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1.
Iran J Allergy Asthma Immunol ; 18(2): 131-142, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066249

RESUMO

The Chronic granulomatous disease (CGD) is a primary immunodeficiency that characterized by mutations in phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in deficient antimicrobial activity of phagocytic cells and recurrent childhood infections. Hematopoietic stem cell transplantation (HSCT) is a curative option for patients with human leukocyte antigen (HLA) matched donor, when conventional cares and therapies fail. However, in many cases when the patients have not an HLA-matched donor, they need to a method to recapitulate the function of the affected gene within the patient's own cells. Gene therapy is a promising approach for CGD. While, the success of retroviral or lentiviral vectors in gene therapy for CGD has been hampered by random integration and insertional activation of proto-oncogenes. These serious adverse events led to improvement and generations of viral vectors with increased safety characteristics. Gene therapy continues to progress and the advent of new technologies, such as engineered endonucleases that have shown a great promise for the treatment of genetic disease. This review focuses on the application of gene therapy for the CGD, the limitations encountered in current clinical trials, advantages and disadvantages of endonucleases in gene correction and modeling with CRISPR/Cas9 approach.


Assuntos
Terapia Genética , Doença Granulomatosa Crônica/terapia , Infecção/terapia , Mutação/genética , NADPH Oxidases/genética , Animais , Sistemas CRISPR-Cas , Ensaios Clínicos como Assunto , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Infecção/genética , Fagocitose/genética
2.
Scand J Immunol ; 90(1): e12767, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30963593

RESUMO

One of the components of NADPH oxidase is p47-phox, encoded by NCF1 gene. This study aims to find new genetic changes and clinical features in 38 Iranian patients with autosomal recessive chronic granulomatous disease (AR-CGD) caused by NCF1 gene defect. Patients who had abnormal NBT and DHR-1,2,3 assay with loss of p47-phox in Western blotting were included in this study. After recording demographic and clinical data, PCR amplification was performed followed by direct sequencing for all exons and exon-intron boundaries. The most common form of CGD in Iran was AR-CGD due to consanguinity marriages. Among patients with AR-CGD, NCF1 deficiency was found to be more common than other forms. Cutaneous involvements (53%), pulmonary infections (50%) and lymphadenopathy (29%) were more prevalent than other clinical manifestations of CGD. Mutation analysis of NCF1 gene identified five different mutations. Homozygous delta GT deletion (c.75_76delGT) was the most frequent mutation and was detected in more than 63% of families. Six families had a nonsense mutation in exon 7 (c.579G > A). Two novel mutations were found in exon 4 in two families, including a missense mutation (c.328C > T) and a nine-nucleotide deletion (c.331_339delTGTCCCCAC). Genetic detection of these mutations may result in early diagnosis and prevention of possible complications of the disease. This could be useful for timely decision-making for haematopoietic stem cell transplantation and for carrier detection as well as prenatal diagnosis of next children in the affected families. Our findings might help to predict outcomes, raise awareness and help effective treatment in these patients.


Assuntos
Doença Granulomatosa Crônica/genética , Linfonodos/patologia , Mutação/genética , NADPH Oxidases/genética , Infecções Respiratórias/genética , Pele/patologia , Adolescente , Adulto , Criança , Análise Mutacional de DNA , Diagnóstico Precoce , Feminino , Humanos , Irã (Geográfico) , Masculino , Reação em Cadeia da Polimerase , Adulto Jovem
3.
Int Arch Allergy Immunol ; 179(1): 62-73, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30904913

