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1.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

2.
J Clin Immunol ; 38(7): 794-803, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30264381

RESUMO

PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

3.
Biomedica ; 38(0): 30-42, 2018 05 01.
Artigo em Espanhol | MEDLINE | ID: mdl-29809327

RESUMO

INTRODUCTION: Type III glycogen storage disease (GSD III) is an autosomal recessive disorder in which a mutation in the AGL gene causes deficiency of the glycogen debranching enzyme. The disease is characterized by fasting hypoglycemia, hepatomegaly and progressive myopathy. Molecular analyses of AGL have indicated heterogeneity depending on ethnic groups. The full spectrum of AGL mutations in Colombia remains unclear. OBJECTIVE: To describe the clinical and molecular characteristics of ten Colombian patients diagnosed with GSD III. MATERIALS AND METHODS: We recruited ten Colombian children with a clinical and biochemical diagnosis of GSD III to undergo genetic testing. The full coding exons and the relevant exon-intron boundaries of the AGL underwent Sanger sequencing to identify mutation. RESULTS: All patients had the classic phenotype of the GSD III. Genetic analysis revealed a mutation p.Arg910X in two patients. One patient had the mutation p.Glu1072AspfsX36, and one case showed a compound heterozygosity with p.Arg910X and p.Glu1072AspfsX36 mutations. We also detected the deletion of AGL gene 3, 4, 5, and 6 exons in three patients. The in silico studies predicted that these defects are pathogenic. No mutations were detected in the amplified regions in three patients. CONCLUSION: We found mutations and deletions that explain the clinical phenotype of GSD III patients. This is the first report with a description of the clinical phenotype and the spectrum of AGL mutations in Colombian patients. This is important to provide appropriate prognosis and genetic counseling to the patient and their relatives.

4.
Biomédica (Bogotá) ; 38(supl.1): 30-42, mayo 2018. tab, graf
Artigo em Espanhol | LILACS-Express | ID: biblio-950952

RESUMO

Resumen Introducción. La enfermedad por almacenamiento de glucógeno de tipo III es una alteración autosómica recesiva, en la cual las mutaciones del gen AGL causan una deficiencia en la enzima desramificadora de glucógeno. Se caracteriza por hipoglucemia, hepatomegalia y miopatías progresivas. El análisis molecular del gen AGL ha evidenciado mutaciones que difieren según la población estudiada. En la actualidad, no existen reportes que describan mutaciones en el AGL de pacientes colombianos con esta condición. Objetivo. Describir las características clínicas y moleculares de diez pacientes colombianos con enfermedad por almacenamiento del glucógeno de tipo III. Materiales y métodos. Se analizaron diez pacientes pediátricos colombianos con la enfermedad y se hizo su estudio genético mediante la secuenciación de las regiones que codifican y las intrónicas circundantes del gen AGL con el método de Sanger. Resultados. Todos los pacientes tenían el fenotipo clásico de la enfermedad. El estudio genético reveló la mutación p.Arg910X en dos pacientes. Uno presentó la mutación p.Glu1072AspfsX36 y otro resultó heterocigoto compuesto con las mutaciones p.Arg910X y p.Glu1072AspfsX36. Asimismo, en tres pacientes se detectó la deleción de los exones 4, 5 y 6 del gen AGL. Los estudios de simulación computacional predijeron que estos defectos eran patogénicos. En tres pacientes no se encontraron mutaciones en las regiones amplificadas. Conclusión. Se encontraron mutaciones y deleciones que explican el fenotipo clínico de los pacientes. Este es el primer reporte en el que se describe el fenotipo clínico y el espectro de mutaciones en el gen AGL de pacientes colombianos, lo cual es importante para ofrecer un apropiado pronóstico, y asesoría genética al paciente y a su familia.


