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J Allergy Clin Immunol ; 140(1): 232-241, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28011069


BACKGROUND: Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants. OBJECTIVE: We estimated variations in the CCD/DBD of STAT1. METHODS: We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. RESULTS: Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases. CONCLUSION: The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.

Alanina/genética , Infecções por Mycobacterium/genética , Fator de Transcrição STAT1/genética , Bioensaio , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Mutagênese , Mutação , Domínios Proteicos
J Clin Immunol ; 35(8): 769-76, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26563159


Mutations in the FAS gene are the most common cause of Autoimmune Lymphoproliferative Syndrome (ALPS), and the majority of them affect the intracellular domain of FAS protein, particularly the region termed death domain. However, approximately one third of these mutations affect the extracellular region of FAS and most are stop codons, with very few missense changes having been described to date. We previously described 7 patients with a FAS missense extracellular mutation, C107Y, two in homozygozity and 5 in heterozygosity. We investigated here the mechanistic effects of this mutation and observed that the homozygous patients did not show any FAS surface expression, while the heterozygous patients had diminished receptor expression. Aiming to understand why a missense mutation was abolishing receptor expression, we analyzed intracellular FAS protein trafficking using fluorescent fusion proteins of wild type FAS, two missense extracellular mutants (FAS-C107Y and FAS-C104Y) and one missense change localized in the intracellular region, FAS-D260E. The FAS-C107Y and FAS-C104Y mutants failed to reach the cell surface, being retained at the endoplasmic reticulum, unlike the WT or the FAS-D260E which were clearly expressed at the plasma membrane. These results support haploinsufficiency as the underlying mechanism involved in the pathogenesis of ALPS caused by extracellular FAS missense mutations.

Síndrome Linfoproliferativa Autoimune/genética , Mutação de Sentido Incorreto/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/genética , Receptor fas/metabolismo , Alelos , Apoptose/genética , Argentina , Espaço Extracelular , Células HEK293 , Haploinsuficiência , Humanos , Linhagem , Engenharia de Proteínas , Transporte Proteico/genética , Receptor fas/genética
J Clin Immunol ; 32(6): 1197-203, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22752343


UNLABELLED: Mutations in the Fas gene (TNFRSF6) are the most common causes of Autoimmune Lymphoproliferative Syndrome (ALPS-FAS). PURPOSE: In Argentina almost a third of patients with ALPS-FAS present a missense mutation affecting the extracellular cysteine rich domain 2 of Fas, p.Cys107Tyr (C107Y). This change was found in homozygous state in 2 patients from a consanguineous family, and heterozygously, in 3 other patients from 3 unrelated families. In these families, 12 relatives were identified as healthy carriers of the mutation. We sought to test the hypothesis that this mutation actually represents a single haplotype of TNFRSF6. METHODS: DNAs from ALPS-C107Y patients and their families, as well as from 150 Argentinean control subjects were sequenced for the known higher frequency single nucleotide polymorphisms (SNPs) of TNFRSF6. The C107Y-carriers were also genotyped at 5 microsatellites proximal to the Fas gene locus. RESULTS: All C107Y alleles presented a unique intragenic haplotype that could be restricted to this group. Extent of haplotype sharing and variability of microsatellite alleles in C107Y chromosomes support the presence of a single haplotype block including the mutation and encompassing 2.395 Mb. CONCLUSIONS: A founder effect for C107Y has been evidenced in this work and the most common recent ancestor to the patients probably lived 350 years ago. This constitutes the first report of a founder event in ALPS.

Síndrome Linfoproliferativa Autoimune/genética , Efeito Fundador , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Receptor fas/genética , Adolescente , Adulto , Alelos , Argentina , Síndrome Linfoproliferativa Autoimune/patologia , Sequência de Bases , Estudos de Casos e Controles , Criança , Feminino , Haplótipos , Heterozigoto , Homozigoto , Humanos , Masculino , Repetições de Microssatélites , Dados de Sequência Molecular , Linhagem , Estrutura Terciária de Proteína , Análise de Sequência de DNA
J Leukoc Biol ; 92(2): 301-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22595141


This study tested the hypothesis that the IFN-γ R1 287-YVSLI-91 intracellular motif regulates its endocytosis. IFN-γ exerts its biological activities by interacting with a specific cell-surface RC composed of two IFN-γ R1 and two IFN-γ R2 chains. Following IFN-γ binding and along with the initiation of signal transduction, the ligand and IFN-γ R1 are internalized. Two major types of consensus-sorting signals are described in receptors, which are rapidly internalized from the plasma membrane to intracellular compartments: tyrosine-based and dileucine-based internalization motifs. Transfection of HEK 293 cells and IFN-γ R1-deficient fibroblasts with WT and site-directed, mutagenesis-generated mutant IFN-γ R1 expression vectors helped us to identify region IFN-γ R1 287-YVSLI-291 as the critical domain required for IFN-γ-induced IFN-γ R1 internalization and Y287 and LI290-291 as part of a common structure essential for receptor endocytosis and function. This new endocytosis motif, YxxLI, shares characteristics of tyrosine-based and dileucine-based internalization motifs and is highly conserved in IFN-γ Rs across species. The IFN-γ R1 270-LI-271 dileucine motif, previously thought to be involved in this receptor endocytosis, showed to be unnecessary for receptor endocytosis.