RESUMO

Recurrent severe bacterial and fungal infections are characteristic features of the rare genetic immunodeficiency disorder chronic granulomatous disease (CGD). The disease usually manifests within the first years of life with an incidence of 1 in approximately 200,000 live births. The incidence is higher in Iran and Morocco where it reaches 1.5 per 100,000 live births. Mutations have been described in the 5 subunits of NADPH oxidase, mostly in gp91phox and p47phox, with fewer mutations reported in p67phox, p22phox, and p40phox. These mutations cause loss of superoxide production in phagocytic cells. CYBB, the gene encoding the large gp91phox subunit of the transmembrane component cytochrome b558 of the NADPH oxidase complex, is localized on the X-chromosome. Genetic defects in CYBB are responsible for the disease in the majority of male CGD patients. CGD is associated with the development of granulomatous reactions in the skin, lungs, bones, and lymph nodes, and chronic infections may be seen in the liver, gastrointestinal tract, brain, and eyes. There is usually a history of repeated infections, including inflammation of the lymph glands, skin infections, and pneumonia. There may also be a persistent runny nose, inflammation of the skin, and inflammation of the mucous membranes of the mouth. Gastrointestinal problems can also occur, including diarrhea, abdominal pain, and perianal abscesses. Infection of the bones, brain abscesses, obstruction of the genitourinary tract and/or gastrointestinal tract due to the formation of granulomatous tissue, and delayed growth are also symptomatic of CGD. The prevention of infectious complications in patients with CGD involves targeted prophylaxis against opportunistic microorganisms such as Staphylococcus aureus, Klebsiella spp., Salmonella spp. and Aspergillus spp. In this review, we provide an update on organ involvement and the association with specific isolated microorganisms in CGD patients.


Assuntos
Infecções Bacterianas/etiologia , Doença Granulomatosa Crônica/complicações , Micoses/etiologia , Autoimunidade , Criança , Pré-Escolar , Feminino , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Humanos , Lactente , Abscesso Hepático/etiologia , Pneumopatias/etiologia , Masculino , NADPH Oxidases/genética , Dermatopatias/etiologia
4.
Pediatr Allergy Immunol ; 30(3): 378-386, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30716179

RESUMO

BACKGROUND: Chronic granulomatous disease (CGD) is a rare disease in China, and very little large-scale studies have been conducted to date. We aimed to investigate the clinical and genetic features of CGD in Chinese pediatric patients. METHODS: Pediatric patients with CGD from Beijing Children's Hospital, Capital Medical University, China, were enrolled from January 2006 to December 2016. RESULTS: A total of 159 pediatric patients with CGD were enrolled. The median age of clinical onset was 1.4 months, and 73% (116/159) had clinical onset symptoms before the 1 year of age. The most common site of invasion was the lungs. The lymph nodes, liver, and skin were more frequently invaded in X-linked (XL) CGD patients than in autosomal recessive (AR) CGD patients (P < 0.05). Approximately 64% (92/144) of the pediatric patients suffered from abnormal response to BCG vaccination. The most frequent pathogens were Aspergillus and Mycobacterium tuberculosis. Gene analysis indicated that 132 cases (89%, 132/147) harbored CYBB pathogenic variants, 7 (5%, 7/147) carried CYBA pathogenic variants, 4 (3%, 4/147) had NCF1 pathogenic variants, and 4 (3%, 4/147) had NCF2 pathogenic variants. The overall mortality rate in this study was 43%, particularly the patients were males, with CYBB mutant and did not receive HSCT treatment. CONCLUSIONS: Chronic granulomatous disease is a rare disease affecting Chinese children; however, it is often diagnosed at a later age, and thus, the mortality rate is relatively high. The prevalence and the severity of disease in XL-CGD are higher than AR-CGD.


Assuntos
Doença Granulomatosa Crônica/diagnóstico , NADPH Oxidases/genética , Adolescente , Anti-Infecciosos/uso terapêutico , Grupo com Ancestrais do Continente Asiático/genética , Criança , Pré-Escolar , China , Feminino , Testes Genéticos/métodos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/mortalidade , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Estudos Retrospectivos
5.
J Pediatric Infect Dis Soc ; 7(suppl_1): S2-S5, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746675

RESUMO

Chronic Granulomatous Disease is one of the classic primary immunodeficiencies of childhood. While the incidence and severity of bacterial and fungal infections have been greatly reduced in this patient population, much remains to be learned about the pathophysiology of the disease, particularly for autoinflammatory manifestations. In this review, we examine the epidemiology, pathophysiology, and genetic basis for CGD.