Abstract Introduction: Type III glycogen storage disease (GSD III) is an autosomal recessive disorder in which a mutation in the AGL gene causes deficiency of the glycogen debranching enzyme. The disease is characterized by fasting hypoglycemia, hepatomegaly and progressive myopathy. Molecular analyses of AGL have indicated heterogeneity depending on ethnic groups. The full spectrum of AGL mutations in Colombia remains unclear. Objective: To describe the clinical and molecular characteristics of ten Colombian patients diagnosed with GSD III. Materials and methods: We recruited ten Colombian children with a clinical and biochemical diagnosis of GSD III to undergo genetic testing. The full coding exons and the relevant exon-intron boundaries of the AGL underwent Sanger sequencing to identify mutation. Results: All patients had the classic phenotype of the GSD III. Genetic analysis revealed a mutation p.Arg910X in two patients. One patient had the mutation p.Glu1072AspfsX36, and one case showed a compound heterozygosity with p.Arg910X and p.Glu1072AspfsX36 mutations. We also detected the deletion of AGL gene 3, 4, 5, and 6 exons in three patients. The in silico studies predicted that these defects are pathogenic. No mutations were detected in the amplified regions in three patients. Conclusion: We found mutations and deletions that explain the clinical phenotype of GSDIII patients. This is the first report with a description of the clinical phenotype and the spectrum of AGLmutations in Colombian patients. This is importantto provide appropriate prognosis and genetic counseling to the patient and their relatives.

5.
J Clin Immunol ; 37(7): 732-738, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28865061

RESUMO

PURPOSE: Mendelian susceptibility to mycobacterial disease is a rare clinical condition characterized by a predisposition to infectious diseases caused by poorly virulent mycobacteria. Other infections such as salmonellosis and candidiasis are also reported. The purpose of this article is to describe a young boy affected with various infectious diseases caused by Mycobacterium tuberculosis complex, Salmonella sp, Klebsiella pneumonie, Citrobacter sp., and Candida sp, complicated with severe enteropathy and transient hypogammaglobulinemia. METHODS: We reviewed medical records and performed flow cytometry staining for lymphocyte populations, lymphocyte proliferation in response to PHA, and intracellular IFN-γ production in T cell PHA blasts in the patient and a healthy control. Sanger sequencing was used to confirm the genetic variants in the patient and relatives. RESULTS: Genetic analysis revealed a bi-allelic mutation in IL12RB1 (C291Y) resulting in complete IL-12Rß1 deficiency. Functional analysis demonstrated the lack of intracellular production of IFN-γ in CD3+ T lymphocytes from the patient in response to rhIL-12p70. CONCLUSIONS: To our knowledge, this is the third patient with MSMD due to IL-12Rß1 deficiency complicated with enteropathy and hypogammaglobulinemia and the first case of this disease to be described in Colombia.


Assuntos
Agamaglobulinemia/genética , Candidíase/genética , Enterite/genética , Infecções por Bactérias Gram-Negativas/genética , Receptores de Interleucina-12/deficiência , Receptores de Interleucina-12/genética , Agamaglobulinemia/tratamento farmacológico , Vacina BCG , Candidíase/tratamento farmacológico , Farmacorresistência Bacteriana , Enterite/tratamento farmacológico , Predisposição Genética para Doença , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Lactente , Mutação , Mycobacterium tuberculosis
6.
Mol Biochem Parasitol ; 163(1): 28-38, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18940204

RESUMO

It is increasingly accepted that single-celled organisms, such as Leishmania parasites, are able to undergo a cell death process that resembles apoptosis in metazoans and is induced by a variety of stimuli. However, the molecular mechanisms that participate and regulate this death process are still very poorly described, and very few of the participating molecules have been identified. Because DNA degradation is probably the most frequently characterized event during programmed cell death in Leishmania parasites, we have focused on identifying a candidate nuclease responsible for this effect during the cell death process. The results presented herein demonstrate that Leishmania infantum promastigotes express a nuclease similar to the endonuclease G of higher eukaryotes which, according to its predicted structure, belongs to the beta beta alpha metal superfamily of nucleases. Its cation dependence resembles that of the EndoGs present in other organisms and, similarly to them, it is inhibited by moderate concentrations of K+ or Na+. L. infantum EndoG contains a signal peptide that causes its translocation to the mitochondrion where it is maintained under normal growth conditions. However, under the pressure of a death stimulus such as edelfosine treatment, L. infantum EndoG is released from the single mitochondrion and translocates to the nucleus, where it is thought to participate in the process of DNA degradation that is associated with programmed cell death. Our results also demonstrate that overexpression of the nuclease in edelfosine-treated promastigotes causes a significant increase in the percentage of TUNEL-positive parasites.


