STAT1 Gain-of-function Research Articles

RECURRENT APHTHOUS STOMATITIS AND ORAL CANDIDIASIS DUE TO STAT1 MUTATION

<h3>Introduction</h3> Chronic mucocutaneous candidiasis (CMC) often presents with chronic <i>Candida</i> infections of the skin, nails or mucous membranes in the absence of invasive fungal infections. Various genetic defects are associated with CMC. Here, we describe a case of recurrent aphthous stomatitis and chronic oral candidiasis due to gain-of-function (GOF) mutation in STAT1. <h3>Case Description</h3> A 21-year-old male presented to the Allergy and Immunology clinic with long-standing history of recurrent aphthous ulcers and oral candidiasis. He had previously undergone evaluation as a child with normal immunoglobulin levels, normal antibody response to vaccines and normal delayed-type hypersensitivity skin test to <i>Candida</i> antigen, but low absolute NK cells. Targeted genetic testing was negative for APECED and IPEX. He continued to experience frequent aphthous ulcers and oral thrush that can be debilitating at times, with one episode resulting in hospital admission due to severe mucosal pain and PO intolerance. Repeat immunology evaluation was similar to prior. He had low absolute NK cells, and NK cell function study using chromium release assay was notably decreased. A comprehensive primary immunodeficiency next generation sequencing panel identified a pathogenic heterozygous c.800C>T (p.A267V) mutation in STAT1. <h3>Discussion</h3> While fungal infection of the oral mucosa is a main clinical feature of CMC, aphthous stomatitis can be found frequently in patients with STAT1 GOF mutations as well. These patients also frequently have decreased NK cells and NK cell function. It is important to be aware of these additional phenotypes in the evaluation of a patient with CMC.

Immunoprofiling of monocytes in STAT1 gain-of-function chronic mucocutaneous candidiasis.

Patients with STAT1 gain-of-function (GOF) mutations suffer from an inborn error of immunity hallmarked by chronic mucocutaneous candidiasis (CMC). The pathogenesis behind this complex and heterogeneous disease is still incompletely understood. Beyond the well-recognized Th17 failure, linked to the STAT1/STAT3 dysbalance-driven abrogation of antifungal defense, only little is known about the consequences of augmented STAT1 signaling in other cells, including, interestingly, the innate immune cells. STAT1-mediated signaling was previously shown to be increased in STAT1 GOF CD14+ monocytes. Therefore, we hypothesized that monocytes might represent important co-orchestrators of antifungal defense failure, as well as various immunodysregulatory phenomena seen in patients with STAT1 GOF CMC, including autoimmunity. In this article, we demonstrate that human STAT1 GOF monocytes are characterized by proinflammatory phenotypes and a strong inflammatory skew of their secretory cytokine profile. Moreover, they exhibit diminished CD16 expression, and reduction of classical (CD14++C16-) and expansion of intermediate (CD14++16+) subpopulations. Amongst the functional aberrations, a selectively enhanced responsiveness to TLR7/8 stimulation, but not to other TLR ligands, was noted, which might represent a contributing mechanism in the pathogenesis of STAT1 GOF-associated autoimmunity. Importantly, some of these features extend to STAT1 GOF monocyte-derived dendritic cells and to STAT1 GOF peripheral myeloid dendritic cells, suggesting that the alterations observed in monocytes are, in fact, intrinsic due to STAT1 mutation, and not mere bystanders of chronic inflammatory environment. Lastly, we observe that the proinflammatory bias of STAT1 GOF monocytes may be ameliorated with JAK inhibition. Taken together, we show that monocytes likely play an active role in both the microbial susceptibility and autoimmunity in STAT1 GOF CMC.

Systems Immunology Analyses of STAT1 Gain-of-Function Immune Phenotypes Reveal Heterogeneous Response to IL-6 and Broad Immunometabolic Roles for STAT1.

