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1.
Zhonghua Er Ke Za Zhi ; 57(8): 631-635, 2019 Aug 02.
Artigo em Chinês | MEDLINE | ID: mdl-31352750

RESUMO

Objective: To investigate the clinical and genotypic manifestations of X-linked neutropenia caused by gain-of-function mutation in WAS gene. Methods: The clinical history of two patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene in Shenzhen Children's Hospital were analyzed."X-linked neutropenia" and "WAS mutation" were used as key words to search related literatures published from January 2000 to December 2018 in CNKI,Wanfang, and Pubmed databases. Results: The first case was male,1 year old, admitted for 1 year of neutropenia combined with 5 days of cough and 3 days of fever. Persistent neutropenia (0.1×10(9)-0.3×10(9)/L) was reported before admission and during hospitalization (0.4×10(9)-0.5×10(9)/L). The patient was treated with Ciprofloxacin, cefoperazone sulbactam and Vancomycin,and relieved from fever after 4 weeks of hospitalization,yet the neutropenia (0.1×10(9)-0.6×10(9)/L) continued after discharge. Variant in WAS gene (c.T869C (p.I290T) ) was identified, and the percentage of WAS protein on lymphocyte was 97.7%. The second case was male, 42 days old,admitted for fever and neutropenia (0.5×10(9)/L). Similarly,he relieved from fever after 4 weeks of treatment with amoxicillin sulbactam,vancomysin,meropenem,rifampin and isoniacid,yet was discharged with continued neutropenia. Variant in WAS gene (c.T881C (p.I294T)) was identified and the percentage of WAS protein on lymphocyte was 92%. Published literature reported four variants,including I290T, L270P, S272P and I294T, as the pathogenic mutation of X-linked neutropenia in 18 patients from five families. Neutropenia (0.1×10(9)-1.0×10(9)/L) were reported in 15 patients,while normal neutrophil number was found in the rest. Recurrent infection,mainly pneumonia and otitis media,was the most common clinical manifestation. Conclusions: Neutropenia is the prominent presentation in the patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene, but it unnecessarily correlates with the clinical severity in terms of infection. Gene sequencing should be considered for the male patients with persistent neutropenia.


Assuntos
Cromossomos Humanos X , Mutação com Ganho de Função/genética , Neutropenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Febre/etiologia , Humanos , Lactente , Masculino , Mutação
2.
Mol Immunol ; 114: 30-40, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336247

RESUMO

Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.


Assuntos
Mutação com Ganho de Função/genética , Mutação de Sentido Incorreto/genética , Domínios Proteicos/genética , Fator de Transcrição STAT1/genética , Candidíase Mucocutânea Crônica/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Células Cultivadas , Citocinas/genética , Células HeLa , Heterozigoto , Humanos , Interferon gama/genética , Fosforilação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Transcrição Genética/genética
3.
Pediatr Rheumatol Online J ; 17(1): 38, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286971

