RESUMO
UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.
Assuntos
Lúpus Eritematoso Sistêmico , Receptor 7 Toll-Like , Camundongos , Animais , Humanos , Receptor 7 Toll-Like/genética , Autoimunidade/genética , Receptor Toll-Like 9/metabolismo , Receptor 8 Toll-Like , Receptor 3 Toll-Like/metabolismo , Lúpus Eritematoso Sistêmico/genética , Proteínas de Membrana TransportadorasRESUMO
Mutations in SAMHD1, encoding SAM and HD domain-containing protein 1, cause Aicardi-Goutières syndrome (AGS) 5, an infancy-onset autoinflammatory disease characterized by neurodegeneration and chronic activation of type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts and peripheral blood mononuclear cells from three AGS patients with biallelic SAMHD1 mutations. These cell lines provide a valuable source to study disease mechanisms and to assess therapeutic molecules.
Assuntos
Células-Tronco Pluripotentes Induzidas , Interferon Tipo I , Proteínas Monoméricas de Ligação ao GTP , Humanos , Proteína 1 com Domínio SAM e Domínio HD/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação/genética , Interferon Tipo I/genética , Interferon Tipo I/metabolismoRESUMO
Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.
Assuntos
Células-Tronco Pluripotentes Induzidas , Interferon Tipo I , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Exodesoxirribonucleases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mutação/genética , Interferon Tipo I/genética , Citocinas , AntiviraisRESUMO
Importance: Familial chilblain lupus is a monogenic autosomal dominant form of cutaneous lupus erythematosus that in most cases is caused by mutations in the 3 prime repair exonuclease 1 (TREX1). Familial chilblain lupus presents in early childhood with cold-induced painful erythematous infiltrates leading to mutilation and is associated with systemic involvement illustrated by an elevated type I interferon (IFN) signature in the skin and blood. Effective treatment is currently not available. Objectives: To evaluate the clinical response to the Janus kinase inhibitor baricitinib in familial chilblain lupus and assess the effect of cold on patient fibroblasts. Design, Setting, and Participants: In this case series, 3 patients with familial chilblain lupus due to TREX1 mutation underwent treatment with baricitinib for 3 months. Interventions: Doses of baricitinib, 4 mg, were administered daily for 3 months. Main Outcomes and Measures: Reduction of cutaneous lupus lesions was measured by the revised cutaneous lupus area and severity index, pain due to skin and joint involvement was assessed by visual analog scale, type I IFN signature in blood was determined by polymerase chain reaction, and the in vitro response of fibroblasts to cold exposure was analyzed. Results: All 3 patients (2 women and 1 man; mean [SD] age, 51 [24] years) showed a significant improvement of cutaneous lupus lesions with suppression of systemic type I IFN activation. One patient had a complete remission regarding pain and, in 2 patients, pain associated with joint inflammation was partially reduced. No severe adverse reactions were reported. Exposure of patient fibroblasts to cold induced a stress response and enhanced senescence along with induction of IFN-stimulated gene in vitro. Conclusions and Relevance: These findings demonstrate the therapeutic efficacy of Janus kinase inhibition in a monogenic form of lupus among 3 patients and provide mechanistic insight into the process of disease exacerbation by cold in TREX1-deficient cells. This finding may be relevant to other type I IFN-mediated disorders and implicates Janus kinase inhibition as a potential therapeutic option also for multifactorial cutaneous lupus erythematosus.
Assuntos
Azetidinas/uso terapêutico , Pérnio/tratamento farmacológico , Pérnio/genética , Exodesoxirribonucleases/genética , Inibidores de Janus Quinases/uso terapêutico , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Lúpus Eritematoso Cutâneo/genética , Fosfoproteínas/genética , Sulfonamidas/uso terapêutico , Adulto , Pérnio/diagnóstico , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Lúpus Eritematoso Cutâneo/diagnóstico , Masculino , Mutação , Linhagem , Prognóstico , Purinas , Pirazóis , Medição de Risco , Estudos de Amostragem , Resultado do TratamentoRESUMO
Single cell gel electrophoresis or comet assay enables the quantification of DNA damage such as single-strand or double-strand breaks on a single cell level. Here, we describe a variant of this method for the detection of ribonucleotides embedded in genomic DNA. Briefly, cells are embedded in agarose on a microscopic slide, lysed under high salt and alkaline conditions and then subjected to in situ treatment with E. coli RNase HII which nicks 5' to a ribonucleotide within the context of a DNA duplex thereby converting genomic ribonucleotides into strand breaks. After unwinding of genomic DNA using a highly alkaline buffer, electrophoresis under mild alkaline conditions is performed resulting in formation of comets due to migration of fragmented DNA toward the anode. Following SYBR Gold staining comets can be visualized by fluorescence microscopy. In this setting, the length and the intensity of comets formed reflect the level of genomic ribonucleotides present in a given cell.