Patients with STAT1 gain-of-function (GOF) pathogenic variants have enhanced or prolonged STAT1 phosphorylation following cytokine stimulation and exhibit increased yet heterogeneous susceptibility to infections, autoimmunity, and cancer. Although disease phenotypes are diverse and other genetic factors contribute, how STAT1 GOF affects cytokine sensitivity and cell biology remains poorly defined. In this study, we analyzed the immune and immunometabolic profiles of two patients with known pathogenic heterozygous STAT1 GOF mutation variants. A systems immunology approach of peripheral blood cells from these patients revealed major changes in multiple immune cell compartments relative to healthy adult and pediatric donors. Although many phenotypes of STAT1 GOF donors were shared, including increased Th1 cells but decreased class-switched B cells and plasmacytoid dendritic cell populations, others were heterogeneous. Mechanistically, hypersensitivity for cytokine-induced STAT1 phosphorylation in memory T cell populations was particularly evident in response to IL-6 in one STAT1 GOF patient. Immune cell metabolism directly influences cell function, and the STAT1 GOF patients shared an immunometabolic phenotype of heightened glucose transporter 1 (GLUT1) and carnitine palmitoyl transferase 1A (CPT1a) expression across multiple immune cell lineages. Interestingly, the metabolic phenotypes of the pediatric STAT1 GOF donors more closely resembled or exceeded those of healthy adult than healthy age-similar pediatric donors, which had low expression of these metabolic markers. These results define new features of STAT1 GOF patients, including a differential hypersensitivity for IL-6 and a shared increase in markers of metabolism in many immune cell types that suggests a role for STAT1 in metabolic regulation of immunity.

Ex vivo effect of JAK inhibition on JAK-STAT1 pathway hyperactivation in patients with dominant-negative STAT3 mutations.

STAT1 gain-of-function (GOF) and dominant-negative (DN) STAT3 syndromes share clinical manifestations including infectious and inflammatory manifestations. Targeted treatment with Janus-kinase (JAK) inhibitors shows promising results in treating STAT1 GOF-associated symptoms while management of DN STAT3 patients has been largely supportive. We here assessed the impact of ruxolitinib on the JAK-STAT1/3 pathway in DN STAT3 patients' cells. Using flow cytometry, immunoblot, qPCR, and ELISA techniques, we examined the levels of basal STAT1 and phosphorylated STAT1 (pSTAT1) of cells obtained from DN STAT3, STAT1 GOF patients, and healthy donors following stimulation with type I/II interferons (IFNs) or interleukin (IL)-6. We also describe the impact of ruxolitinib on cytokine-induced STAT1 signaling in these patients. DN STAT3 and STAT1 GOF resulted in a similar phenotype characterized by increased STAT1 and pSTAT1 levels in response to IFNα (CD3+ cells) and IFNγ (CD14+ monocytes). STAT1-downstream gene expression and C-X-C motif chemokine 10 secretion were higher in most DN STAT3 patients upon stimulation compared to healthy controls. Ex vivo treatment with the JAK1/2-inhibitor ruxolitinib reduced cytokine responsiveness and normalized STAT1 phosphorylation in DN STAT3 and STAT1 GOF patient' cells. In addition, ex vivo treatment was effective in modulating STAT1 downstream signaling in DN STAT3 patients. In the absence of effective targeted treatment options for AD-HIES at present, modulation of the JAK/STAT1 pathway with JAK inhibitors may be further explored particularly in those AD-HIES patients with autoimmune and/or autoinflammatory manifestations.

Impact of JAK Inhibitors in Pediatric Patients with STAT1 Gain of Function (GOF) Mutations—10 Children and Review of the Literature