Assuntos
Doença Granulomatosa Crônica , Mutação , Criança , Feminino , Doença Granulomatosa Crônica/epidemiologia , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/fisiopatologia , Humanos , Masculino , NADPH Oxidases/fisiologia
6.
J Pediatric Infect Dis Soc ; 7(suppl_1): S40-S44, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29746676

RESUMO

Chronic granulomatous disease is a rare and potentially fatal disorder of neutrophil function. Beyond current medical management and hematopoietic stem cell transplantation, new methods of gene therapy that use lentiviral vectors or gene editing might extend curative therapies to patients who lack a suitable transplantation donor while eliminating the risk of graft-versus-host disease. Furthermore, new therapies focused on altering the biology of phagolysosomes might offer novel targeted treatments for inflammatory complications in patients with chronic granulomatous disease.


Assuntos
Terapia Genética , Doença Granulomatosa Crônica/terapia , Vetores Genéticos , Doença Granulomatosa Crônica/genética , Humanos , Lentivirus , Mutação com Perda de Função
7.
Immunol Invest ; 47(3): 221-228, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29388853

RESUMO

BACKGROUND: Primary immunodeficiency diseases are a group of genetic disorders that lead to increased propensity to a variety of infections, sometimes with fatal outcomes. METHOD: In this study, whole-exome sequencing (WES) was used to identify mutations in two patients suspected of having primary immunodeficiency. Sanger sequencing was used to confirm the results in the patients and their family. RESULT: One patient was diagnosed as X-linked severe combined immunodeficiency (X-SCID) and another patient as X-linked chronic granulomatous disease (X-CGD) by WES. Sequencing analysis of IL2RG gene revealed a novel mutation (c.794T>A, p.I265N) and CYBB gene revealed a missense mutation (c.935T>A, p.M312K). DISCUSSION AND CONCLUSION: This study identifies one novel mutation in the IL2RG gene and another, previously described mutation in the CYBB genes. It is the first report establishing a diagnosis of X-SCID and X-CGD using WES in Chinese patients.


Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Mutação , NADPH Oxidase 2/genética , Alelos , Substituição de Aminoácidos , Grupo com Ancestrais do Continente Asiático/genética , Biomarcadores , China , Análise Mutacional de DNA , Genótipo , Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/genética , Humanos , Imunofenotipagem , Lactente , Subunidade gama Comum de Receptores de Interleucina/química , Masculino , Modelos Moleculares , NADPH Oxidase 2/química , Linhagem , Fenótipo , Relação Estrutura-Atividade , Sequenciamento Completo do Genoma , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/diagnóstico , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
9.
J Clin Pathol ; 71(5): 425-435, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28970295

RESUMO

BACKGROUND: Primary immunodeficiency disorders (PID) include a wide spectrum of inherited disorders characterised by functional abnormalities of one or more components of the immune system. Recent updates from the genomic data have contributed significantly to its better understanding with identification of new entities. Diagnosis is always challenging due to their variable clinical presentation. With the evolution of molecular diagnosis, many of these children are being diagnosed early and offered appropriate therapy. However, in developing countries, early diagnosis is still not being made: as a result these patients succumb to their disease. Autopsy data on PID is notably lacking in the literature with histopathological evaluation of PID being limited to rare case reports. OBJECTIVE: To analyse the clinical, immunologic (including mutational) and morphologic features at autopsy in 10 proven and suspected cases of primary immunodeficiency disorders diagnosed at our Institute over the past decade. METHODS: Study includes a detailed clinico-pathological analysis of 10 proven and suspected cases of primary immunodeficiency disorders. RESULTS: A varied spectrum of infectious and non-infectious complications were identified in these cases of which fungal infections were found to be more frequent compared with viral or bacterial infections. Rare and novel morphological findings, like granulomatous involvement of the heart in a patient with chronic granulomatous disease, systemic amyloidosis in a teenage girl with X-linked agammaglobulinemia, are highlighted which is distinctly lacking in the literature. CONCLUSIONS: The present study is perhaps the first autopsy series on PID. Even in the molecular era, such analysis is still important, as correlation of pathological features with clinical symptoms provides clues for a timely diagnosis and appropriate therapeutic intervention.


