Successful Therapy of a Patient with a Novel STAT1 Gain of Function Mutation and Life-Threatening Cytopenias with Janus Kinase Inhibitor Ruxolitinib

Abstract Monoallelic gain of function (GOF) mutations in the human Signal Transducer and Activator of Transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity. The immunodeficiency is caused by impaired IL-17 immunity and typically presents with chronic mucocutaneous candidiasis, variably associated with other opportunistic infections. The autoimmune manifestations due to exaggerated responsiveness to interferon are more diverse and include SLE, thyroiditis, hepatitis, and alopecia areata. The phenotypic spectrum of STAT1 GOF mutations is extremely wide, ranging from intermittent localized disease to chronic systemic illness and fatality. Allogeneic bone marrow transplantation has been attempted in severely affected patients but outcomes have been poor, leaving a void for alternate long-term strategies to control the disease and maintain remission. Recently, we diagnosed a patient suffering from chronic mucocutaneous candidiasis and life-threatening autoimmune cytopenias with a novel monoallelic mutation in the linker domain of STAT1 (c.1633G>A; p.E545K). The mutation caused a profound increase in STAT1 phosphorylation in response to type 1 and 2 interferon without affecting dephosphorylation kinetics, which is mechanistically distinct from all reported STAT1 GOF mutations to date. The potential of naïve patient CD4+ T cells to become IFN- γ or IL-17 producing cells was investigated under TH0 (anti-CD3/28), TH1 (anti-CD3/28, IL-12) and TH17 (anti-CD3/28, IL6, IL-23, TGF-β1) conditions and revealed that patient CD4+ T cells are biased to produce IFN-γ irrespective of polarizing conditions, and resistant to TH17 induction upon stimulation with IL6, IL-23 and TGF-β1. In addition, the patient's proportion of T follicular helper cells (TFH) relative to regulatory T cells (Treg) was increased. Treatment with the Janus Kinase (JAK) 1/2 inhibitor Ruxolitinib reduced the hyperresponsiveness to interferon, normalized the TH1 response, enabled naïve T cells to differentiate into TH17 cells and decreased the TFH to Treg ratio. Under therapeutic dose Ruxolitinib the patient maintained clinical remission with respect to both autoimmunity and mucocutaneous candidiasis. Conclusion: Clinical vigilance for an underlying immune dysregulation syndrome due to abnormal JAK-STAT signaling is critical when evaluating patients with autoimmunity combined with opportunistic infections as JAK-inhibitors represents an effective targeted therapy for long-term disease control even in severely affected patients for whom hematopoietic stem cell transplantation is not available. Disclosures Off Label Use: Ruxolitinib use for STAT1 GOF mutation.

Download Full-text

Human gain-of-function STAT1 mutation disturbs IL-17 immunity in mice

International Immunology ◽

10.1093/intimm/dxz079 ◽

2019 ◽

Vol 32 (4) ◽

pp. 259-272 ◽

Cited By ~ 3

Author(s):

Moe Tamaura ◽

Naoko Satoh-Takayama ◽

Miyuki Tsumura ◽

Takaharu Sasaki ◽

Satoshi Goda ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Molecular Mechanisms ◽

Improve Patient Care ◽

Chronic Mucocutaneous Candidiasis ◽

Gain Of Function ◽

Human Phenotype ◽

Ifn Γ ◽

High Level ◽

Stat1 Protein

Abstract Gain-of-function (GOF) mutations in the gene for signal transducer and activator of transcription 1 (STAT1) account for approximately one-half of patients with chronic mucocutaneous candidiasis (CMC) disease. Patients with GOF-STAT1 mutations display a broad variety of infectious and autoimmune manifestations in addition to CMC, and those with severe infections and/or autoimmunity have a poor prognosis. The establishment of safe and effective treatments based on a precise understanding of the molecular mechanisms of this disorder is required to improve patient care. To tackle this problem, we introduced the human R274Q GOF mutation into mice [GOF-Stat1 knock-in (GOF-Stat1R274Q)]. To investigate the immune responses, we focused on the small intestine (SI), which contains abundant Th17 cells. Stat1R274Q/R274Q mice showed excess phosphorylation of STAT1 in CD4+ T cells upon IFN-γ stimulation, consistent with the human phenotype in patients with the R274Q mutation. We identified two subpopulations of CD4+ T cells, those with ‘normal’ or ‘high’ level of basal STAT1 protein in Stat1R274Q/R274Q mice. Upon IFN-γ stimulation, the ‘normal’ level CD4+ T cells were more efficiently phosphorylated than those from WT mice, whereas the ‘high’ level CD4+ T cells were not, suggesting that the level of STAT1 protein does not directly correlate with the level of pSTAT1 in the SI. Inoculation of Stat1R274Q/R274Q mice with Candida albicans elicited decreased IL-17-producing CD4+RORγt+ cells. Stat1R274Q/R274Q mice also excreted larger amounts of C. albicans DNA in their feces than control mice. Under these conditions, there was up-regulation of T-bet in CD4+ T cells. GOF-Stat1R274Q mice thus should be a valuable model for functional analysis of this disorder.