Assuntos
Eletroforese , Genômica , Ribonucleotídeos , Análise de Célula Única , Ensaio Cometa , DNA , Eletroforese/métodos , Genômica/métodos , Microscopia de Fluorescência , Análise de Célula Única/métodosRESUMO
OBJECTIVES: Familial chilblain lupus is a monogenic form of cutaneous lupus erythematosus caused by loss-of-function mutations in the nucleases TREX1 or SAMHD1. In a family without TREX1 or SAMHD1 mutation, we sought to determine the causative gene and the underlying disease pathology. METHODS: Exome sequencing was used for disease gene identification. Structural analysis was performed by homology modelling and docking simulations. Type I interferon (IFN) activation was assessed in cells transfected with STING cDNA using an IFN-ß reporter and Western blotting. IFN signatures in patient blood in response to tofacitinib treatment were measured by RT-PCR of IFN-stimulated genes. RESULTS: In a multigenerational family with five members affected with chilblain lupus, we identified a heterozygous mutation of STING, a signalling molecule in the cytosolic DNA sensing pathway. Structural and functional analyses indicate that mutant STING enhances homodimerisation in the absence of its ligand cGAMP resulting in constitutive type I IFN activation. Treatment of two affected family members with the Janus kinase (JAK) inhibitor tofacitinib led to a marked suppression of the IFN signature. CONCLUSIONS: A heterozygous gain-of-function mutation in STING can cause familial chilblain lupus. These findings expand the genetic spectrum of type I IFN-dependent disorders and suggest that JAK inhibition may be of therapeutic value.
Assuntos
Pérnio/genética , Lúpus Eritematoso Cutâneo/genética , Proteínas de Membrana/genética , Adulto , Western Blotting , Pérnio/tratamento farmacológico , Pérnio/imunologia , Pérnio/patologia , Família , Feminino , Grécia , Humanos , Interferon Tipo I/imunologia , Interferon beta/imunologia , Lúpus Eritematoso Cutâneo/tratamento farmacológico , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Cutâneo/patologia , Masculino , Angioscopia Microscópica , Simulação de Acoplamento Molecular , Mutação , Linhagem , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologiaRESUMO
OBJECTIVE: Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy resembling congenital viral infection that is characterized by basal ganglia calcifications, loss of white matter, cerebrospinal fluid (CSF) lymphocytosis, and elevated interferon-alpha levels in the CSF. Studies have shown that AGS is an autosomal-recessive disease linked to mutations in 5 genes, encoding the 3'-repair DNA exonuclease 1 (TREX1), the 3 subunits of ribonuclease H2 (RNASEH2A-C), and sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1). In this study we further characterized the phenotypic spectrum of this disease. METHODS: Clinical and laboratory data were obtained from 26 patients fulfilling the clinical diagnostic criteria for AGS. Genomic DNA was screened for mutations in all 5 AGS genes by direct sequencing, and sera were analyzed for autoantibodies. RESULTS: In 20 patients with AGS, 20 mutations, 12 of which were novel, were identified in all 5 AGS genes. Clinical and laboratory investigations revealed a high prevalence of features (some not previously described in patients with AGS) that are commonly seen in patients with systemic lupus erythematosus (SLE), such as thrombocytopenia, leukocytopenia, antinuclear antibodies, erythematous lesions, oral ulcers, and arthritis, which were observed in 12 (60%) of 20 patients with AGS. Moreover, the coexistence of AGS and SLE, was for the first time, demonstrated in 2 patients with molecularly proven AGS. CONCLUSION: These findings expand the phenotypic spectrum of lupus erythematosus in AGS and provide further insight into its disease mechanisms by showing that activation of the innate immune system as a result of inherited defects in nucleic acid metabolism could lead to systemic autoimmunity.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Encefalopatias/genética , Exodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfoproteínas/genética , Ribonuclease H/genética , Adolescente , Adulto , Doenças Autoimunes do Sistema Nervoso/epidemiologia , Doenças Autoimunes do Sistema Nervoso/patologia , Encefalopatias/epidemiologia , Encefalopatias/patologia , Criança , Pré-Escolar , Distonia/epidemiologia , Distonia/genética , Feminino , Predisposição Genética para Doença/epidemiologia , Humanos , Lactente , Lúpus Eritematoso Sistêmico/epidemiologia , Lúpus Eritematoso Sistêmico/patologia , Imageamento por Ressonância Magnética , Masculino , Hipotonia Muscular/epidemiologia , Hipotonia Muscular/genética , Mutação de Sentido Incorreto , Fenótipo , Polimorfismo de Nucleotídeo Único , Prevalência , Proteína 1 com Domínio SAM e Domínio HD , Adulto JovemRESUMO
TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE.