RESUMO

BACKGROUND: Autosomal dominant gain of function mutations in caspase recruitment domain family member 14 (CARD14) is a rare condition associated with plaque-type psoriasis, generalized pustular psoriasis, palmoplantar pustular psoriasis and pityriasis rubra pilaris. Recently, a new CARD14 -associated phenotype defined as CAPE (CARD14-associated papulosquamous eruption) with clinical features of both psoriasis and pityriasis rubra pilaris was reported. We describe a family carrying a novel heterozygous mutation in CARD14 gene, with childhood-onset erythrodermic psoriasis requiring an unusual extremely high dose (up to 2 mg/kg every 8 weeks) of ustekinumab to achieve disease remission. CASE PRESENTATION: We describe a large family with three pairs of twins presenting a clinical phenotype characterized by childhood-onset erythrodermic psoriasis; in some family members is also reported psoriatic arthritis. The two probands presented poor clinical response to topic and systemic therapy with antihistamine, steroid, retinoids, cyclosporine and etanercept. After exclusion of the most common genes associated to autoinflammatory diseases (IL36RN, IL1RN, MVK, TNFRSF1A, NLRP3, NLRP12, MEFV, NOD2, PSMB8, PSTPIP1, LPIN2) we approached a new gene search by subjecting to Whole Exome Sequencing (WES) analysis five members of the family. A novel heterozygous mutation (c.446 T > G, leading to the missense amino acid substitution p.L149R) in the exon 4 of the CARD14 gene was identified in all affected members. Increasing dosages (up to 2 mg/kg every 8 weeks) of ustekinumab, a human monoclonal antibody targeting interleukin-12 (IL-12) and interleukin-23 (IL-23), allowed the complete control of the clinical manifestations, with an evident reduction of circulating Th17 and Th22 CD4+ T cell subsets. CONCLUSIONS: We describe the association of mutations of the CARD14 gene with an erythrodermic psoriasis pedigree, underlying the necessity to investigate CARD14 mutations in childhood-onset psoriasis cases and confirming the presence of CARD14 causative mutations also in erythrodermic psoriasis form, as recently reported. Also in pediatric age, ustekinumab represents a powerful therapeutic option for this rare condition, that is usually refractory to other treatments. In young children, high and frequent dosages allowed a complete control of the clinical manifestations without any severe side effects, with a long-term follow-up.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Fármacos Dermatológicos/uso terapêutico , Mutação com Ganho de Função/genética , Guanilato Ciclase/genética , Proteínas de Membrana/genética , Psoríase/tratamento farmacológico , Psoríase/genética , Ustekinumab/uso terapêutico , Criança , Dermatite Esfoliativa/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Linhagem , Gêmeos Dizigóticos , Sequenciamento Completo do Exoma
4.
Medicine (Baltimore) ; 98(18): e15329, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31045771

RESUMO

RATIONALE: Gain of function (GOF) mutations in PIK3CD gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, cytomegalovirus and/or epstein-Barr virus (EBV) viremia, and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. PATIENT CONCERNS: We report a patient who was diagnosed with systemic lupus erythematosus (SLE) at a young age and was recently found to carry heterozygous mutations in PIK3CD. The patient not only presented with recurrent sinopulmonary infections, CD4 lymphopenia, lymphadenopathy, EBV viremia, and elevated serum IgM, but also met classification criteria of SLE based on persistent proteinuria and hematuria, leukopenia and anemia, low level of serum complement, and positive autoantibody for antinuclear antibodies. DIAGNOSES: Activated PI3Kδ syndrome. INTERVENTIONS: Oral prednisolone and hydroxychloroquine combined with mycophenolate mofetil was given to the patient. He was currently receiving intravenous immunoglobulin per month in association with hydroxychloroquine, low-dose prednisolone, and mycophenolate mofetil. OUTCOMES: At present, the level of complement restored to normal, hematuria and proteinuria disappeared, and liver function returned to normal. LESSONS: SLE may be a novel phenotype of GOF mutation in PI3CKD gene (GOF PIK3CD).


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação com Ganho de Função/genética , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Lúpus Eritematoso Sistêmico/genética , Adolescente , Anticorpos Antinucleares/sangue , Grupo com Ancestrais do Continente Asiático/genética , Classe I de Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas do Sistema Complemento/análise , Proteínas do Sistema Complemento/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/imunologia , Glucocorticoides/uso terapêutico , Herpesvirus Humano 4/imunologia , Humanos , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Ácido Micofenólico/administração & dosagem , Ácido Micofenólico/uso terapêutico , Fenótipo , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico
5.
PLoS Genet ; 15(5): e1008139, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31050682

RESUMO

Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Espermatogônias/metabolismo , Células-Tronco Germinativas Adultas/fisiologia , Animais , Mutação com Ganho de Função/genética , Mutação em Linhagem Germinativa/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Mutação/genética , Idade Paterna , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Seleção Genética/genética , Transdução de Sinais/genética , Espermatogônias/fisiologia , Espermatozoides/metabolismo , Testículo/metabolismo
6.
Cells ; 8(5)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052273