IntroductionSince the first description of gain of function (GOF) mutations in signal transducer and activator of transcription (STAT) 1, more than 300 patients have been described with a broad clinical phenotype including infections and severe immune dysregulation. Whilst Jak inhibitors (JAKinibs) have demonstrated benefits in several reported cases, their indications, dosing, and monitoring remain to be established.MethodsA retrospective, multicenter study recruiting pediatric patients with STAT1 GOF under JAKinib treatment was performed and, when applicable, compared with the available reports from the literature.ResultsTen children (median age 8.5 years (3–18), receiving JAKinibs (ruxolitinib (n = 9) and baricitinib (n = 1)) with a median follow-up of 18 months (2–42) from 6 inborn errors of immunity (IEI) reference centers were included. Clinical profile and JAKinib indications in our series were similar to the previously published 14 pediatric patients. 9/10 (our cohort) and 14/14 patients (previous reports) showed partial or complete responses. The median immune deficiency and dysregulation activity scores were 15.99 (5.2–40) pre and 7.55 (3–14.1) under therapy (p = 0.0078). Infection, considered a likely adverse event of JAKinib therapy, was observed in 1/10 patients; JAKinibs were stopped in 3/10 children, due to hepatotoxicity, pre-HSCT, and absence of response.ConclusionsOur study supports the potentially beneficial use of JAKinibs in patients with STAT1 GOF, in line with previously published data. However, consensus regarding their indications and timing, dosing, treatment duration, and monitoring, as well as defining biomarkers to monitor clinical and immunological responses, remains to be determined, in form of international prospective multicenter studies using established IEI registries.

DNA-Binding domain mutations confer severe outcome at an early age among STAT1 gain-of-function patients.

STAT1gain-of-function (GOF) is an immune dysregulatory disorder with poorly studied genotype-phenotype correlation, impeding prognostication and early intervention. Given previous mechanistic studies, as well as anecdotal clinical reports, we sought to systematically determine whether DNA-binding domain (DBD) mutations in STAT1 result in a different phenotype than mutations in other gene domains. Negative prognostic features previously identified by the International STAT1 GOF Study Group (invasive infections, intracranial aneurysms, and malignancy), as well as other clinical features and mortality, were compared within a cohort of 30 patients with STAT1 GOF diagnosed at our center, consisting of 9 patients with DBD mutations and 21 patients with non-DBD mutations. We subsequently re-analyzed mortality data from a large, previously-published 274-patient cohort by the International STAT1 GOF Study Group. While no differences were noted with respect to malignancy or symptomatic aneurysms, invasive /opportunistic infections were substantially more common among DBD patients, as were sinopulmonary infections, bronchiectasis, enteropathy, endocrinopathies, lymphoproliferative manifestations, and recurrent fevers/HLH. DBD patients also had a lower probability of survival and younger age of mortality compared with non-DBD patients. Our re-evaluation of the published data from the International STAT1 GOF Study Group revealed a similar finding of earlier mortality among patients harboring DBD mutations. We report that STAT1 GOF patients with DBD mutations may be regarded as a unique subgroup, impacted more by early-onset profound combined immunodeficiency and with earlier mortality. These findings may impact clinical decision making with respect to early intervention, and in particular hematopoietic stem cell transplant considerations, in such patients.

Epstein-Barr Virus associated gastric carcinoma in a patient with germline STAT3 gain-of-function mutation

Abstract Introduction/Objective Signal transducer and activator of transcription 3 (STAT3) is a transcription factor involved in inflammation, proliferation, differentiation and survival. STAT3 gain-of-function (GoF) disorders are characterized by immune dysregulation and present with polyendocrinopathy, enteropathy, X-linked syndrome (IPEX)- or autoimmune lymphoproliferative syndrome (ALPS)-like features. While patients with STAT-3 GoF are known to have an increased risk for hematologic malignancies, neither solid tumors nor an increased risk for EBV-associated disorders have been described. We report the first case of an EBV-associated solid organ tumor in a patient with STAT-3 GoF. Methods/Case Report A 21-year-old male with germline mutation in STAT3 (variant p. M329K) presented with early satiety, abdominal pain and worsening of chronic anemia. Serology showed high EBV DNA PCR levels. Endoscopy showed multiple nodular lesions in the stomach, which were biopsied to reveal EBV-associated high-grade dysplasia and intramucosal adenocarcinoma. Initiation of chemotherapy with a poor response led to a total gastrectomy. Gross examination of the specimen showed a 7.9 x 6.5 x 1.8 cm tan-brown, exophytic mass in the posterior wall of the body and antrum, involving the greater curvature. Histology revealed an adenocarcinoma with tubulovillous morphology extending into the lamina propria, without invasion into the muscularis mucosa or submucosa. EBER in-situ hybridization was diffusely positive in the tumor cells. The background mucosa showed severe chronic active and atrophic gastritis with intestinal metaplasia and low-grade dysplasia. All the seventy examined lymph nodes were negative for metastasis. Helicobacter-like organisms were not seen. Results (if a Case Study enter NA) NA Conclusion This is the first report of a solid tumor in a patient with STAT3 GoF mutation. The role of the patient’s underlying immune dysregulation disorder in the development of EBV-associated gastric adenocarcinoma is unclear and warrants further investigation. The case also highlights the importance of a close clinical follow-up in this patient population, as unexpected malignancies can develop at younger ages.

