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1.
Front Immunol ; 10: 2000, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507602

RESUMO

Interleukin-2-inducible T cell kinase (ITK) is critical for T cell signaling and cytotoxicity, and control of Epstein-Barr virus (EBV). We identified a patient with a novel homozygous missense mutation (D540N) in a highly conserved residue in the kinase domain of ITK who presented with EBV-positive lymphomatoid granulomatosis. She was treated with interferon and chemotherapy and her disease went into remission; however, she has persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Molecular modeling predicts that the mutation increases the flexibility of the ITK kinase domain impairing phosphorylation of the protein. Stimulation of her T cells resulted in reduced phosphorylation of ITK, PLCγ, and PKC. The CD8 T cells were moderately impaired for cytotoxicity and degranulation. Importantly, addition of magnesium to her CD8 T cells in vitro restored cytotoxicity and degranulation to levels similar to controls. Supplemental magnesium in patients with mutations in another protein important for T cell signaling, MAGT1, was reported to restore EBV-specific cytotoxicity. Our findings highlight the critical role of ITK for T cell activation and suggest the potential for supplemental magnesium to treat patients with ITK deficiency.

2.
J Mol Diagn ; 20(5): 686-702, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29936259

RESUMO

Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory.

3.
PLoS Genet ; 13(9): e1007021, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28961250

RESUMO

Knowledge of biological relatedness between samples is important for many genetic studies. In large-scale human genetic association studies, the estimated kinship is used to remove cryptic relatedness, control for family structure, and estimate trait heritability. However, estimation of kinship is challenging for sparse sequencing data, such as those from off-target regions in target sequencing studies, where genotypes are largely uncertain or missing. Existing methods often assume accurate genotypes at a large number of markers across the genome. We show that these methods, without accounting for the genotype uncertainty in sparse sequencing data, can yield a strong downward bias in kinship estimation. We develop a computationally efficient method called SEEKIN to estimate kinship for both homogeneous samples and heterogeneous samples with population structure and admixture. Our method models genotype uncertainty and leverages linkage disequilibrium through imputation. We test SEEKIN on a whole exome sequencing dataset (WES) of Singapore Chinese and Malays, which involves substantial population structure and admixture. We show that SEEKIN can accurately estimate kinship coefficient and classify genetic relatedness using off-target sequencing data down sampled to ~0.15X depth. In application to the full WES dataset without down sampling, SEEKIN also outperforms existing methods by properly analyzing shallow off-target data (~0.75X). Using both simulated and real phenotypes, we further illustrate how our method improves estimation of trait heritability for WES studies.


Assuntos
Bases de Dados Genéticas , Genética Populacional/métodos , Genoma Humano , Análise de Sequência de DNA , Grupo com Ancestrais do Continente Asiático/genética , Biologia Computacional , Exoma , Estudos de Associação Genética , Genótipo , Técnicas de Genotipagem , Humanos , Desequilíbrio de Ligação , Modelos Genéticos , Software
4.
J Exp Med ; 214(7): 1949-1972, 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28606988

RESUMO

MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-ß/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.


Assuntos
Helicase IFIH1 Induzida por Interferon/genética , Mutação , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/virologia , Rhinovirus/fisiologia , Antivirais/farmacologia , Sequência de Bases , Células Cultivadas , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica/efeitos dos fármacos , Genes Recessivos/genética , Heterozigoto , Homozigoto , Interações Hospedeiro-Patógeno , Humanos , Helicase IFIH1 Induzida por Interferon/deficiência , Interferons/farmacologia , Masculino , Linhagem
5.
Nat Immunol ; 18(7): 813-823, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28530713

RESUMO

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized.