RESUMO

SH3 domain-binding protein 2 (SH3BP2) is an adaptor protein that is predominantly expressed in immune cells, and it regulates intracellular signaling. We had previously reported that a gain-of-function mutation in SH3BP2 exacerbates inflammation and bone loss in murine arthritis models. Here, we explored the involvement of SH3BP2 in a lupus model. Sh3bp2 gain-of-function (P416R knock-in; Sh3bp2KI/+) mice and lupus-prone B6.MRL-Faslpr mice were crossed to yield double-mutant (Sh3bp2KI/+Faslpr/lpr) mice. We monitored survival rates and proteinuria up to 48 weeks of age and assessed renal damage and serum anti-double-stranded DNA antibody levels. Additionally, we analyzed B and T cell subsets in lymphoid tissues by flow cytometry and determined the expression of apoptosis-related molecules in lymph nodes. Sh3bp2 gain-of-function mutation alleviated the poor survival rate, proteinuria, and glomerulosclerosis and significantly reduced serum anti-dsDNA antibody levels in Sh3bp2KI/+Faslpr/lpr mice. Additionally, B220+CD4-CD8- T cell population in lymph nodes was decreased in Sh3bp2KI/+Faslpr/lpr mice, which is possibly associated with the observed increase in cleaved caspase-3 and tumor necrosis factor levels. Sh3bp2 gain-of-function mutation ameliorated clinical and immunological phenotypes in lupus-prone mice. Our findings offer better insight into the unique immunopathological roles of SH3BP2 in autoimmune diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação com Ganho de Função/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Animais , Anticorpos Antinucleares/sangue , Apoptose , Linfócitos B/imunologia , Caspase 3/metabolismo , Diferenciação Celular , Células Dendríticas/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Linfonodos/patologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Fagocitose , Fenótipo , Análise de Sobrevida , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor fas/metabolismo
7.
Cell ; 177(3): 597-607.e9, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31002796

RESUMO

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of ß-arrestin recruitment to MC4R, rather than canonical Gαs-mediated cyclic adenosine-monophosphate production, explained 88% of the variance in the association of MC4R variants with BMI. While most MC4R variants caused loss of function, a subset caused gain of function; these variants were associated with significantly lower BMI and lower odds of obesity, type 2 diabetes, and coronary artery disease. Protective associations were driven by MC4R variants exhibiting signaling bias toward ß-arrestin recruitment and increased mitogen-activated protein kinase pathway activation. Harnessing ß-arrestin-biased MC4R signaling may represent an effective strategy for weight loss and the treatment of obesity-related cardiometabolic diseases.


Assuntos
Mutação com Ganho de Função/genética , Obesidade/patologia , Receptor Tipo 4 de Melanocortina/genética , Transdução de Sinais , Adulto , Idoso , Índice de Massa Corporal , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , AMP Cíclico/metabolismo , Bases de Dados Factuais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/metabolismo , Polimorfismo de Nucleotídeo Único , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/metabolismo , beta-Arrestinas/metabolismo
8.
J Hum Genet ; 64(6): 511-519, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30853710

RESUMO

ORAI1 encodes a calcium channel essential in the store-operated calcium entry mechanism. A previous genetic association study identified a rare in-frame insertion variant of ORAI1 conferring Kawasaki disease (KD). To deepen our understanding of the involvement of rare variants of ORAI1 in KD pathogenesis, we investigated 3812 patients with KD and 2644 healthy individuals for variations in the protein-coding region of ORAI1. By re-sequencing the study participants' DNA, 27 variants with minor allele frequencies (MAFs) < 0.01 that had not been examined in the previous study were identified. Although no significant association with KD was observed either in single-variant analyses or in a collapsing method analysis of the 27 variants, stratification by MAFs, variant types, and predicted deleteriousness revealed that six rare, deleterious, missense variants (MAF < 0.001, CADD C-score ≥ 20) were exclusively present in KD patients, including three refractory cases (OR = ∞, P = 0.046). The six missense variants include p.Gly98Asp, which has been demonstrated to result in gain of function leading to constitutive Ca2+ entry. Conversely, five types of frameshift variants, all identified near the N terminus and assumed to disrupt ORAI1 function, showed an opposite trend of association (OR = 0.35, P = 0.24). These findings support our hypothesis that genetic variations causing the upregulation of the Ca2+/NFAT pathway confer susceptibility to KD. Our findings also provide insights into the usefulness of stratifying the variants based on their MAFs and on the direction of the effects on protein function when conducting association studies using the gene-based collapsing method.


Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Síndrome de Linfonodos Mucocutâneos/genética , Proteína ORAI1/genética , Cálcio/metabolismo , Pré-Escolar , Feminino , Mutação com Ganho de Função/genética , Frequência do Gene , Humanos , Lactente , Masculino , Síndrome de Linfonodos Mucocutâneos/patologia , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética
9.
Dev Cell ; 48(6): 765-779.e7, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30773489

RESUMO

Specialized adult somatic cells, such as cardiomyocytes (CMs), are highly differentiated with poor renewal capacity, an integral reason underlying organ failure in disease and aging. Among the least renewable cells in the human body, CMs renew approximately 1% annually. Consistent with poor CM turnover, heart failure is the leading cause of death. Here, we show that an active version of the Hippo pathway effector YAP, termed YAP5SA, partially reprograms adult mouse CMs to a more fetal and proliferative state. One week after induction, 19% of CMs that enter S-phase do so twice, CM number increases by 40%, and YAP5SA lineage CMs couple to pre-existing CMs. Genomic studies showed that YAP5SA increases chromatin accessibility and expression of fetal genes, partially reprogramming long-lived somatic cells in vivo to a primitive, fetal-like, and proliferative state.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/fisiologia , Cromatina/metabolismo , Coração/crescimento & desenvolvimento , Organogênese , Fosfoproteínas/metabolismo , Potenciais de Ação , Animais , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Ciclo Celular , Proteínas de Ciclo Celular , Linhagem da Célula , Proliferação de Células , Diploide , Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função/genética , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/anatomia & histologia , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organogênese/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-1/metabolismo , Transgenes
10.
Proc Natl Acad Sci U S A ; 116(9): 3536-3545, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808747

RESUMO

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Animais , Caderinas/genética , Mutação com Ganho de Função/genética , Gastrulação/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/genética , Síndrome de Noonan/fisiopatologia , Ligação Proteica , Peixe-Zebra/genética
11.
Nat Med ; 25(4): 583-590, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804514

RESUMO

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression. Heterozygous loss-of-function point mutations of miRNA genes are associated with several human congenital disorders1-5, but neomorphic (gain-of-new-function) mutations in miRNAs due to nucleotide substitutions have not been reported. Here we describe a neomorphic seed region mutation in the chondrocyte-specific, super-enhancer-associated MIR140 gene encoding microRNA-140 (miR-140) in a novel autosomal dominant human skeletal dysplasia. Mice with the corresponding single nucleotide substitution show skeletal abnormalities similar to those of the patients but distinct from those of miR-140-null mice6. This mutant miRNA gene yields abundant mutant miR-140-5p expression without miRNA-processing defects. In chondrocytes, the mutation causes widespread derepression of wild-type miR-140-5p targets and repression of mutant miR-140-5p targets, indicating that the mutation produces both loss-of-function and gain-of-function effects. Furthermore, the mutant miR-140-5p seed competes with the conserved RNA-binding protein Ybx1 for overlapping binding sites. This finding may explain the potent target repression and robust in vivo effect by this mutant miRNA even in the absence of evolutionary selection of miRNA-target RNA interactions, which contributes to the strong regulatory effects of conserved miRNAs7,8. Our study presents the first case of a pathogenic gain-of-function miRNA mutation and provides molecular insight into neomorphic actions of emerging and/or mutant miRNAs.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Mutação com Ganho de Função/genética , MicroRNAs/genética , Animais , Sequência de Bases , Condrócitos/metabolismo , Feminino , Homozigoto , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , MicroRNAs/metabolismo , Linhagem , Fenótipo , Transcriptoma/genética
12.
Gene ; 697: 57-66, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-30796966

