Mutations in STAT1 may also cause autosomal-dominant or autosomal-recessive diseases. STAT1 is heavily involved in IFN-α/β, IFN-γ, IFN-λ, IL-6, IL-21 and IL-27 signaling [85]. IFN-α/β, IFN-γ and IL-27 are known as typical cytokines that hinder the development of so-called TH17 cells producing IL-17A, IL-17F, and IL-22. STAT1 has been described to have activating gain-of-function but also LOF mutations.
Since its first description in 2011 by Van de Veerdonk et al. [86], the STAT1-GOF phenotype, characterized by immunodeficiency and autoimmunity, has been well described: The most prominent symptom of patients is the presence of chronic mucocandidiasis (CMC), which can be observed in about 90% of all patients before the age of 10 years (early-onset CMC), yet the lifetime risk of CMC is nearly 100% [51]. The main pathogen causing mucocutaneous infections is Candida albicans, although invasive infections with Candida albicans, Cryptococcus spp., Pneumocystis jirovecii, Aspergillus spp., and Penicillium marneffei can also be observed in approximately 20% of cases. Overall, around half of all CMC cases appear to be attributable to STAT1-GOF mutations [87–89], highlighting the importance of CMC as a diagnostic criterion for STAT1-GOF mutation. In addition to fungal pathogens, patients with STAT1-GOF mutations are susceptible to recurrent lower respiratory tract bacterial infections (LRTI). Infections with S. aureus, Streptococcus spp, Pseudomonas aeruginosa, or H. influenzae can - in some circumstances - lead to long-lasting LRTI that can result in severe pneumonia, bronchitis, interstitial pneumonia, or more persistently, bronchiectasis. Mycobacterial diseases such as tuberculosis have been described as well as skin diseases and adenitis; these may occur after infections with Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium genavense, environmental mycobacteria or BCG vaccine [51]. Viral infections with herpesviridae (herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus) have also been commonly described [51]. On the basis of immune dysregulation, patients may develop an autoimmune phenotype, which may present as autoimmune thyroid disease, autoimmune cytopenia, vitiligo, psoriasis, alopecia, type I diabetes, autoimmune hepatitis, enteropathy, systemic lupus erythematosus, polyendocrinopathy, or an IPEX-like syndrome. STAT1-GOF mutations are associated with an increased incidence of certain tumour entities, some of which may be explained by persistent mucocutaneous fungal infections that develop into squamous cell carcinoma (cutaneous, oral, laryngeal, oesophageal, gastrointestinal cancer). STAT1-GOF patients also present with melanoma, lymphoma, leukaemia, prostate cancer, papillary thyroid cancer [51]. Aneurysms occur three times more frequently than in the normal population (10.7%) and may remain asymptomatic but might burst, especially intracerebrally [51, 90]. Chronic (fungal) infections with the development of drug resistance, malignancies, and aneurysms associated with haemorrhages (particularly in the brain), are predictors of poor outcome in STAT1-GOF patients [87].
In light of the characteristic symptoms, treating physicians should consider the differential diagnosis STAT1-GOF in patients with CMC alone, CMC with lower respiratory tract infections, or CMC with autoimmune thyroid disease. Altered lymphocyte subpopulations can be determined by lymphopanel analyses (see below). In this context, various genetic and functional tests are useful for strengthening the diagnosis of STAT-GOF, e.g., subcellular distribution or levels of STAT1, pSTAT1, ISGF3 (Interferon-stimulated gene factor 3), γ-activated factor (GAF) in unstimulated and in IFNα-, IFNγ-, or IL-27-stimulated conditions. Also, gene transcription induced by interferons or in vitro luciferase STAT1 reporter assays may be helpful for diagnosing STAT1-GOF. Eventually, DNA sequencing can provide information about the exact genotypic variation of the patient. Integrating such findings could serve to identify possible genotype-phenotype correlations. According to Zhang et al. the median onset of symptoms in STAT1-GOF patients is one year, but the median diagnosis does not occur until 6.2 years, implying a noteworthy delay of diagnosis [51].