Assuntos
Doenças Autoimunes/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Síndromes de Imunodeficiência/genética , Corticosteroides/uso terapêutico , Adulto , Doenças Autoimunes/complicações , Colite/complicações , Colite/genética , Colite/patologia , Feminino , Febre/complicações , Febre/tratamento farmacológico , Febre/genética , Haploinsuficiência , Heterozigoto , Humanos , Síndromes de Imunodeficiência/complicações , Linfopenia/complicações , Linfopenia/genética , Masculino , Pessoa de Meia-Idade , Mutação , Pancitopenia/complicações , Pancitopenia/tratamento farmacológico , Pancitopenia/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Recidiva , Infecções Respiratórias/complicações , Infecções Respiratórias/diagnóstico por imagem , Infecções Respiratórias/genética , Esplenomegalia/complicações , Esplenomegalia/genética , Síndrome , Tomografia Computadorizada por Raios X , Adulto Jovem
6.
Nat Genet ; 48(12): 1564-1569, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27749843

RESUMO

Elevated basal serum tryptase levels are present in 4-6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase-encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.


Assuntos
Dor Crônica/genética , Doenças do Tecido Conjuntivo/genética , Variações do Número de Cópias de DNA/genética , Disautonomia Familiar/genética , Gastroenteropatias/genética , Prurido/genética , Dermatopatias/genética , Triptases/sangue , Triptases/genética , Adolescente , Adulto , Idoso , Criança , Dor Crônica/sangue , Dor Crônica/enzimologia , Doenças do Tecido Conjuntivo/sangue , Doenças do Tecido Conjuntivo/enzimologia , Disautonomia Familiar/sangue , Disautonomia Familiar/enzimologia , Feminino , Gastroenteropatias/sangue , Gastroenteropatias/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Prurido/sangue , Prurido/enzimologia , Dermatopatias/sangue , Dermatopatias/enzimologia , Adulto Jovem
7.
Biomed Res Int ; 2016: 8356435, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27088091

RESUMO

Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined.


Assuntos
Anticorpos Monoclonais/genética , Formação de Anticorpos/genética , RNA/genética , Animais , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetinae , Cricetulus , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Análise de Sequência de RNA
8.
Science ; 349(6246): 436-40, 2015 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-26206937

RESUMO

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doenças Autoimunes/tratamento farmacológico , Antígeno CTLA-4/deficiência , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunoconjugados/uso terapêutico , Abatacepte , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Doenças Autoimunes/metabolismo , Antígeno CTLA-4/genética , Criança , Cloroquina/farmacologia , Imunodeficiência de Variável Comum/metabolismo , Endossomos/metabolismo , Feminino , Fatores de Transcrição Forkhead/análise , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/metabolismo , Ativação Linfocitária , Lisossomos/metabolismo , Masculino , Proteólise , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto Jovem
9.
Blood ; 125(4): 591-9, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25359994

RESUMO

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.


Assuntos
Doenças Autoimunes/genética , Doenças Genéticas Inatas/genética , Transtornos Linfoproliferativos/genética , Fator de Transcrição STAT3/genética , Adolescente , Adulto , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Criança , Pré-Escolar , Feminino , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Lactente , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Masculino , Mutação , Fosforilação/genética , Fosforilação/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia
10.
J Allergy Clin Immunol ; 133(5): 1400-9, 1409.e1-5, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24589341

RESUMO

BACKGROUND: Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. OBJECTIVE: We sought to define a genetic syndrome of severe atopy, increased serum IgE levels, immune deficiency, autoimmunity, and motor and neurocognitive impairment. METHODS: Eight patients from 2 families with similar syndromic features were studied. Thorough clinical evaluations, including brain magnetic resonance imaging and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T-cell cytokine production were measured. Whole-exome sequencing was performed to identify disease-causing mutations. Immunoblotting, quantitative RT-PCR, enzymatic assays, nucleotide sugar, and sugar phosphate analyses, along with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of glycans, were used to determine the molecular consequences of the mutations. RESULTS: Marked atopy and autoimmunity were associated with increased T(H)2 and T(H)17 cytokine production by CD4(+) T cells. Bacterial and viral infection susceptibility were noted along with T-cell lymphopenia, particularly of CD8(+) T cells, and reduced memory B-cell numbers. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurologic abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced uridine diphosphate-N-acetyl-D-glucosamine, along with decreased O- and N-linked protein glycosylation in patients' cells. These results define a new congenital disorder of glycosylation. CONCLUSIONS: Autosomal recessive hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability, and hypomyelination.


