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1.
J Genet Couns ; 29(1): 78-87, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31701594

RESUMO

Genomic sequencing (GS), such as whole genome and exome sequencing, is rapidly being integrated into pediatric critical care settings. Results are being used to make high impact decisions including declarations of futility, withdrawal of care, and rationing of scarce resources. In this qualitative study, we conducted interviews with clinicians involved in the care of critically ill children with congenital heart disease (CHD) to investigate their views on implementation of GS into clinical practice. Interviews were transcribed and inductively analyzed for major themes using grounded theory and thematic analysis. Three major themes emerged surrounding the use of genomic information in the high-stakes, time pressured decision making that characterizes clinical care of critically ill children with CHD: (a) that clinicians felt they did not have sufficient training to accurately assess genetic results despite pressure to incorporate results into clinical decisions; (b), that they desire knowledge support from genetic specialists, such as genetic counselors, who both understand the critical care context and are available within the time constraints of critical care clinical pressures; and (c), that clinicians feel a pressing need for increased genetics education to be able to safely and appropriately incorporate GS results into clinical decisions Our data suggest that genetics specialists may need a stronger presence in the pediatric critical care setting.

2.
Nature ; 577(7788): 109-114, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31827280

RESUMO

Activation of RIPK1 controls TNF-mediated apoptosis, necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325, respectively, by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However, the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation, apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore, we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis, but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast, patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species, resulting in resistance to necroptosis and ferroptosis. Together, these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1, and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells, as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts.

3.
World J Pediatr ; 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31587140

RESUMO

BACKGROUND: A20, a protein encoded by the tumor necrosis factor alpha-induced protein 3 gene (TNFAIP3), plays a vital role in the negative regulation of inflammation and immunity. Loss-of-function mutation in TNFAIP3 leads to a new described autoinflammatory disease-haploinsufficiency of A20 (HA20). Since HA20 was first described in 2016, a number of new cases have been described in this literature, however, the disease and its pathogenesis are poorly understood. This review seeks to improve clinical recognition of this disorder, and promote both earlier diagnosis and initiation of targeted therapies to improve patients' outcomes. METHODS: We reviewed 26 papers about A20 and HA20, and we summarized genetic variants and clinical manifestations of a total of 61 reported patients from 26 families identified to have a genetic diagnosis of germline pathogenic variants in TNFAIP3/A20. Additionally, we discussed the pathogenesis and treatment of HA20. RESULTS: A total of 24 pathogenic variants of A20 had been reported. There was significant clinical heterogeneity, even among those with the same variants in TNFAIP3. Prior to receiving a molecular diagnosis of HA20, patients had been diagnosed with Behcet's disease, rheumatoid arthritis, rheumatic fever, juvenile idiopathic arthritis, systemic lupus erythematosus, and even adult-onset Stills' disease. The patients with HA20 that presented with inflammatory signatures in NF-κB signaling were mostly responsive to treatment. CONCLUSIONS: HA20 is a monogenic autoinflammatory disease with highly variable clinical manifestations. This extensive heterogeneity makes it difficult to set a clinical diagnostic criteria, and genetic sequencing is necessary for a definitive diagnosis of HA20.

4.
CRISPR J ; 2(5): 324-330, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31599684

RESUMO

Researchers are exploring the use of gene-editing technologies to prevent and/or treat genetic conditions in humans. Stakeholder views, including those of patient and family populations, are important in the ongoing bioethical discussion. We conducted 27 semi-structured interviews with parents of people with trisomy 21 (T21; N = 10), trisomy 18 (T18; N = 8), and trisomy 13 (T13; N = 9)-conditions not previously studied in regard to attitudes toward hypothetical gene editing. While many discussions focus on the morality of gene editing, parents in our study focused on quality of life and concerns about changing their children's identity. All participants prioritized ameliorating life-threatening health issues when those were present; many also emphasized increasing their children's communication and cognitive ability. These results suggest that patient populations with the lived experience of genetic conditions have unique concerns that may differ from broader discourse.

6.
Front Immunol ; 9: 1361, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29963054

RESUMO

Objective: Describe the clinical characteristics and histopathology findings in a family with two siblings affected with deficiency of adenosine deaminase 2 (DADA2). Both patients presented in childhood with polyarthritis and developed significant neurological and gastrointestinal features of DADA2 in ear, including variable degrees of immunologic and hematologic manifestations. Methods: Adenosine Deaminase 2 (ADA2; also known as cat eye syndrome chromosome region, candidate 1 gene; CECR1) exon sequencing and serum ADA2 levels were performed to confirm the diagnosis of DADA2. Comparison of serum adenosine deaminase 2 levels was made to DADA2 patients, carriers, and healthy controls in Patient 2. Autopsy specimens from brain and liver tissues were submitted for analysis. Results: Both patients were found to carry a previously reported rare intronic missense mutation predicted to affect the transcript splicing (c.973-2A > G; rs139750129) and an unreported missense mutation p.Val458Asp (c.1373T > A; V458D). Both brothers started therapy with a tumor necrosis factor inhibitor following the molecular diagnosis of DADA2 with good response and were eventually tapered off prednisone. However, Patient 1 died 18 months later due to complications of end-stage liver disease. His autopsy showed evidence for nodular hyperplasia of the liver often seen in common variable immunodeficiency (CVID) and numerous small, old infarcts throughout the brain that had not been demonstrated on prior MRI/MRA imaging. Conclusion: These cases emphasize the importance of recognition of DADA2 in adults, compare CNS imaging modalities to pathologic findings and suggest similarities in liver pathology between DADA2 and CVID. MRI may not be most sensitive method to identify small subcortical infarcts in patients suspected to have DADA2.

