Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib.

INTRODUCTION: The signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity. METHODS: In our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation. RESULTS: STAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment. CONCLUSION: In STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Establishment of three Joubert syndrome-derived induced pluripotent stem cell (iPSC) lines harbouring compound heterozygous mutations in CC2D2A gene.

We have developed Joubert syndrome (JS)-derived induced pluripotent stem cell (iPSC) lines from dermal fibroblasts biopsied from a female patient harbouring novel compound heterozygous mutations in CC2D2A gene. The newly established iPSC lines provide tremendous promises for development of JS-derived neuronal cell lines to uncover the molecular and cellular mechanisms underlying the pathogenesis of JS and to develop therapeutic interventions for treatment of JS.

Generation of induced pluripotent stem cell (iPSC) lines from a Joubert syndrome patient with compound heterozygous mutations in C5orf42 gene.

We have generated new disease-specific induced pluripotent stem cell (iPSC) lines from skin fibroblasts obtained from a female patient with Joubert syndrome (JS) caused by compound heterozygous mutations in C5orf42 gene. The generated iPSCs offer an unprecedented opportunity to obtain iPSC-derived neurons to investigate the pathogenesis of JS in vitro and to develop therapeutic strategies.

Biomarkers and Precision Therapy for Primary Immunodeficiencies: An In Vitro Study Based on Induced Pluripotent Stem Cells From Patients.

Ataxia telangiectasia (AT) and Aicardi-Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients' cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate-adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.

Generation of three isogenic induced Pluripotent Stem Cell lines (iPSCs) from fibroblasts of a patient with Aicardi Goutières Syndrome carrying a c.2471G>A dominant mutation in IFIH1 gene.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Crohn's disease.

Crohn's disease is a debilitating and incurable chronic inflammatory bowel disease, affecting millions of individuals worldwide, with an increasing frequency. Surgery must be applicable in half of the cases often with a disabling course, and pharmacological treatments may have adverse complications. We generated three isogenic clones of iPSCs from peripheral blood mononuclear cells (PBMCs) of a patient with Crohn's Disease under pharmacological treatment without adverse effects. Sendai virus based vector was used and the iPSCs were characterized for genetic uniqueness, genomic integrity, pluripotency, and differentiation ability. These iPSCs will be a powerful tool to develop tailored therapies.