Assuntos
Doenças Autoimunes/genética , Transtornos Cognitivos/genética , Imunodeficiência de Variável Comum/genética , Doenças Genéticas Inatas/genética , Hipersensibilidade/genética , Mutação , Fosfoglucomutase/genética , Doenças Autoimunes/enzimologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Criança , Pré-Escolar , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/imunologia , Transtornos Cognitivos/patologia , Imunodeficiência de Variável Comum/enzimologia , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Família , Feminino , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/patologia , Humanos , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Masculino , Linhagem , Fosfoglucomutase/imunologia , Fosfoglucomutase/metabolismo , Células Th17/enzimologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/enzimologia , Células Th2/imunologia , Células Th2/patologia , Adulto Jovem
11.
PLoS One ; 6(7): e21503, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21750714

RESUMO

Small interfering RNAs (siRNAs) are routinely used to reduce mRNA levels for a specific gene with the goal of studying its function. Several studies have demonstrated that siRNAs are not always specific and can have many off-target effects. The 3' UTRs of off-target mRNAs are often enriched in sequences that are complementary to the seed-region of the siRNA. We demonstrate that siRNA off-targets can be significantly reduced when cells are treated with a dose of siRNA that is relatively low (e.g. 1 nM), but sufficient to effectively silence the intended target. The reduction in off-targets was demonstrated for both modified and unmodified siRNAs that targeted either STAT3 or hexokinase II. Low concentrations reduced silencing of transcripts with complementarity to the seed region of the siRNA. Similarly, off-targets that were not complementary to the siRNA were reduced at lower doses, including up-regulated genes that are involved in immune response. Importantly, the unintended induction of caspase activity following treatment with a siRNA that targeted hexokinase II was also shown to be a concentration-dependent off-target effect. We conclude that off-targets and their related phenotypic effects can be reduced for certain siRNA that potently silence their intended target at low concentrations.


Assuntos
Hexoquinase/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Bioorg Med Chem Lett ; 18(17): 4872-5, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18691886

RESUMO

Relationships between physicochemical drug properties and toxicity were inferred from a data set consisting of animal in vivo toleration (IVT) studies on 245 preclinical Pfizer compounds; an increased likelihood of toxic events was found for less polar, more lipophilic compounds. This trend held across a wide range of types of toxicity and across a broad swath of chemical space.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Preparações Farmacêuticas/química , Relação Estrutura-Atividade , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Ratos
13.
Protein Sci ; 13(1): 190-202, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14691234

RESUMO

Protein interfaces are thought to be distinguishable from the rest of the protein surface by their greater degree of residue conservation. We test the validity of this approach on an expanded set of 64 protein-protein interfaces using conservation scores derived from two multiple sequence alignment types, one of close homologs/orthologs and one of diverse homologs/paralogs. Overall, we find that the interface is slightly more conserved than the rest of the protein surface when using either alignment type, with alignments of diverse homologs showing marginally better discrimination. However, using a novel surface-patch definition, we find that the interface is rarely significantly more conserved than other surface patches when using either alignment type. When an interface is among the most conserved surface patches, it tends to be part of an enzyme active site. The most conserved surface patch overlaps with 39% (+/- 28%) and 36% (+/- 28%) of the actual interface for diverse and close homologs, respectively. Contrary to results obtained from smaller data sets, this work indicates that residue conservation is rarely sufficient for complete and accurate prediction of protein interfaces. Finally, we find that obligate interfaces differ from transient interfaces in that the former have significantly fewer alignment gaps at the interface than the rest of the protein surface, as well as having buried interface residues that are more conserved than partially buried interface residues.


Assuntos
Ligação Proteica , Proteínas/química , Sequência de Aminoácidos , Aminoácidos/química , Sítios de Ligação , Análise por Conglomerados , Sequência Conservada , Cristalografia por Raios X , Bases de Dados Factuais , Dimerização , Entropia , Enzimas/química , Enzimas/metabolismo , Evolução Molecular , Genômica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solventes/metabolismo , Propriedades de Superfície
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