Download Full-text

A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-γ-mediated responses in chronic mucocutaneous candidiasis

Molecular Immunology ◽

10.1016/j.molimm.2015.09.014 ◽

2015 ◽

Vol 68 (2) ◽

pp. 597-605 ◽

Cited By ~ 13

Author(s):

Laura Martinez-Martinez ◽

Maria Teresa Martinez-Saavedra ◽

Pablo Fuentes-Prior ◽

Maria Barnadas ◽

Maria Victoria Rubiales ◽

...

Keyword(s):

Chronic Mucocutaneous Candidiasis ◽

Gain Of Function ◽

Mucocutaneous Candidiasis ◽

Ifn Γ

Download Full-text

Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

Journal of Experimental Medicine ◽

10.1084/jem.20110958 ◽

2011 ◽

Vol 208 (8) ◽

pp. 1635-1648 ◽

Cited By ~ 514

Author(s):

Luyan Liu ◽

Satoshi Okada ◽

Xiao-Fei Kong ◽

Alexandra Y. Kreins ◽

Sophie Cypowyj ◽

...

Keyword(s):

Autosomal Recessive ◽

Coiled Coil ◽

Cellular Responses ◽

Chronic Mucocutaneous Candidiasis ◽

Loss Of Function ◽

Gain Of Function ◽

Mucocutaneous Candidiasis ◽

Coiled Coil Domain ◽

Whole Exome ◽

Ifn Γ

Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity.

Download Full-text

Risks of Ruxolitinib in STAT1 Gain-of-Function-Associated Severe Fungal Disease

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx202 ◽

2017 ◽

Vol 4 (4) ◽

Cited By ~ 23

Author(s):

Ofer Zimmerman ◽

Berenice Rösler ◽

Christa S Zerbe ◽

Lindsey B Rosen ◽

Amy P Hsu ◽

...

Keyword(s):

Broad Spectrum ◽

Fungal Infections ◽

Fungal Disease ◽

Janus Kinase ◽

Chronic Mucocutaneous Candidiasis ◽

Jak Inhibitor ◽

Gain Of Function ◽

Mucocutaneous Candidiasis

Abstract Heterozygous STAT1 gain-of-function (GOF) mutations are associated with chronic mucocutaneous candidiasis and a broad spectrum of infectious, inflammatory, and vascular manifestations. We describe therapeutic failures with the Janus Kinase (JAK) inhibitor ruxolitinib in 2 STAT1 GOF patients with severe invasive or cutaneous fungal infections.

Download Full-text

STAT1 Gain-of-Function in Patients with Chronic Mucocutaneous Candidiasis Can be Detected By the Excessive Phosphorylation of STAT1 in Peripheral Blood Monocytes

Blood ◽

10.1182/blood.v124.21.4111.4111 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4111-4111

Author(s):

Yoko Mizoguchi ◽

Miyuki Tsumura ◽

Satoshi Okada ◽

Osamu Hirata ◽

Shizuko Minegishi ◽

...