Assuntos
Apoptose , Núcleo Celular/enzimologia , Endodesoxirribonucleases/metabolismo , Expressão Gênica , Leishmania infantum/enzimologia , Mitocôndrias/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/química , Núcleo Celular/genética , Desoxirribonucleases/química , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Humanos , Leishmania infantum/química , Leishmania infantum/citologia , Leishmania infantum/genética , Mitocôndrias/química , Mitocôndrias/genética , Modelos Moleculares , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência
7.
Biomedica ; 24(3): 262-72, 2004 Sep.
Artigo em Espanhol | MEDLINE | ID: mdl-15551878

RESUMO

The NADPH oxidase system plays a central role in the antimicrobial activity of phagocytes. This system is initiated by the translocation of cytosolic proteins p67phox, p47phox and p40phox to be in close contact with membrane flavocytochrome b558. This event begins the electron transfer from cytosolic NADPH to molecular oxygen to produce superoxide anions. Herein, a functional analysis is presented of p67phox polymorphisms identified from healthy humans. Mutations were generated in the p67phox cDNA by site-directed mutagenesis and then transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox from stable transgenes. The changes Va1166lle, Pro329Ser and His389Gln correspond to possible polymorphisms identified in healthy individuals revealed a functional activity similar to COSphox cells transiently transfected with WT p67phox; therefore, these modifications are not associated with genetic deficiencies in NADPH oxidase. In conclusion, the COSphox system represents an easily transfectable model for analysis of NADPH oxidase function in intact cells. The analysis of mutant derivatives of p67phox provides insight into molecular mechanisms by which this subunit regulates the NADPH oxidase.


Assuntos
NADPH Oxidases/genética , Fosfoproteínas/genética , Animais , Células COS , Cercopithecus aethiops , Humanos , Fagócitos/fisiologia , Polimorfismo Genético , Superóxidos/metabolismo
8.
Biomédica (Bogotá) ; 24(3): 262-272, sept. 2004. ilus
Artigo em Espanhol | LILACS | ID: lil-422502

RESUMO

El sistema NADPH oxidasa de las células fagocíticas cumple una función importante durante la respuesta antimicrobiana del organismo. La activación de este sistema está precedida por la translocación de las proteínas citosólicas p67 phox , p47 phox y p40 phox hacia la membrana para ponerse en contacto con el flavocitocromo b 558 , lo que induce la generación del anión superóxido, un precursor de agentes microbicidas oxidantes. El presente trabajo presenta un análisis funcional del sistema NADPH oxidasa basado en los hallazgos de polimorfismos encontrados en el gen de p67 phox de individuos sanos. Para esto se generaron mutaciones en el cADN que codifica la p67 phox y se expresaron en el sistema de células COS phox . Los datos obtenidos en el presente trabajo indican que los cambios Val 166 .Ile, Pro 329 .Ser y His 389 .Gln no generan alteraciones en el funcionamiento de la p67 phox cuando su función se analizó en el sistema transgénico basado en células COS-7. Por lo tanto, estos polimorfismos no generan ningún riesgo genético de producir deficiencias en la activación del sistema NADPH oxidasa. Además, se demuestra que el modelo de células COS phox representa un nuevo sistema celular, fácilmente transfectable que permite estudiar la función del sistema NADPH oxidasa de las células fagocíticas y sus particularidades genéticas. Finalmente, los hallazgos con estos polimorfismos nos permiten avanzar en el conocimiento sobre los mecanismos moleculares involucrados en la activación del sistema NADPH oxidasa células fagocíticas


Assuntos
Células COS , NADPH Oxidases , Polimorfismo Genético , Proteínas/genética , Transfecção , Transformação Genética
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