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J Allergy Clin Immunol ; 143(1): 173-181.e10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30248356


BACKGROUND: Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. OBJECTIVE: We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. METHODS: Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. RESULTS: In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. CONCLUSIONS: Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.

Proteínas Adaptadoras de Sinalização CARD , Dermatite Atópica , Guanilato Ciclase , Queratinócitos , Mutação com Perda de Função , Proteínas de Membrana , Mutação de Sentido Incorreto , Transdução de Sinais/genética , Adolescente , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Feminino , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Índice de Gravidade de Doença , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
J Allergy Clin Immunol ; 143(4): 1482-1495, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30170123


BACKGROUND: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. OBJECTIVES: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

Nat Genet ; 49(8): 1192-1201, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28628108


Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Proteínas Adaptadoras de Sinalização CARD/genética , Dermatite Atópica/genética , Mutação em Linhagem Germinativa , Guanilato Ciclase/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Dermatite Atópica/imunologia , Feminino , Genes Dominantes , Glutamina/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Antígenos de Histocompatibilidade Menor/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Linhagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo
J Allergy Clin Immunol ; 132(6): 1388-96, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24184145


BACKGROUND: Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. OBJECTIVE: Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. METHODS: We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. RESULTS: Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. CONCLUSION: This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.

Degranulação Celular/genética , Hipersensibilidade Alimentar/epidemiologia , Síndrome de Job/epidemiologia , Mastócitos/imunologia , Fator de Transcrição STAT3/fisiologia , Adolescente , Adulto , Idoso , Animais , Células Cultivadas , Criança , Pré-Escolar , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Feminino , Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoglobulina E/metabolismo , Incidência , Lactente , Síndrome de Job/genética , Síndrome de Job/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transgenes/genética , Adulto Jovem