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1.
Anticancer Res ; 41(9): 4295-4304, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475049

RESUMO

BACKGROUND/AIM: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults. The aim of this study was to elucidate the molecular pathogenesis of sporadic RCC in Taiwan. MATERIALS AND METHODS: Fifteen patients with RCC were screened for mutations in the von Hippel-Lindau (VHL) gene by PCR and Sanger sequencing. The methylation status of promoters of 24 tumor suppressor genes by methylation sensitive multiplex ligation-dependent probe amplification analysis was also determined. RESULTS: Inactivation of the VHL gene was observed in 5 cases: three missense somatic mutations, one promoter methylation, and one small deletion. In RCCs, methylation was most frequently observed in APC (100%), CDKN2B (92.9%), CASP8, MLH1_167, and KLLN (85.7.4%), but not in FHIT, MLH1_463, DAPK1, or HIC1 (0%). CONCLUSION: In addition to VHL inactivation, promoter methylation of APC may be a universal pathognomonic event in the tumorigenesis of RCC and a candidate diagnostic and therapeutic biomarker.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Carcinoma de Células Renais/diagnóstico , Metilação de DNA , Neoplasias Renais/diagnóstico , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Detecção Precoce de Câncer , Epigênese Genética , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Mutação de Sentido Incorreto , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Deleção de Sequência
2.
Arch Oral Biol ; 130: 105246, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34454376

RESUMO

OBJECTIVE: To investigate whether null variants of Glutathione S-transferase Mu 1 (GSTM1) and GST Theta 1 (GSTT1) in infants and mothers, as well as maternal exposures to environmental factors, contribute to the risk of non-syndromic cleft lip with or without palate (NSCL/P) in a Mexican population. DESIGN: We performed a matched pair case-control study, including 98 cases and 98 controls and their mothers. Sociodemographic information and environmental exposures were collected by a questionnaire. Null variants of GSTM1 and GSTT1 were assessed by multiplex Polymerase Chain Reaction (PCR). Odds ratios (OR) and their 95 % confidence intervals (CI) were calculated to estimate risks. The interaction of genetic variables with smoking and adjusted ORs were evaluated by binary logistic regression. RESULTS: Homozygous null GSTM1 was associated with the risk of NSCL/P when present in mothers (OR = 2.45, 95 % CI 1.23-4.86) or infants (OR = 2.98, 95 % CI 1.45-6.14). A higher risk was also found when children carried the homozygous null GSTT1 (OR = 4.89, 95 % CI 2.42-9.87). In mothers, this variant showed a crude risk of 9.17 (95 % CI 3.95-21.29), which increased to OR = 13.81 (95 % CI 1.63-117.09) upon interaction with frequent passive smoking (5-7 days/week). Sociodemographic and other environmental exposures were not significantly associated with the risk of NSCL/P. CONCLUSIONS: Maternal and infant GSTT1 and GSTM1 homozygous null genotypes were associated with a higher risk of NSCL/P, and the results suggest an interaction of the maternal GSTT1-null/null genotype with frequent passive smoking.


Assuntos
Fissura Palatina , Glutationa Transferase , Estudos de Casos e Controles , Criança , Fissura Palatina/genética , Feminino , Predisposição Genética para Doença , Genótipo , Glutationa Transferase/genética , Homozigoto , Humanos , Exposição Materna , Polimorfismo Genético , Fatores de Risco , Deleção de Sequência
3.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445396

RESUMO

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Deleção de Sequência , Pareamento de Bases , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , RNA/química , Ribonuclease III/genética
4.
Nucleic Acids Res ; 49(15): 8732-8742, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365511

RESUMO

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Reparo do DNA , Edição de Genes , Animais , Células Cultivadas , Simulação por Computador , Humanos , Camundongos , Plasmídeos/genética , Deleção de Sequência , Translocação Genética
5.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
6.
Methods Mol Biol ; 2351: 321-334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382198

RESUMO

Loss-of-function experiments are essential for the functional investigation of cis-regulatory elements (CREs), such as transcriptional enhancers. This can be achieved with CRISPR-Cas9 using pairs of single guide RNAs (sgRNAs) to target the flanking regions of a CRE. Here, I describe a single-step protocol to rapidly and inexpensively generate vectors co-expressing two sgRNAs, which allows re-usage of gRNAs oligonucleotides from one experimental design to another. This protocol is applicable to cloning sgRNAs into virtually any CRISPR-Cas9 backbone that allows cloning using Golden Gate, by adapting the primer design.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Vetores Genéticos/genética , Sequências Reguladoras de Ácido Nucleico , Deleção de Sequência , Clonagem Molecular , Ordem dos Genes , Marcação de Genes/métodos , Humanos , Células Secretoras de Insulina/metabolismo , RNA Guia/química , RNA Guia/genética
7.
Nat Commun ; 12(1): 4922, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389729

RESUMO

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Perda de Heterozigosidade/genética , Deleção de Sequência , Células Cultivadas , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Metilação de DNA , Expressão Gênica , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Fator de Crescimento Insulin-Like II/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
8.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208159

RESUMO

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V-FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.