Assuntos
Amiloidose/patologia , Doença Granulomatosa Crônica/patologia , Hospedeiro Imunocomprometido , Síndromes de Imunodeficiência/patologia , Infecções Oportunistas/patologia , Amiloidose/genética , Amiloidose/imunologia , Amiloidose/mortalidade , Autopsia , Biópsia , Causas de Morte , Criança , Pré-Escolar , Análise Mutacional de DNA , Países em Desenvolvimento , Diagnóstico Precoce , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/mortalidade , Humanos , Imuno-Histoquímica , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/mortalidade , Índia , Lactente , Recém-Nascido , Masculino , Mutação , Infecções Oportunistas/genética , Infecções Oportunistas/imunologia , Infecções Oportunistas/mortalidade , Fenótipo , Valor Preditivo dos Testes , Prognóstico
10.
J Infect Chemother ; 24(3): 224-227, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29132925

RESUMO

Hepatic abscess in chronic granulomatous disease (CGD) is very refractory and frequently requires multiple surgeries with frequent morbidities. Although surgical interventions are often required, patients are often not able to have surgery for various reasons. We present the case of a 21-year-old man with recurrent hepatic abscess in CGD. We could not provide surgical interventions due to the lack of a fluid cavity and the patient's refusal, and therefore we administered transcatheter arterial antimicrobial and steroid therapy. He did not have any exacerbation for more than 18 months after the final transcatheter treatment. This is the first reported case of successful transcatheter arterial antimicrobial and steroid therapy for refractory hepatic abscess in CGD. Although the patient's burden and medical cost were not inconsequential, this case shows that the transcatheter arterial antimicrobial and steroid therapy may be a treatment option for patients who are not candidates for surgical interventions.


Assuntos
Anti-Infecciosos/uso terapêutico , Cateterismo Periférico/métodos , Doença Granulomatosa Crônica/tratamento farmacológico , Abscesso Hepático/tratamento farmacológico , Esteroides/uso terapêutico , Doença Granulomatosa Crônica/genética , Humanos , Fígado/patologia , Abscesso Hepático/diagnóstico por imagem , Masculino , NADPH Oxidase 2/genética , Recidiva , Adulto Jovem
11.
Pediatr Hematol Oncol ; 35(5-6): 341-349, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30633606

RESUMO

Chronic granulomatous disease (CGD) is a hereditary immunodeficiency syndrome caused by a defect in the NADPH oxidase complex, which is essential for bactericidal function of phagocytes. Approximately 70% of patients with CGD have a mutation in the CYBB gene on the X chromosome, resulting in defective expression of gp91phox, one of the membrane-bound subunits of NADPH oxidase. Although most patients with X-linked CGD are males, owing to transmission of this disease as an X-linked recessive trait, there are female patients with X-linked CGD. Here, we report the case of a teenage girl with X-linked CGD associated with a heterozygous mutation in exon 5 of the CYBB gene (c.389G > C; R130P), which causes skipping of exon 5, resulting in a premature stop codon in exon 6 of CYBB. Accurate pro-mRNA splicing for mature mRNA formation is regulated by several splicing mechanisms that are essential for appropriate recognition of exonic sequences. The c.389G > C mutation disrupts exonic-splicing regulator sequences, thereby resulting in the aberrant skipping of exon 5 in the CYBB transcript of the patient. The patient showed an extremely skewed (≥96%) X inactivation pattern of the HUMARA locus; this inactivation is thought to be responsible for the development of CGD not only in neutrophils but also in monocytic, T-cell, and B-cell lineages and in CD34-positive immature hematopoietic cells. Our case and other reports indicate that the onset of X-linked CGD in female patients tends to occur later in life, and that the symptoms tend to be milder as compared to male patients.