Chronic demodicosis in patients with immune dysregulation: An unexpected infectious manifestation of Signal transducer and activator of transcription (STAT)1 gain-of-function.

Signal transducer and activator of transcription (STAT)1 heterozygous gain-of-function (GOF) mutations are known to induce immune dysregulation and chronic mucocutaneous candidiasis (CMCC). Previous reports suggest an association between demodicosis and STAT1 GOF. However, immune characterization of these patients is lacking. Here, we present a retrospective analysis of patients with immune dysregulation and STAT1 GOF who presented with facial and ocular demodicosis. In-depth immune phenotyping and functional studies were used to characterize the patients. We identified five patients (three males) from two non-consanguineous Jewish families. The mean age at presentation was 11.11 (range=0.58-24) years. Clinical presentation included CMCC, chronic demodicosis and immune dysregulation in all patients. Whole-exome and Sanger sequencing revealed a novel heterozygous c.1386C>A; p.S462R STAT1 GOF mutation in four of the five patients. Immunophenotyping demonstrated increased phosphorylated signal transducer and activator of transcription in response to interferon-α stimuli in all patients. The patients also exhibited decreased T cell proliferation capacity and low counts of interleukin-17-producing T cells, as well as low forkhead box protein 3+ regulatory T cells. Specific antibody deficiency was noted in one patient. Treatment for demodicosis included topical ivermectin and metronidazole. Demodicosis may indicate an underlying primary immune deficiency and can be found in patients with STAT1 GOF. Thus, the management of patients with chronic demodicosis should include an immunogenetic evaluation.

Enhanced osteoclastogenesis in patients with MSMD due to impaired response to IFN-γ

Patients with Mendelian susceptibility to mycobacterial disease (MSMD) experience recurrent and/or persistent infectious diseases associated with poorly virulent mycobacteria. Multifocal osteomyelitis is among the representative manifestations of MSMD. The frequency of multifocal osteomyelitis is especially high in patients with MSMD etiologies that impair cellular response to IFN-γ, such as IFN-γR1, IFN-γR2, or STAT1 deficiency. This study sought to characterize the mechanism underlying multifocal osteomyelitis in MSMD. GM colonies prepared from bone marrow mononuclear cells from patients with autosomal dominant (AD) IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 gain of function (GOF) and from healthy controls were differentiated into osteoclasts in the presence or absence of IFN-γ. The inhibitory effect of IFN-γ on osteoclastogenesis was investigated by quantitative PCR, immunoblotting, tartrate-resistant acid phosphatase staining, and pit formation assays. Increased osteoclast numbers were identified by examining the histopathology of osteomyelitis in patients with AD IFN-γR1 deficiency or AD STAT1 deficiency. In the presence of receptor activator of nuclear factor kappa-B ligand and M-CSF, GM colonies from patients with AD IFN-γR1 deficiency, AD STAT1 deficiency, or STAT1 GOF differentiated into osteoclasts, similar to GM colonies from healthy volunteers. IFN-γ concentration-dependent inhibition of osteoclast formation was impaired in GM colonies from patients with AD IFN-γR1 deficiency or AD STAT1 deficiency, whereas it was enhanced in GM colonies from patients with STAT1 GOF. Osteoclast differentiation is increased in AD IFN-γR1 deficiency and AD STAT1 deficiency due to an impaired response to IFN-γ, leading to excessive osteoclast proliferation and, by inference, increased bone resorption in infected foci, which may underlie multifocal osteomyelitis.

Novel STAT-3 gain-of-function variant with hypogammaglobulinemia and recurrent infection phenotype.