Endocitose/imunologia , Leucina/química , Leucina/metabolismo , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Sequência Conservada/imunologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/fisiologia , Receptores de Interferon/genética , Tirosina/química , Tirosina/metabolismo
Medicine (Baltimore) ; 89(6): 381-402, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21057261


Interleukin-12 receptor ß1 (IL-12Rß1) deficiency is the most common form of Mendelian susceptibility to mycobacterial disease (MSMD). We undertook an international survey of 141 patients from 102 kindreds in 30 countries. Among 102 probands, the first infection occurred at a mean age of 2.4 years. In 78 patients, this infection was caused by Bacille Calmette-Guérin (BCG; n = 65), environmental mycobacteria (EM; also known as atypical or nontuberculous mycobacteria) (n = 9) or Mycobacterium tuberculosis (n = 4). Twenty-two of the remaining 24 probands initially presented with nontyphoidal, extraintestinal salmonellosis. Twenty of the 29 genetically affected sibs displayed clinical signs (69%); however 8 remained asymptomatic (27%). Nine nongenotyped sibs with symptoms died. Recurrent BCG infection was diagnosed in 15 cases, recurrent EM in 3 cases, recurrent salmonellosis in 22 patients. Ninety of the 132 symptomatic patients had infections with a single microorganism. Multiple infections were diagnosed in 40 cases, with combined mycobacteriosis and salmonellosis in 36 individuals. BCG disease strongly protected against subsequent EM disease (p = 0.00008). Various other infectious diseases occurred, albeit each rarely, yet candidiasis was reported in 33 of the patients (23%). Ninety-nine patients (70%) survived, with a mean age at last follow-up visit of 12.7 years ± 9.8 years (range, 0.5-46.4 yr). IL-12Rß1 deficiency is characterized by childhood-onset mycobacteriosis and salmonellosis, rare recurrences of mycobacterial disease, and more frequent recurrence of salmonellosis. The condition has higher clinical penetrance, broader susceptibility to infections, and less favorable outcome than previously thought.

Subunidade beta 1 de Receptor de Interleucina-12/deficiência , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Citocinas/sangue , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Subunidade beta 1 de Receptor de Interleucina-12/genética , Masculino , Pessoa de Meia-Idade , Infecções por Micobactéria não Tuberculosa/epidemiologia , Infecções por Micobactéria não Tuberculosa/genética , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Análise de Sobrevida
J Clin Virol ; 31(1): 58-65, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15288615


BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplant recipients that is caused by suppression of T-cell function. OBJECTIVE: Evaluation of the diagnostic value of EBV DNA load in non-fractionated whole blood samples (n = 297) from 110 pediatric transplant patients by real-time PCR. RESULTS AND CONCLUSIONS: Patients with PTLD had a median viral load of 1.08 x 10(5) copies/ml blood (n = 24), which was significantly higher compared with patients without PTLD (median: 50 copies/ml blood, n = 273, P < 0.0001). From receiver operating characteristic (ROC) curve analysis we obtained a cut-off value of 6215 copies/ml blood with a sensitivity of 95.8%, specificity of 71.4%, negative predictive value (NPV) of 99.5% and positive predictive value (PPV) of 22.8%. Thus, real-time PCR proved to be more useful in ruling out than in indicating the presence of PTLD. Further analysis showed that patients without PTLD but developing a post-transplant EBV-primary infection had associated high viral loads that were indistinguishable from those of the PTLD group (statistically not significant). Similarly, the presence of clinical symptoms of disease in patients without PTLD was associated with higher viral loads than in patients that were asymptomatic (P < 0.0001), but the difference was much less significant when compared with the PTLD group of patients (P = 0.0391). These patients who had a high viral load may benefit from a close follow-up of the viral burden.

Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/isolamento & purificação , Transtornos Linfoproliferativos/virologia , Transplante de Órgãos , Adolescente , Argentina , Criança , Pré-Escolar , DNA Viral/sangue , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Carga Viral