Assuntos
Cromossomos Humanos X/genética , Éxons , Doença Granulomatosa Crônica , NADPH Oxidase 2 , Mutação Puntual , RNA Mensageiro , Inativação do Cromossomo X , Criança , Feminino , Loci Gênicos , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/patologia , Humanos , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo
12.
BMC Med Genet ; 18(1): 127, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29132304

RESUMO

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by the defect of NADPH oxidase. Mutations in CYBB or CYBA gene may result in membrane subunits, gp91phox or p22phox, expression failure respectively and NADPH oxidase deficiency. Previous study showed that three variants, c.214 T > C (rs4673), c.521 T > C (rs1049254) and c.*24G > A (rs1049255), in CYBA gene form a haplotype, which are associated with decreased reactive oxygen species generation. The study aims to confirm the three above mentioned variants are benign and report a novel mutation in CYBB gene. METHODS: A patient with CGD and his family members were enrolled in the study. NADPH oxidase activity and gp91phox protein expression of neutrophils were analyzed by flow cytometry. Direct sequencing was used to detect CYBB and CYBA gene mutations. RESULTS: The patient was diagnosed with CGD according to clinical and immune phenotype. The case has a novel homozygous mutation in CYBB gene and the above mentioned three variants in CYBA gene. The mutation in CYBB gene was confirmed to be pathogenic, and the three variants in CYBA gene to be benign. CONCLUSIONS: The study not only reported a novel mutation in CYBB, which results in CGD, but also confirmed the above mentioned three variants in CYBA are benign.


Assuntos
Doença Granulomatosa Crônica/diagnóstico , Doença Granulomatosa Crônica/genética , NADPH Oxidases/genética , Antibacterianos/uso terapêutico , Sequência de Bases , Doença Granulomatosa Crônica/tratamento farmacológico , Haplótipos , Homozigoto , Humanos , Lactente , Masculino , Mutação , NADPH Oxidase 2/genética , Neutrófilos/metabolismo , Linhagem , Fenótipo
13.
Blood Cells Mol Dis ; 66: 50-57, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886419

RESUMO

In a male patient suffering from X-linked chronic granulomatous disease (CGD) we found a c.389G>T mutation in exon 5 of the CYBB gene. We have analyzed why 95% of the transcripts of this gene lacked exon 5, leading to a frameshift and premature termination codon. The mutation was located in a region comprising three putative exonic splicing enhancer binding sites, for SRSF1, SRFS2 and SRFS6, according to the ESEfinder Tool (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi). With the Analyser Splice Tool we calculated the probability of skipping of exon 5 in CYBB mRNA, and by means of Sroogle the number of putative binding motifs for splicing enhancer and splicing silencer proteins (http://astlab.tau.ac.il/index.php). These analyses clarify why this exon was skipped in the majority of the mRNA. The normally spliced transcript contains an amino acid change p.Arg130Leu. This poorly expressed transcript gives rise to a protein with low expression but presumably normal activity, leading to a respiratory burst activity in the patient's neutrophils of about 15% of normal.