7.
Ann Rheum Dis ; 77(4): 612-619, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29358286

RESUMO

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.


Assuntos
Anemia Sideroblástica/genética , Anti-Inflamatórios/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Síndromes de Imunodeficiência/genética , Mutação , Nucleotidiltransferases/genética , RNA de Transferência/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Anemia Sideroblástica/sangue , Criança , Pré-Escolar , Citocinas/sangue , Citocinas/genética , Deficiências do Desenvolvimento/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Humanos , Imunofenotipagem , Masculino , Linhagem , Fenótipo , Fator de Necrose Tumoral alfa/análise , Sequenciamento Completo do Exoma
8.
Arthritis Rheumatol ; 69(9): 1832-1839, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28544690

RESUMO

OBJECTIVE: To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease. METHODS: We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples. RESULTS: We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients' blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects. CONCLUSION: Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants.


Assuntos
Alelos , Complemento C1r/deficiência , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Complemento C1r/genética , Consanguinidade , Exoma , Feminino , Genótipo , Humanos , Interferon Tipo I/sangue , Leucócitos Mononucleares/citologia , Lúpus Eritematoso Sistêmico/sangue , Masculino , Neutrófilos/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Índice de Gravidade de Doença , Turquia
9.
Mol Biol Cell ; 28(1): 41-53, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27852897

RESUMO

Negatively regulating key signaling pathways is critical to development and altered in cancer. Wnt signaling is kept off by the destruction complex, which is assembled around the tumor suppressors APC and Axin and targets ß-catenin for destruction. Axin and APC are large proteins with many domains and motifs that bind other partners. We hypothesized that if we identified the essential regions required for APC:Axin cooperative function and used these data to design a minimal ß-catenin-destruction machine, we would gain new insights into the core mechanisms of destruction complex function. We identified five key domains/motifs in APC or Axin that are essential for their function in reconstituting Wnt regulation. Strikingly, however, certain APC and Axin mutants that are nonfunctional on their own can complement one another in reducing ß-catenin, revealing that the APC:Axin complex is a highly robust machine. We used these insights to design a minimal ß-catenin-destruction machine, revealing that a minimized chimeric protein covalently linking the five essential regions of APC and Axin reconstitutes destruction complex internal structure, size, and dynamics, restoring efficient ß-catenin destruction in colorectal tumor cells. On the basis of our data, we propose a new model of the mechanistic function of the destruction complex as an integrated machine.


Assuntos
Proteína Axina/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteína Axina/fisiologia , Linhagem Celular Tumoral , Drosophila/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteínas Repressoras/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/fisiologia
10.
PLoS One ; 11(8): e0160509, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27486871

RESUMO

Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.


Assuntos
Clonagem Molecular/métodos , DNA/isolamento & purificação , Plasmídeos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Técnicas Bacteriológicas/métodos , Linhagem Celular , Comércio , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Humanos , Proteínas/genética , Proteínas/metabolismo , Transfecção/métodos
11.
Proc Natl Acad Sci U S A ; 113(36): 10127-32, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27559085

RESUMO

Systemic autoinflammatory diseases are caused by mutations in genes that function in innate immunity. Here, we report an autoinflammatory disease caused by loss-of-function mutations in OTULIN (FAM105B), encoding a deubiquitinase with linear linkage specificity. We identified two missense and one frameshift mutations in one Pakistani and two Turkish families with four affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated OTULIN had decreased enzyme activity relative to cells transfected with WT OTULIN, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients' fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-κB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the supernatants of stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies.


Assuntos
Alelos , Endopeptidases/genética , Fibroblastos/patologia , Doenças Hereditárias Autoinflamatórias/genética , Leucócitos Mononucleares/patologia , Mutação , Idade de Início , Criança , Pré-Escolar , Consanguinidade , Citocinas/genética , Citocinas/imunologia , Dermatite/fisiopatologia , Endopeptidases/deficiência , Endopeptidases/imunologia , Insuficiência de Crescimento/fisiopatologia , Feminino , Febre/fisiopatologia , Fibroblastos/enzimologia , Fibroblastos/imunologia , Regulação da Expressão Gênica , Células HEK293 , Doenças Hereditárias Autoinflamatórias/diagnóstico , Doenças Hereditárias Autoinflamatórias/enzimologia , Doenças Hereditárias Autoinflamatórias/patologia , Humanos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/imunologia , Masculino , NF-kappa B/genética , NF-kappa B/imunologia , Paniculite/fisiopatologia , Linhagem , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/imunologia
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