Assuntos
Exodesoxirribonucleases/genética , Lúpus Eritematoso Cutâneo/genética , Mutação , Fosfoproteínas/genética , Regiões 3' não Traduzidas/genética , Endossomos/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Mutação da Fase de Leitura , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lúpus Eritematoso Cutâneo/enzimologia , Mutação de Sentido Incorreto , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We recently described a novel autosomal-dominant genodermatosis, termed familial chilblain lupus, and mapped its genetic locus to chromosome 3p21. Familial chilblain lupus manifests in early childhood with ulcerating acral skin lesions and is associated with arthralgias and circulating antinuclear antibodies. In this study, we report the identification of a heterozygous missense mutation (D18N) in TREX1 encoding the 3'-5'repair exonuclease 1 in affected individuals of the family with chilblain lupus. The homodimeric TREX1 is the most abundant intracellular DNase in mammalian cells. We have recently shown that TREX1 plays a role in apoptotic single-stranded DNA damage induced by the killer lymphocyte protease granzyme A. D18N affects a highly conserved amino acid residue critical for catalytic activity. Recombinant mutant TREX1 homodimers are enzymatically inactive, while wild type/mutant heterodimers show residual exonucleolytic activity, suggesting a heterozygous loss of function. Lymphoblastoid cells carrying the D18N mutation are significantly less sensitive to granzyme A-mediated cell death, suggesting a novel role for this caspase-independent form of apoptosis in the pathogenesis of familial chilblain lupus. Our findings also warrant further investigation of TREX1 in common forms of lupus erythematosus.
Assuntos
Apoptose , Exodesoxirribonucleases/metabolismo , Granzimas/metabolismo , Lúpus Eritematoso Cutâneo/metabolismo , Mutação de Sentido Incorreto , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Análise Mutacional de DNA , Dimerização , Exodesoxirribonucleases/genética , Predisposição Genética para Doença , Heterozigoto , Humanos , Lúpus Eritematoso Cutâneo/enzimologia , Lúpus Eritematoso Cutâneo/genética , Lúpus Eritematoso Cutâneo/patologia , Glicoproteínas de Membrana/metabolismo , Linhagem , Perforina , Fosfoproteínas/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Fatores de RiscoRESUMO
Maturity-onset diabetes of the young is a genetically heterogeneous autosomal dominant form of diabetes mellitus, characterized by an early age at onset and a primary defect in beta-cell function. Forty families with a clinical presentation suggestive of MODY were screened for the most common MODY subtypes caused by mutations in the genes encoding glucokinase (GCK, MODY2) and hepatocyte nuclear 1-alpha (HNF1A/TCF1, MODY3). Overall, 14 mutations were found (35%) giving a relative frequency of 22.5% and 12.5% for MODY2 and MODY3, respectively. Five of the nine GCK mutations identified were novel and included two deletions, two nonsense, and one splice site mutation. The GCK splice donor mutation was shown to result in an aberrant transcript owing to the recruitment of a cryptic splice site. The translated protein is predicted to contain an in frame insertion of nine amino acids. Among the five HNF1A mutations identified, three were novel comprising one missense mutation, one deletion, and one insertion. In addition, several novel polymorphisms within GCK were identified and their allele frequencies estimated. Knowledge of the genetic cause of MODY has significant impact on therapeutic decision making and may help to identify family members at risk for diabetes.