The symptoms of the patients can be partly explained by the changes in immune cell subpopulations. 87% of patients with STAT1-GOF mutations have a deficiency of TH17 cells, which is due to a poor development of IL-17A, IL-17F, and IL-22 producing cells. Increased responses to IFN-α/β, IFN-γ, IL-27, which are all STAT1-dependent repressors of IL-17 producing cells, hinder the development of those cells in mice and humans [91]. Interestingly, the lack of TH17 cells, which are necessary for preventing CMC, cannot be observed in all patients, whereas almost all patients develop CMC. While the production of IL-6, IL-17A, IL-22 is impaired, the production of IL-4 is increased in comparison to healthy individuals [92]. Other lymphocyte subpopulations are also reduced: diminished numbers of CD4+ and CD8+ T cells are observed (23% of patients have low T cells, 30.8% have low CD4+cells, 17.9% have CD8+ low, in some cases both CD4+ and CD8+ are reduced); CD19+ or CD20+ B cell numbers are low in 23.9%, and most patients have reduced numbers of CD19+CD27+ memory B cells. CD16+CD56+ NK cells are low in 32% and show a reduced NK cell toxicity. In addition, humoral immunodeficiency with low serum IgG, IgA or IgM levels was described in 6.5%, 18.3%, and 6.3% of patients, respectively [51].
The underlying genetic causes for STAT1-GOF are activating mutations in mostly the coiled-coil domain but also in the DNA-binding domain, linker domain [93] or SH2 domain [94]. More than 110 missense mutations and one deletion have been described [51]. The resulting mechanisms for hyperactivation of STAT1 are the subject of current research and are not yet fully understood in their complexity. Various, sometimes opposing, hypotheses exist for the increased molecular activity of the STAT1 protein. Easily understood are results that imply an impaired cycle of phosphorylation or dephosphorylation, where either the phosphorylation of STAT1 occurs faster or the nuclear dephosphorylation is slowed down [85]. In experiments with the protein kinase inhibitor staurosporine, a persistently high level of pSTAT1 was observed, supporting the latter hypothesis. This is opposed by findings that observed a normal or even faster rate of dephosphorylation in CD14+ monocytes with concomitant high levels of total STAT1 expression [93, 95–98]. These findings are complemented by the observation of high STAT1 mRNA levels [95, 99, 100]. Other studies suggest premature nuclear import with normal phosphorylation/dephosphorylation rate [101], increased nuclear accumulation (R247W), decreased mobility (R321, N571I), or immobility in the nucleus (T419R) as the cause of STAT1 hyperactivation [102]. There also exists the concept that DNA binding specificity decreases with certain STAT1-GOF mutations as fewer GAS motifs are detectable in promoters of STAT1-GOF-specific genes. The ratio of GAS present to GAS absent in promoters of STAT1-regulated genes decreased significantly for the STAT1-T419R variant in ChiP-Seq experiments [102]. In RNA-Seq analyses, dysregulated gene transcription caused by the STAT1 variants resulted in different mutation-specific fingerprints and offers a possible explanation for the wide range of phenotypic variation. Reasonably well described is the observation that GOF mutations in the coiled-coil domain and DNA-binding domain impair the stabilization of the STAT1 homodimer in an antiparallel conformation. Physiologically, conversion from parallel conformation to antiparallel conformation is required for dephosphorylation at pY701 by phosphatases. By remaining in the parallel conformation and lacking access to pY701, STAT1-GOF homodimers remain phosphorylated and become resistant to dephosphorylation [103, 104].