Assuntos
Doença Granulomatosa Crônica/genética , Mutação , NADPH Oxidase 2/genética , Processamento de RNA/genética , Códon sem Sentido , Éxons , Humanos , Masculino , Neutrófilos/metabolismo , Fosfoproteínas/genética , Explosão Respiratória , Fatores de Processamento de Serina-Arginina/genética
14.
Int J Immunogenet ; 44(6): 314-321, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28941186

RESUMO

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme. The enzyme is at least composed of membrane-bound subunits gp91-phox and p22-phox (also named cytochrome b558 ), and cytosolic ones p40-phox, p47-phox and p67-phox. A defect in the enzyme activity leads to impaired intracellular killing of phagocytic cells. The CYBA gene encoding p22-phox is located on chromosome 16q24. In this study, new genetic changes of CYBA gene in 22 Iranian patients with autosomal recessive-CGD (AR-CGD) were identified. Twenty-two patients with CGD were referred to Immunology, Asthma and Allergy Research Institute (IAARI) and enrolled in this study based on defect in NADPH oxidase activity, demographic data and clinical histories. All patients had p22-phox deficiency based on Western blotting. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs), and PCR followed by direct sequencing was performed to find p22-phox mutations. Mutation analysis of CYBA revealed 12 different mutations, including three novel mutations: one was deletion of exon 1, and two were point mutations in exon 3 (c.136G>A (p.Gly46Ser)), and exon 6 (c.388C>T (p.Gln130X)). Three new mutations of CYBA gene in four of 22 Iranian patients with AR-CGD were found. These three novel mutations can partly complete the database of Human Gene Mutation Database (HGMD) and other related ones. It can also be helpful for further prenatal diagnosis in the affected families. Given that currently bone marrow transplantation is considered to be the curative treatment for patients with CGD, finding mutations will also be useful for timely decision-making in bone marrow transplantation.


Assuntos
Doença Granulomatosa Crônica/genética , Mutação/genética , NADPH Oxidases/genética , Adolescente , Sequência de Bases , Western Blotting , Criança , Pré-Escolar , DNA/genética , Demografia , Éxons/genética , Feminino , Humanos , Lactente , Irã (Geográfico) , Masculino
16.
Pediatr Infect Dis J ; 36(12): 1165-1166, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28650935

RESUMO

Granulibacter bethesdensis is a Gram-negative bacillus described as a pathogen exclusively in patients with chronic granulomatous disease, a phagocytic disorder that impairs the ability to clear catalase-producing organisms. Granulibacter usually causes chronic and recurrent lymphadenopathies. We report the fatal case of a 4-year-old boy with chronic granulomatous disease, who presented with sepsis after a few days of abdominal pain and diarrhea.


Assuntos
Acetobacteraceae , Infecções por Bactérias Gram-Negativas , Doença Granulomatosa Crônica , Sepse , Pré-Escolar , Coagulação Intravascular Disseminada , Evolução Fatal , Doença Granulomatosa Crônica/complicações , Doença Granulomatosa Crônica/genética , Humanos , Masculino
17.
Arthritis Rheumatol ; 69(8): 1647-1660, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28471497

RESUMO

OBJECTIVE: We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE). METHODS: We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus. Clinical disease, serology, and immunopathology were evaluated. RESULTS: NCF-2-null mice on the B6 background were susceptible to Aspergillus fumigatus pneumonia characteristic of chronic granulomatous disease, but did not develop systemic lupus disease. In contrast, NCF-2-null and even NCF-2-haploinsufficient mice on the NZM 2328 background developed accelerated full-blown lupus with significantly accelerated lupus kidney disease. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments, increased expression of type I interferon-responsive genes, and generation of neutrophil extracellular traps, which were observed even in the absence of NADPH oxidase activity. CONCLUSION: Just as patients with chronic granulomatous disease who lack NADPH oxidase rarely develop SLE, NCF-2-null mice on a nonautoimmune background were susceptible to a chronic granulomatous disease-like opportunistic infection but did not develop lupus. In contrast, on a lupus-prone background, even haploinsufficiency of NCF-2 accelerated the development of full-blown lupus disease. This establishes an interaction between reduced oxidase activity and other lupus-predisposing genes, paralleling human SLE-associated variants predicted to have only reduced NADPH oxidase activity.