Signal transducer and activator of transcription 3 (STAT‐3) gain‐of‐function (GOF) syndrome is an early‐onset monogenic inborn error of immunity characterized by multi‐organ autoimmune disorders, growth failure and lymphoproliferation. We describe that STAT‐3 GOF syndrome may be presented with hypogammaglobulinemia and recurrent severe upper and lower respiratory tract infections. In addition, the patient had lymphoproliferation, short stature and interstitial lung disease. Chest computerized tomography examinations showed mild bronchiectasis with areas of non‐fibrosing alveolar‐interstitial disease and maldevelopment of bilateral first ribs. Using Sanger sequencing, we revealed a novel c.508G>C, p.D170H STAT‐3 variant affecting the coiled coil domain of STAT‐3. Functional studies confirmed that p.D170H was a GOF variant, as shown by increased phosphorylated STAT‐3 (pSTAT‐3) and STAT‐3 transcriptional activity. Our observation suggests that STAT‐3 GOF syndrome can manifest in early childhood with hypogammaglobulinemia and recurrent severe respiratory tract infections. We suggest that patients with lymphoproliferation, hypogammaglobulinemia and severe recurrent infections should be screened for STAT‐3 variants, even if autoimmune manifestations are missing.

The paradigm of hematological malignant versus non-malignant manifestations, driven by primary immunodeficiencies: a complex interplay.

Hematological malignancies (HM) developed on underlying primary immunodeficiencies (PID) are rare and of unusual features. Differentiating between malignant and non-malignant lymphoproliferation in cases of pediatric hematology and oncology and revealing their molecular predisposition demonstrate the complex interplay between PID and HM. We retrospectively studied a case series of seven pediatric patients, all with PID with manifestations raising suspicion for HM or hypereosinophilic syndrome (HES) or confirmed HM of lymphoid origin. Combined immunodeficiency (CID) without detection of a known mutated gene or with ataxia-telangiectasia (AT), STAT3 gain of function (GOF), DOCK8 deficiency, and CTLA4 deficiency were diagnosed in three, one, one, one, and one patient, respectively. Acute lymphoblastic leukemia and Hodgkin lymphoma followed by second primary Burkitt lymphoma were diagnosed in one patient with CID each, while lymphomatoid granulomatosis in one patient with AT. Lymphoproliferative disease occurred in STAT3 GOF, CTLA4 deficiency and CID, one patient each, and idiopathic HES in DOCK8 deficiency (median age at presentation of PID or any hematological manifestation: four years). Four patients underwent hematopoietic cell transplantation (HCT) for STAT3 GOF, DOCK8 deficiency and CID in one, one, and two cases, respectively (median age: 10years). At the last follow-up, all transplanted patients were alive. Reporting on patients' phenotype, genotype and course of disease shed light on the prevalence, characteristics, and pathophysiology of HM complicating PID. Discriminating the non-yet malignant lymphoproliferation from its malignant equivalent on the same pathophysiology background proved of additional value. Outcomes of PID after HCT, herein reported, are favorable.

P672 A tertiary multicenter cohort of patients with chronic intestinal pseudo-obstruction and Crohn’s disease: a rare association with a high prevalence of monogenic disorders