Assuntos
Apoptose/genética , Peixes-Gato/genética , Citocromos c/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Citocromos c/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência
9.
Front Immunol ; 12: 608604, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248927

RESUMO

Background and Objectives: Atypical hemolytic uremic syndrome (aHUS) is mostly attributed to dysregulation of the alternative complement pathway (ACP) secondary to disease-causing variants in complement components or regulatory proteins. Hereditary aHUS due to C3 disruption is rare, usually caused by heterozygous activating mutations in the C3 gene, and transmitted as autosomal dominant traits. We studied the molecular basis of early-onset aHUS, associated with an unusual finding of a novel homozygous activating deletion in C3. Design Setting Participants & Measurements: A male neonate with eculizumab-responsive fulminant aHUS and C3 hypocomplementemia, and six of his healthy close relatives were investigated. Genetic analysis on genomic DNA was performed by exome sequencing of the patient, followed by targeted Sanger sequencing for variant detection in his close relatives. Complement components analysis using specific immunoassays was performed on frozen plasma samples from the patient and mother. Results: Exome sequencing revealed a novel homozygous variant in exon 26 of C3 (c.3322_3333del, p.Ile1108_Lys1111del), within the highly conserved thioester-containing domain (TED), fully segregating with the familial disease phenotype, as compatible with autosomal recessive inheritance. Complement profiling of the patient showed decreased C3 and FB levels, with elevated levels of the terminal membrane attack complex, while his healthy heterozygous mother showed intermediate levels of C3 consumption. Conclusions: Our findings represent the first description of aHUS secondary to a novel homozygous deletion in C3 with ensuing unbalanced C3 over-activation, highlighting a critical role for the disrupted C3-TED domain in the disease mechanism.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Sequência de Bases/genética , Complemento C3/genética , Deleção de Sequência , Síndrome Hemolítico-Urêmica Atípica/congênito , Síndrome Hemolítico-Urêmica Atípica/etiologia , Pré-Escolar , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento , Genes Recessivos , Homozigoto , Humanos , Masculino , Sequenciamento Completo do Exoma
10.
Nat Commun ; 12(1): 4605, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326328

RESUMO

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Espermatogênese/fisiologia , Animais , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cristalografia por Raios X/métodos , Feminino , Recombinação Homóloga , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Meiose , Camundongos , Modelos Animais , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência
11.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281288

RESUMO

PURPOSE: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. METHODS: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat-/- and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. RESULTS: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat-/- animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat-/- animals. Lrat-/- animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat-/- animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. CONCLUSIONS: Our Lrat-/- rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.


Assuntos
Aciltransferases/deficiência , Aciltransferases/genética , Retinite Pigmentosa/genética , Animais , Comportamento Animal , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Oftalmoscopia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Transgênicos , Retinite Pigmentosa/diagnóstico por imagem , Retinite Pigmentosa/fisiopatologia , Deleção de Sequência , Tomografia de Coerência Óptica , Visão Ocular
12.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199599

RESUMO

Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2'-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol.


Assuntos
Hepatopatias Alcoólicas/genética , Fígado/efeitos dos fármacos , Receptor CB1 de Canabinoide/genética , Receptores de Estrogênio/genética , Receptores da Transferrina/genética , Álcoois/farmacologia , Animais , Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerídeos/farmacologia , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Ferro/metabolismo , Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Camundongos , Regiões Promotoras Genéticas , Receptor CB1 de Canabinoide/agonistas , Deleção de Sequência/genética , Transferrina/genética , Transferrina/metabolismo
13.
Nat Commun ; 12(1): 4228, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244484

RESUMO

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.


Assuntos
Neoplasias Encefálicas/genética , Encéfalo/patologia , Desoxiadenosinas/metabolismo , Glioblastoma/genética , Purina-Núcleosídeo Fosforilase/deficiência , Tionucleosídeos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Encéfalo/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Desoxiadenosinas/análise , Feminino , Secções Congeladas , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Homozigoto , Humanos , Metabolômica , Metionina Adenosiltransferase/metabolismo , Terapia de Alvo Molecular/métodos , Medicina de Precisão/métodos , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Deleção de Sequência , Tionucleosídeos/análise , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206573

RESUMO

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Assuntos
RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , DNA Espaçador Ribossômico , Endorribonucleases , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
15.
Nat Commun ; 12(1): 4193, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234122

RESUMO

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/genética , Carcinoma Nasofaríngeo/imunologia , Neoplasias Nasofaríngeas/imunologia , Evasão Tumoral/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/imunologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/terapia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Regulação Viral da Expressão Gênica/imunologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/metabolismo , Camundongos , NF-kappa B/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/terapia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/terapia , Neoplasias Nasofaríngeas/virologia , Nasofaringe/imunologia , Nasofaringe/patologia , Nasofaringe/cirurgia , Nasofaringe/virologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Deleção de Sequência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Evasão Tumoral/efeitos dos fármacos , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199056