Assuntos
Haploinsuficiência/genética , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/genética , NADPH Oxidases/genética , Animais , Peptídeos Catiônicos Antimicrobianos , Aspergillus fumigatus , Linfócitos B/imunologia , Catelicidinas/imunologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Armadilhas Extracelulares/imunologia , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Doença Granulomatosa Crônica/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Rim/imunologia , Rim/patologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Aspergilose Pulmonar/genética , Reação em Cadeia da Polimerase em Tempo Real , Linfócitos T/imunologia
18.
Sci Rep ; 7: 44187, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28287132

RESUMO

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.


Assuntos
Sistemas CRISPR-Cas , Terapia Genética/métodos , Vetores Genéticos , Doença Granulomatosa Crônica , Sequência de Bases , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Células HL-60 , Humanos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Deleção de Sequência
19.
Hum Gene Ther ; 28(7): 565-575, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28264583

RESUMO

Chronic granulomatous disease (CGD) is characterized by defects in the production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for the treatment of CGD in human hematopoietic cells requires preclinical transplant models. The use of the lymphocyte-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized for targeted knockout of mouse Cybb on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting Cybb exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at least one Cybb allele based on sequence analysis of tail snip DNA. A female mouse heterozygous for a 235-bp deletion in Cybb exon 1 was bred to an NSG male to establish the X-CGD NSG mouse strain, NSG.Cybb[KO]. Resulting male offspring with the 235 bp deletion were found to be defective for production of ROS by neutrophils and other phagocytes, and demonstrated increased susceptibility to spontaneous bacterial and fungal infections with granulomatous inflammation. The establishment of the phagocyte-defective NSG.Cybb[KO] mouse model enables the in vivo assessment of gene and cell therapy strategies for treating CGD in human hematopoietic cell transplants without obfuscation by functional mouse phagocytes, and may also be useful for modeling other phagocyte disorders in humanized NSG mouse xenografts.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Terapia Genética , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , NADPH Oxidase 2/genética , Transplante de Células-Tronco , Animais , Sequência de Bases , Linhagem da Célula , Modelos Animais de Doenças , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução Genética
20.
J Immunol Res ; 2017: 8745254, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28251166

RESUMO

Chronic Granulomatous Disease (CGD) is a rare inherited primary immunodeficiency, which is characterized by recurrent infections due to defective phagocyte NADPH oxidase enzyme. Nowadays, little is known about Chinese CGD patients. Here we report 48 CGD patients in our single center study, which is the largest cohort study from Mainland China. The ratio of male to female was 11 : 1. The mean onset age was 0.29 years old, and 52% patients had an onset within the 1st month of life. The mean diagnosis age was 2.24 years old. 11 patients (23%) had died with an average age of 2.91 years old. 13 patients (28%) had positive family histories. The most prevalent infectious sites were the lungs (77%), followed by gastrointestinal tract (54%), lymph nodes (50%), and skin (46%). In addition, septicopyemia, thrush, and hepatosplenomegaly were also commonly observed, accounting for 23%, 23%, and 40% of the cases. Lesions due to BCG vaccination occurred in more than half of the patients. X-linked CGD due to CYBB gene mutations accounted for 75% of the cases, and 11 of them were novel mutations. Autosomal recessive inheritance accounted for 6% patients, including 1 patient with CYBA, 1 with NCF1, and 1 with NCF2 gene mutations.


Assuntos
Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/fisiopatologia , Mutação , Adolescente , Adulto , Idoso , Vacina BCG/efeitos adversos , Criança , Pré-Escolar , China/epidemiologia , Estudos de Coortes , Feminino , Trato Gastrointestinal/microbiologia , Testes Genéticos , Doença Granulomatosa Crônica/congênito , Doença Granulomatosa Crônica/epidemiologia , Humanos , Síndromes de Imunodeficiência/genética , Lactente , Pulmão/microbiologia , Linfonodos/microbiologia , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , NADPH Oxidase 2 , NADPH Oxidases/genética , Pele/microbiologia , Adulto Jovem
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