Abstract Background Chronic intestinal pseudo-obstruction (CIPO) is a rare condition that is not commonly associated with Crohn’s disease (CD). We performed a cohort study aiming at identifying clinical, immunological and genetic features as well as response to conventional CD treatments of patients co-affected with CIPO and CD. Methods We conducted an observational retrospective cohort study in two tertiary CIPO and IBD centers. Patients with parenteral nutrition-dependent CIPO and features of CD including large intestinal or perianal ulcers, strictures, abscesses and fistula with pathology findings compatible with CD, were included. We used flow-cytometry and targeted-next generation sequencing to identify immune defects and monogenic causes in 129 predefined genes responsible for monogenic enteropathies Results Eight unrelated patients were studied. 5 had a myogenic phenotype and 3 had a neurogenic form with histological exam for all patients. CD involved small bowel in all cases whereas one patient had ileocolonic involvement. Two patients had perianal complex fistula. Seven patients had a stricturing form and 1 had an inflammatory-only behavior. No patient had a penetrating form. All patients were diagnosed with CIPO prior to CD. Median age at CIPO diagnosis was 11.5 years old (IQR 0,31.5) while it was 22.5 (IQR 19,29) at CD diagnosis. At CD diagnosis, mean fecal calprotectin level was 551 ug/g (range 390 - 1200) and citrulline averaged 23.6 umol/l (range 15–35 umol/l). Histopathological analyses of intestinal specimens revealed ulcerations for half of the patients, increase in intraepithelial lymphocytes in 3, granuloma and cryptitis in 1 patient. Abnormalities in peripheral lymphocytes’ subsets were found in 2 patients. 5 patients were ultimately diagnosed with monogenic forms of enteropathy: 2 in ACTG2, with an autosomal dominant inheritance, 1 with a STAT1gain of function, (GOF) and two with recessive inheritance: TYMP. All patients received steroids with clinical response in 2. 6 patients received antiTNF, with only one sustained remission. Two patients received vedolizumab and 2 were treated with ustekinumab. Except for antiTNF in 1 patient, common CD treatments did not lead to any improvement. Of note, antiJAK therapy in a patient with STAT1GOF failed to induce remission. Conclusion Herein is described the first cohort of extensively genetically and immunologically explored patients co-affected with CIPO and CD. Immune defects were common and monogenic forms were found with a high prevalence. Rates of response to biologic treatment were very low. Identification of monogenic cause through immunological and genetic explorations is warranted in patient with CIPO-CD association as it could lead to targeted therapy and genetic counseling.

STAT Immunodeficiency Disorders and Fungal Infection Susceptibility

Invasive fungal diseases (IFD) or complicated mucocutaneous fungal infections can occur secondary to both primary and acquired immunodeficiency disorders. The signal transducer and activator of transcription (STAT) proteins are critical transcription factors involved in a wide range of processes including the immune response. This review highlights the immune pathways, clinical phenotypes and current management options of fungal infections in patient with mutations in STAT1 and STAT3, the most relevant STAT molecules involved in the host response to fungal pathogens. STAT1 gain of function (GOF) and STAT3 loss of function (LOF) (cause of autosomal dominant Hyper IgE syndrome) mutations have been associated with both chronic mucocutaneous candidiasis (CMC) and invasive fungal infections, not only inhaled moulds, mostly Aspergillus spp., but also Pneumocystis, Cryptococcus and other endemic dimorphic fungi. Significant progress has been made in the insights into the immunopathogenesis of fungal infections in these patients; nevertheless, the concrete mechanisms involved are still not fully elucidated. Early initiation of antifungal therapy followed by long-term secondary prophylaxis is normally required. Some adjunctive therapies have also been employed, although their use has not been completely established. Besides a developing knowledge in STAT1 GOF and STAT3 LOF mutations, additional research is needed. It should aim for a better understanding of the pathogenesis, diagnosis, and management of fungal infections in these patients. The identification of new therapeutic targets should help to improve patient quality of life and outcomes.

Transcriptional Profiling of STAT1 Gain-of-Function Reveals Common and Mutation-Specific Fingerprints.