RESUMO

Palmoplantar keratodermas (PPKs) are characterized by thickness of stratum corneum and epidermal hyperkeratosis localized in palms and soles. PPKs can be epidermolytic (EPPK) or non epidermolytic (NEPPK). Specific mutations of keratin 16 (K16) and keratin 1 (K1) have been associated to EPPK, and NEPPK. Cases of mosaicism in PPKs due to somatic keratin mutations have also been described in scientific literature. We evaluated a patient presenting hyperkeratosis localized monolaterally in the right palmar area, characterized by linear yellowish hyperkeratotic lesions following the Blaschko lines. No other relatives of the patient showed any dermatological disease. Light and confocal histological analysis confirmed the presence of epidermolityic hyperkeratosis. Genetic analysis performed demonstrates the heterozygous deletion NM_006121.4:r.274_472del for a total of 198 nucleotides, in KRT1 cDNA obtained by a palmar lesional skin biopsy, corresponding to the protein mutation NP_006112.3:p.Gly71_Gly137del. DNA extracted from peripheral blood lymphocytes did not display the presence of the mutation. These results suggest a somatic mutation causing an alteration in K1 N-terminal variable domain (V1). The deleted sequence involves the ISIS subdomain, containing a lysine residue already described as fundamental for epidermal transglutaminases in the crosslinking of IF cytoskeleton. Moreover, a computational analysis of the wild-type and V1-mutated K1/K10 keratin dimers, suggests an unusual interaction between these keratin filaments. The mutation taster in silico analysis also returned a high probability for a deleterious mutation. These data demonstrate once again the importance of the head domain (V1) of K1 in the formation of a functional keratinocyte cytoskeleton. Moreover, this is a further demonstration of the presence of somatic mutations arising in later stages of the embryogenesis, generating a mosaic phenotype.


Assuntos
Queratina-10/química , Queratina-1/química , Queratina-1/genética , Nevo/etiologia , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Neoplasias Cutâneas/etiologia , Sequência de Aminoácidos , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Imunofluorescência , Humanos , Imuno-Histoquímica , Queratina-1/metabolismo , Queratina-10/metabolismo , Modelos Moleculares , Nevo/metabolismo , Nevo/patologia , Conformação Proteica , Multimerização Proteica , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203807

RESUMO

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Clivagem do DNA , Reparo do DNA por Junção de Extremidades/genética , Sequência de Bases , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oligonucleotídeos/metabolismo , RNA Guia/genética , Deleção de Sequência/genética
18.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34310272

RESUMO

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5' intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.


Assuntos
Begomovirus/genética , Variação Genética , Manihot/crescimento & desenvolvimento , Manihot/virologia , Doenças das Plantas/virologia , Sequência de Bases , Begomovirus/fisiologia , Códon , DNA Intergênico , DNA Viral/genética , Evolução Molecular , Genoma Viral , Mutação , Polimorfismo de Nucleotídeo Único , Vírus Satélites/genética , Vírus Satélites/fisiologia , Deleção de Sequência , Temperatura , Proteínas Virais/genética
19.
Clin Immunol ; 230: 108812, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34329798

RESUMO

Autoimmune lymphoproliferative syndrome is a primary immunodeficiency caused by variants in FAS-mediated apoptosis related genes and is characterized by lymphadenopathy, splenomegaly and autoimmunity. A total of six different variants in CASP10 have been described as potential causative of disease, although two of them have recently been considered polymorphisms. The high allele frequency of these variants in healthy population in addition to the broad clinical spectrum of the disease difficult the interpretation of their pathogenicity. Here, we describe the clinical and analytical findings of three new patients carrying variants in CASP10 and summarize 12 more cases from the literature. Autoimmune cytopenias, adenopathies and increment of TCRαß+CD4-CD8- cells have been the most common findings, being possibly the FAS-mediated apoptosis pathway the pathogenic mechanism of this disease. The clinical impact and the consequences of CASP10 variants are not fully elucidated, therefore the description of new cases will contribute to solve this issue.


Assuntos
Síndrome Linfoproliferativa Autoimune/enzimologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 10/genética , Variação Genética , Adolescente , Adulto , Substituição de Aminoácidos , Apoptose/genética , Síndrome Linfoproliferativa Autoimune/diagnóstico , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Linhagem , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Deleção de Sequência
20.
Chem Commun (Camb) ; 57(55): 6796-6799, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236364

RESUMO

A rapid dual probe-based fluorimetric assay was developed to detect deletion mutations in circulating tumor DNA using structure-selective isothermal amplification and pattern recognition. This method could detect both homozygous and heterozygous deletion configurations in a one-set experiment and achieved picomolar detection limits with high selectivity within 2 hours. It was promising for point-of-care cancer diagnosis in hospital settings.


Assuntos
DNA Tumoral Circulante/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Deleção de Sequência , Humanos , Limite de Detecção , Testes Imediatos
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