RESUMO

Oncogenes can potentially cause uncontrolled cell growth, leading to cancer development, and these genes are normally mutated and over-expressed in tumor cells. Genomic alteration of oncogenes might result in oncogenesis and promotion of cancer progression. To date, researchers have focused mainly on the roles of oncogenes in particular cancers, but investigation of oncogenes with gain-of-function mutations in multiple cancer types are less represented in the literature. Furthermore, the effect of those gain-of-function are not validated in gene expression level. To meet this demand, we performed a systematic analysis of gene expression in oncogenes to identify the occurrence of gain-of-function mutations in pan-cancer. We identified 33,551 oncogenic mutations in 5000 samples. From our analysis, we identified three tissues with the highest frequency of gain-of-functional oncogenic mutations in hundreds of samples: breast (739 samples), central nervous system (646 samples) and large intestine (498 samples). By further counting the number of occurrences of oncogenes across cancer types, we identified a list cross-cancer mutational signatures of 99 oncogenes highly mutated in >400 samples in breast, central nervous system and large intestine samples. By further overlapping with gene expression data in the matched tumor samples, we further identified 1875 gain-of-functional mutations/events with consistent gene up-regulation in 1031 samples from multiple cancers. This result may offer additional insight into the relationship between gene dosage and oncogenesis and maybe useful in targeted cancer therapy. In summary, this study provides the first globally examining on the genetic alteration of oncogenes across cancer types. Clinical association analysis has shown that these 99 genes have a significant effect on survival.


Assuntos
Mutação com Ganho de Função/genética , Neoplasias/genética , Oncogenes/genética , Carcinogênese , Bases de Dados Genéticas , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Genômica , Humanos , Mutação , Transcriptoma/genética
14.
Brain ; 142(2): 362-375, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601941

RESUMO

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.


Assuntos
Encefalopatias/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Integrases/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neurônios/fisiologia , Prosencéfalo/fisiologia , Animais , Encefalopatias/patologia , Células Cultivadas , Feminino , Mutação com Ganho de Função/genética , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Técnicas de Cultura de Órgãos , Prosencéfalo/patologia
15.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463976

RESUMO

We previously generated STING N153S knock-in mice that have a human disease-associated gain-of-function mutation in STING. Patients with this mutation (STING N154S in humans) develop STING-associated vasculopathy with onset in infancy (SAVI), a severe pediatric autoinflammatory disease characterized by pulmonary fibrosis. Since this mutation promotes the upregulation of antiviral type I interferon-stimulated genes (ISGs), we hypothesized that STING N153S knock-in mice may develop more severe autoinflammatory disease in response to a virus challenge. To test this hypothesis, we infected heterozygous STING N153S mice with murine gammaherpesvirus 68 (γHV68). STING N153S mice were highly vulnerable to infection and developed pulmonary fibrosis after infection. In addition to impairing CD8+ T cell responses and humoral immunity, STING N153S also promoted the replication of γHV68 in cultured macrophages. In further support of a combined innate and adaptive immunodeficiency, γHV68 infection was more severe in Rag1-/- STING N153S mice than in Rag1-/- littermate mice, which completely lack adaptive immunity. Thus, a gain-of-function STING mutation creates a combined innate and adaptive immunodeficiency that leads to virus-induced pulmonary fibrosis.IMPORTANCE A variety of human rheumatologic disease-causing mutations have recently been identified. Some of these mutations are found in viral nucleic acid-sensing proteins, but whether viruses can influence the onset or progression of these human diseases is less well understood. One such autoinflammatory disease, called STING-associated vasculopathy with onset in infancy (SAVI), affects children and leads to severe lung disease. We generated mice with a SAVI-associated STING mutation and infected them with γHV68, a common DNA virus that is related to human Epstein-Barr virus. Mice with the human disease-causing STING mutation were more vulnerable to infection than wild-type littermate control animals. Furthermore, the STING mutant mice developed lung fibrosis similar to that of patients with SAVI. These findings reveal that a human STING mutation creates severe immunodeficiency, leading to virus-induced lung disease in mice.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Fibrose Pulmonar/genética , Imunidade Adaptativa/genética , Animais , Mutação com Ganho de Função/genética , Gammaherpesvirinae/metabolismo , Gammaherpesvirinae/fisiologia , Síndromes de Imunodeficiência , Inflamação/genética , Pulmão/virologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo
16.
Nat Genet ; 51(1): 96-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30478443