STAT1 gain-of-function (GOF) is a primary immunodeficiency typically characterized by chronic mucocutaneous candidiasis (CMC), recurrent respiratory infections, and autoimmunity. Less commonly, also immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX)-like syndromes with CMC, and combined immunodeficiency without CMC have been described. Recently, our group and others have shown that different mutation-specific mechanisms underlie STAT1 GOF in vitro, including faster nuclear accumulation (R274W), and reduced mobility (R321, N574I) to near immobility in the nucleus (T419R) upon IFNγ stimulation. In this work, we evaluated the transcriptomic fingerprint of the aforementioned STAT1 GOF mutants (R274W, R321S, T419R, and N574I) relative to STAT1 wild-type upon IFNγ stimulation in an otherwise isogenic cell model. The majority of genes up-regulated in wild-type STAT1 cells were significantly more up-regulated in cells expressing GOF mutants, except for T419R. In addition to the common interferon regulated genes (IRG), STAT1 GOF mutants up-regulated an additional set of genes, that were in part shared with other GOF mutants or mutation-specific. Overall, R274W and R321S transcriptomes clustered with STAT1 WT, while T419R and N574I had a more distinct fingerprint. We observed reduced frequency of canonical IFNγ activation site (GAS) sequences in promoters of genes up-regulated by all the STAT1 GOF mutants, suggesting loss of DNA binding specificity for the canonical GAS consensus. Interestingly, the T419R mutation, expected to directly increase the affinity for DNA, showed the most pronounced effects on the transcriptome. T419R STAT1 dysregulated more non-IRG than the other GOF mutants and fewer GAS or degenerate GAS promotor sequences could be found in the promoter regions of these genes. In conclusion, our work confirms hyperactivation of common sets of IFNγ-induced genes in STAT1 GOF with additional dysregulation of mutation-specific genes, in line with the earlier observed mutation-specific mechanisms. Binding to more degenerate GAS sequences is proposed as a mechanism toward transcriptional dysregulation in R274W, R321S, and N574I. For T419R, an increased interaction with the DNA is suggested to result in a broader and less GAS-specific response. Our work indicates that multiple routes leading to STAT1 GOF are associated with common and private transcriptomic fingerprints, which may contribute to the phenotypic variation observed in vivo.

PULMONARY HYPERTENSION IN A CHILD WITH STAT3 GAIN OF FUNCTION MUTATION-ASSOCIATED INTERSITIAL LUNG DISEASE

SESSION TITLE: Fellows Pediatrics Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: STAT3 gain of function mutations are a recently described cause of autoimmunity that affect multiple organ systems, including the lungs. CASE PRESENTATION: The patient was first diagnosed with autoimmune lymphoproliferative syndrome (ALPS) at age 2 in the setting of lymphadenopathy, hepatosplenomegaly, & autoimmune cytopenias. Treatment was initially steroids & IVIG; he later started sirolimus as a steroid-sparing agent. No genetic defect consistent with ALPS was identified though he was found to have short telomeres, of unclear significance as he did not fit a dyskeratosis congenita phenotype. Further evaluation revealed a gain of function (GOF) mutation in STAT3. At age 9, he developed dyspnea & syncope; evaluation revealed mild pulmonary hypertension (PH) with positive vasoreactivity testing and CT chest with upper-lobe predominant subpleural cystic lung disease & scattered pulmonary nodules. Immunosuppression was switched from sirolimus to mycophenolate. For the PH, he was prescribed dual oral therapy, with symptomatic improvement. He developed hypoxemia at age 11. VQ scan was notable for markedly heterogeneous perfusion despite homogenous ventilation & PFTs revealed a reduced DLCO with mild restriction. Given concern for worsening pulmonary disease in the setting of the STAT3 mutation, he was started on tocilizumab (in place of mycophenolate) with partial improvement in hypoxemia. At age 12, given persistent oxygen requirement & CT showing new upper lobe predominant mosaic attenuation with groundglass (& overall stable cystic lung disease), he started ruloxitinib with some improvement in his dyspnea & hypoxemia. Since tocilizumab & ruloxitinib were initiated, his TLC & DLCO have normalized. DISCUSSION: In the largest series of patients with STAT3 GOF autoimmunity, 36% were reported to have interstitial lung disease (ILD), though none had cystic lung disease1. Upper-lobe predominant subpleural cystic disease, as seen in our patient, is most reminiscent of that often incidentally found in Trisomy 21, and has not been described in other pediatric populations2. In addition to autoimmunity related to STAT3 GOF involving the lungs, there is evidence that inappropriate activation of STAT3 is involved in the development of fibrosis in patients with UIP/IPF, suggestive of a potential alternative pathway to ILD in these patients3. Pulmonary hypertension has not previously been described as a consequence of STAT3 GOF mutations. Our patient developed PH years before he became hypoxemic, suggestive that his PH is not simply due to chronic hypoxemia. CONCLUSIONS: In this patient, STAT3 GOF mutation was associated with cystic lung disease and pulmonary hypertension, manifestations that have not previously been described. Reference #1: Fabre A, Marchal S, Barlogis V, et al. Clinical aspects of STAT3 gain-of-function germline mutations: a systematic review. J Allergy Clin Immunol Pract. 2019;7(6):1958-69. Reference #2: George M, Amodio J, Lee H. Cystic lung disease in Down syndrome: a case report and literature review. Case Rep Pediatr. 2016;2016:4048501. Reference #3: Pechkovsky DV, Prele CM, Wong J, et al. STAT3-mediated signaling dysregulates lung fibroblast-myofibroblast activation and differentiation in UIP/IPF. AJP. 2012;180(4):1398-1412. DISCLOSURES: No relevant relationships by Sarah Cohen, source=Web Response No relevant relationships by Stephen Kirkby, source=Web Response