Finally, the different mechanisms may all lead to a hyperactivation of STAT1. Increased responses to IFN-α/β, IFN-γ and IL-27 by STAT1 hyperactivation may explain the impaired development of TH17 cells since those cytokines are STAT1-dependent repressors for this development [91] in mice and humans. In addition, in patient cells with STAT1-GOF mutations, increased expression of the adaptive immune system suppressing PD-L1 was detected [105, 106] and thereby may impede TH17 differentiation as observed in a mouse model [107, 108]. Similar to STAT3-GOF mutations, STAT1-induced SOCS1-mediated suppression of STAT5 may occur with STAT1-GOF mutations. Since STAT5 is known to be involved in terminal NK cell differentiation, NK cell toxicity, and perforin expression, this may explain increased viral susceptibility in patients with STAT1-GOF mutations [99, 109, 110]. Based on its hyperactivity and the fact that STAT1 can sequester STAT3 in heterodimers [111–114], it would also be conceivable that an increased STAT1 response inhibits STAT3 by utilizing the process of heterodimerization. Our group is investigating the influence of STAT1 and STAT3 mutations, respectively, on the process of heterodimerization, and how different mutations affect STAT1:STAT3 heterodimer formation. As STAT3 is the key transcription factor for TH17 development (see above), this concept provides an explanation for the high prevalence of CMC in both, patients with STAT3-LOF mutations and patients with STAT1-GOF mutations.
Also, STAT1 GOF mutations seem to be able to have an influence on an epigenetic level. Kaleviste et al. showed that H3K4me3, a marker for activation of gene transcription by chromatin remodelling, was significantly increased in interferon-stimulated genes in patient cells compared to healthy donors, even in the absence of IFNα [94]. These results promote the idea of epigenetic changes due to an increased IFN signal response. It suggests that the increased IFN signature of patients with STAT1-GOF might be epigenetically fixed and that treatment might need to take this into account.
The treatment of patients with STAT1-GOF mutations has advanced considerably over the past 10 years. While the initial focus has been on symptomatic treatment of patients with abatement of infection-related symptoms, treatment options nowadays aim at a more causal approach. Topical or systemic antifungal therapy (preferably triazoles such as fluconazole), prophylactic or ad hoc antibiotic therapy, and immunoglobulin replacement therapy especially for the prophylaxis or treatment of pneumonia, is commonly used. Eczematous dermatitis may be treated with topical steroids or topical application of calcineurin inhibitors.
With the advent of JAK inhibitors (jakinibs) such as ruxolitinib and baricitinib (both inhibiting JAK1 and JAK2), targeted therapeutic options are available for STAT1-GOF patients, and have improved symptoms to even resolution of CMC in 12/20 patients [60, 97, 109, 115–117]. However, due to the possibility of varicella-zoster virus and cytomegalovirus (VZV and CMV) exacerbation, as well as worsening of fungal infections such as CMC and coccidioidomycosis observed in several patients treated with ruxolitinib, aciclovir and antifungal prophylaxis should be considered [60, 93].
In recent studies, ruxolitinib was considered a useful drug for bridging therapy in patients with STAT1-GOF mutations waiting for HSCT to reduce post-transplant complications [110]. Approximately 40% of STAT1-GOF patients have died following HSCT due to secondary graft failure, or gastrointestinal or pulmonary bleedings [51]. Ruxolitinib is considered to reduce the enhanced IFN signalling in blood and tissue cells and therefore the immune dysregulation caused by STAT1-GOF mutations. Mechanistically this could help to down-regulate uncontrolled inflammation and therefore minimize the risk of secondary graft failure. The purpose of the concomitant and bridging therapy is to restore the STAT1-mediated immune response to a normal state until HSCT can correct the underlying genetic defect. The studied patient showed stabilization by therapy with ruxolitinib (improved STAT1 phosphorylation/dephosphorylation, normalized number and response of TfH cells, partly improved health status and IFN signature) but persistent TH17 deficiency. Subsequent HSCT normalized STAT1 phosphorylation/dephosphorylation, dysregulated gene expression, the IFN signature, and T, B, and NK cell function. Further studies are necessary to better understand the challenging problems in transplanting patients with GOF mutations in which - at least theoretically - full conditioning and a 100% donor chimerism needs to be achieved for a sustained therapeutic success.