosis ofSTAT1gain-of-function alleles in patients with chronic mucocutaneous candidiasis

Abstract

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14(+) cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

“…40 In patients with CMCD, STAT1 GOF mutations are associated with a characteristic delay of protein dephosphorylation, resulting in persistent activation of the signaling pathway. 5,18,24,25 In our study the time course analysis of STAT1 phosphorylation in response to IFN-a has shown a similar pattern in NK cells of patients with STAT1 GOF mutations. However, we have also observed that both resting and IL-2-activated NK cells of these patients display increased STAT1 protein levels and higher expression of STAT1 mRNA, suggesting that augmented STAT1 protein levels might concur with the increased response of NK cells to cytokines signaling throughout STAT1.…”

“…13 Fungal infections constitute the most common manifestation of CMCD, but viral infections, including recurrent mucocutaneous infections caused by reactivation of varicella-zoster virus and herpes simplex viruses, debilitating orf infection, or severe invasive infection caused by chicken pox, cytomegalovirus, or EBV have also been reported. [16][17][18][19][20][21] The broad spectrum of infections observed in patients with CMCD suggests that susceptibility to pathogens is not entirely due to the lack of T H 17 cells, but other cell types, particularly NK cells, might play a role in the pathogenesis of CMCD. To define the role of NK cells in the susceptibility of patients with CMCD to intracellular pathogens and viruses, we have investigated NK functional activities and STAT1 and STAT5 signaling in response to cytokines in these patients.…”

“…This observation is in accordance with previous studies showing a characteristic delay of STAT1 dephosphorylation. 5,18,24,25 Moreover, we investigated STAT1 protein levels in primary resting NK and T cells from patients with STAT1 GOF mutations. Surprisingly, flow cytometric analysis of STAT1 protein showed a 3-fold increase in STAT1 protein levels in resting NK and T cells from patients compared with protein levels detected in cells of healthy control subjects (Fig 1, F, and see Fig E1, E).…”

Mechanism-Based Strategies for the Management of Autoimmunity and Immune Dysregulation in Primary Immunodeficiencies

“…Specifically, naïve CD4+ T cells are uniquely biased towards IFN-γ production irrespective of polarizing conditions and expansion of follicular helper T cells relative to Tregs has been shown (51). T cell targeting with cyclosporine has been anecdotally used to treat AIHA in STAT1 -GOF with benefit (52). More recently, a janus kinase (JAK) 1/2 inhibitor (ruxolitinib) was used to treat two distinct cases of STAT1 -GOF with associated autoimmunity including autoimmune cytopenias (51) and refractory alopecia areata (53).…”

Flow Cytometry, a Versatile Tool for Diagnosis and Monitoring of Primary Immunodeficiencies

“…Phospho-flow can be used in the diagnostic evaluation of PIDs associated with defects in the STAT (signal transducer and activator of transcription factor) molecules, e.g., loss-of-function (LOF) STAT1 mutations seen in patients with susceptibility to intracellular bacterial and viral infections (72,73). Similarly, gain-of-function (GOF) mutations seen in the same gene, STAT1, associated with autosomal dominant chronic mucocutaneous candidiasis (CMC) and defective Th17 immunity (74)(75)(76)(77)(78) can also be assessed by changes in phosphorylation by flow cytometry. It is important to recognize that for accurate diagnostic use of phospho-flow assays, only the appropriate cell subset after careful selection of the stimulus (typically cytokines) is evaluated.…”