Three Copies of Four Interferon Receptor Genes Underlie a Mild Type I Interferonopathy in Down Syndrome.

Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

Distinct molecular response patterns of activating STAT3 mutations associate with penetrance of lymphoproliferation and autoimmunity

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations.

STAT1 Gain-of-Function Mutations Cause High Total STAT1 Levels With Normal Dephosphorylation.

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14+ monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14+ monocytes and CD3+ T cells were higher than in healthy donors. There was a strong and positive correlation between CD14+ STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14+ monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.

Interferon signature in patients with STAT1 gain-of-function mutation is epigenetically determined.

STAT1 gain-of-function (GOF) variants lead to defective Th17 cell development andchronic mucocutaneous candidiasis (CMC), but frequently also to autoimmunity. Stimulation of cells with STAT1 inducing cytokines like interferons (IFN) result in hyperphosphorylation and delayed dephosphorylation of GOF STAT1. However, the mechanism how the delayed dephosphorylation exactly causes the increased expression of STAT1-dependent genes, and how the intracellular signal transduction from cytokine receptors is affected, remains unknown. In this study we show that the circulating levels of IFN-α were not persistently elevated in STAT1 GOF patients. Nevertheless, the expression of interferon signature genes was evident even in the patient with low or undetectable serum IFN-α levels. Chromatin immunoprecipitation (ChIP) experiments revealed that the active chromatin mark trimethylation of lysine 4 of histone 3 (H3K4me3), was significantly enriched in areas associated with interferon-stimulated genes in STAT1 GOF cells in comparison to cells from healthy donors. This suggests that the chromatin binding of GOF STAT1 variant promotes epigenetic changes compatible with higher gene expression and elevated reactivity to type I interferons, and possibly predisposes for interferon-related autoimmunity. The results also suggest that epigenetic rewiring may be responsible for treatment failure of Janus kinase 1/2 (JAK1/2) inhibitors in certain patients.

Type 1 Diabetes in STAT Protein Family Mutations: Regulating the Th17/Treg Equilibrium and Beyond.

Improvements in the immunological, molecular, and genetic technologies such as next-generation sequencing have led to an exponential increase in the number of monogenic immune dysregulatory syndromes diagnosed, where type 1 diabetes (T1D) forms part of the autoimmune manifestations. Here, we reviewed the mutations in the signal transducer and activator of transcription (STAT) protein family, namely gain-of-function (GOF) mutations in STAT1 and STAT3 as well as STAT5b deficiency, that show strong association to T1D susceptibility. The equilibrium of T-helper 17 (Th17) and regulatory T cells (Tregs) is often found altered in patients affected by STAT GOF mutations. While the increased number of Th17 cells and the concomitant decrease in Treg cells may explain T1D in STAT3 GOF patients, the reduced number of Th17 cells found in those carrying STAT1 GOF mutations added a new level of complexity on the exact role of Th17 in the pathogenesis of T1D. Here, we describe the possible mechanisms through which STAT3 and STAT1 GOF mutations may perturb the fate and function of Th17 and Tregs and explore how this may lead to the development of T1D. We propose that the study of monogenic diseases, and in particular STAT mutations, may not only improve our understanding of the function of the human immune system but also shed light onto the pathogenic mechanisms of T1D and the genetic variants that confer predisposition to the disease.