RESUMO

DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Nanismo/genética , Mutação com Ganho de Função/genética , Microcefalia/genética , Proteínas do Grupo Polycomb/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Metilases de Modificação do DNA/genética , Feminino , Células HeLa , Histonas/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos/genética , Ligação Proteica/genética , Sequências Reguladoras de Ácido Nucleico/genética
17.
Hum Mol Genet ; 28(1): 96-104, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30239721

RESUMO

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.


Assuntos
Glutaminase/genética , Glutaminase/fisiologia , Adolescente , Animais , Encéfalo/metabolismo , Catarata/genética , Pré-Escolar , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Feminino , Fibroblastos , Mutação com Ganho de Função/genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/fisiologia , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Masculino , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
18.
Methods Mol Biol ; 1864: 295-310, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30415344

RESUMO

With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.


Assuntos
Biotecnologia/métodos , Edição de Genes/métodos , Engenharia Genética/métodos , Nucleases de Dedos de Zinco/metabolismo , Biotecnologia/instrumentação , Mutação com Ganho de Função/genética , Deleção de Genes , Edição de Genes/instrumentação , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Introdução de Genes/instrumentação , Técnicas de Introdução de Genes/métodos , Engenharia Genética/instrumentação , Genoma de Planta/genética , Nucleases de Dedos de Zinco/genética
19.
Mol Neurobiol ; 56(7): 4778-4785, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30387076

RESUMO

The two most common polymorphisms of the human DRD4 gene encode a dopamine D4 receptor (D4R) with four or seven repeats of a proline-rich sequence of 16 amino acids (D4.4R or D4.7R). Although the seven-repeat polymorphism has been repeatedly associated with attention-deficit hyperactivity disorder and substance use disorders, the differential functional properties between D4.4R and D4.7R remained enigmatic until recent electrophysiological and optogenetic-microdialysis experiments indicated a gain of function of D4.7R. Since no clear differences in the biochemical properties of individual D4.4R and D4.7R have been reported, it was previously suggested that those differences emerge upon heteromerization with dopamine D2 receptor (D2R), which co-localizes with D4R in the brain. However, contrary to a gain of function, experiments in mammalian transfected cells suggested that heteromerization with D2R results in lower MAPK signaling by D4.7R as compared to D4.4R. In the present study, we readdressed the question of functional differences of D4.4R and D4.7R forming homomers or heteromers with the short isoform of D2R (D2SR), using a functional bioluminescence resonance energy transfer (BRET) assay that allows the measurement of ligand-induced changes in the interaction between G protein-coupled receptors (GPCRs) forming homomers or heteromers with their cognate G protein. Significant functional and pharmacological differences between D4.4R and D4.7R were only evident upon heteromerization with the short isoform of D2R (D2SR). The most dramatic finding was a significant increase and decrease in the constitutive activity of D2S upon heteromerization with D4.7R and D4.4R, respectively, providing the first clear mechanism for a functional difference between both products of polymorphic variants and for a gain of function of the D4.7R.


Assuntos
Mutação com Ganho de Função/genética , Polimorfismo Genético , Multimerização Proteica , Receptores de Dopamina D4/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Racloprida/farmacologia
20.
Blood ; 133(4): 356-365, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30366922

RESUMO

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands' blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.


Assuntos
Alelos , Mutação com Ganho de Função/genética , Predisposição Genética para Doença , Infarto do Miocárdio/genética , Tirosina/genética , Fator de von Willebrand/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Conformação Proteica , Fatores de Risco , Fator de von Willebrand/química
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