Keyword(s):

Flow Cytometry ◽

Dna Binding ◽

Peripheral Blood ◽

Specific Gene ◽

Chronic Mucocutaneous Candidiasis ◽

Gain Of Function ◽

Transcription Activity ◽

Mucocutaneous Candidiasis ◽

Familial Cases ◽

Ifn Γ

Abstract Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 linage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. STAT1 mutations identified in patients with CMCD are gain-of-function (GOF), gain-of-gamma-activated factor (GAF) DNA binding and gain-of-gamma-activated sequence (GAS) transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased phosphorylation of STAT1 at Tyr701 (pSTAT1). In this paper, we aimed to identify and characterize STAT1 mutations in CMCD patients, and to develop a simple diagnostic assay of CMCD. Five sporadic and five familial cases of CMCD, from a total of 15 patients from 10 kindreds in Japan, are investigated. Six heterozygous missense mutations, including three novel mutations, in CCD and DBD of STAT1 were identified in two sporadic and four familial cases in 10 patients with CMCD. Thus, STAT1 mutations were commonly identified in Japanese patients with CMCD. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are GOF mutations against GAF mediated transcription activity. Next, we studied dephosphorylation of STAT1 using peripheral blood mononuclear cell (PBMCs) from the patients. As the STAT1 GOF mutations are thought to be associated with impairment in dephosphorylation of STAT1, we used staurosporine, the tyrosine kinase inhibitor which inhibits JAK-STAT signaling upstream of STAT1, to clarify the difference between STAT1 WT alleles and STAT1 GOF alleles. If the STAT1 dephosphorylation normally occurs in the nucleus, pSTAT1 should promptly decrease following staurosporine treatment. PBMCs from fourteen healthy individuals and ten patients with CMCD carrying GOF STAT1 mutations were incubated with staurosporine followed by IFN-γ stimulation and analyzed by flow cytometry. Some overlap was observed, but MFI values for pSTAT1 in response to IFN-γ were significantly higher in CD14+ cells from the patients than in those from the controls PBMCs from the patients. This excess phosphorylation persisted after 15 minutes of treatment with staurosporine. Moreover, in these conditions, there was no overlap in MFI of pSTAT1 between the patients and healthy controls. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation and this flow cytometry-based technique is likely to be useful for the rapid assessment of STAT1 function in CMCD patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text

TRAF3IP2 variants in a child with chronic mucocutaneous candidiasis- keeping an open mind!

10.22541/au.162220877.78615359/v1 ◽

2021 ◽

Author(s):

Pilar Blanco Lobo ◽

Wei-Te Lei ◽

Simon Pelham ◽

Paloma Guisado Hernández ◽

Isabel Villaoslada ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Ex Vivo ◽

Luciferase Reporter ◽

Compound Heterozygous ◽

Chronic Mucocutaneous Candidiasis ◽

Reporter System ◽

Mucocutaneous Candidiasis ◽

Ifn Γ ◽

Compound Heterozygous Variants

Background: Inherited chronic mucocutaneous candidiasis (CMC) is often caused by inborn errors of immunity, impairing the response to, or the production of IL-17A and IL-17F. About half of the cases carry STAT1 gain-of-function (GOF) mutations. Only few patients have been reported with mutations of TRAF3IP2, a gene encoding the adaptor ACT1 essential for IL-17-receptor(R) -signaling. We investigated a 10-year-old girl with CMC, carrying a heterozygous variant of STAT1 and compound heterozygous variants of TRAF3IP2. Methods: By flow cytometry STAT1 levels and phosphorylation (CD14+) as well as IL-17A-, IL-22-, IFN-γ- and IL-4-production (memory CD4+ T cells) were determined. ACT1 expression and binding to IL-17RA by western blot and co-immunoprecipitation in HEK-293T cells transfected with plasmids encoding wild-type or mutant HA-tagged ACT1 and Flag-IL-17RA. We evaluated IL-17A response using an NF-κB-driven luciferase reporter system in HEK-293T cells, and by measuring GRO-α secretion by fibroblasts. Results: A likely non-pathogenic STAT1 variant (c.1363G>A/p.V455I) was identified by next generation sequencing., STAT1 expression and phosphorylation upon IFN- were normal. We also found compound heterozygous variants (c.1325A>G/p.D451G and c.1335delA/p.K454fs11*) of TRAF3IP2. By overexpression, despite normal protein expression, and impaired (K454fs11*) or normal (D451G) interaction with IL-17RA, both mutant alleles resulted in an impaired NF-κB-activation. Patient’s fibroblasts displayed abolished GRO-α secretion upon IL-17A. Finally, ex vivo CD4+ T cells showed increased IL-17A, IL-22, and IL-4, and normal-low IFN-γ expression upon stimulation. Conclusion: We identify novel compound heterozygous variants of TRAFP3IP2 causing autosomal recessive ACT1 deficiency in a child with CMC, and provide a review of the current literature.

Download Full-text