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1.
Anticancer Res ; 39(10): 5311-5327, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570425

RESUMO

BACKGROUND/AIM: MiR-221, often described both as an oncogenic microRNA and as a tumour suppressor, targets mRNAs involved in carcinogenesis. While other oncogenic microRNAs showed correlations with prostate cancer cell lines' aggressiveness, miR-221 showed an unusual overexpression in PC3. MATERIALS AND METHODS: CRISPR was used to delete miR-221 from PC3 cells. Analysing the characteristics of PC3miR-221del cells, a reduced growth rate and expression of cell-cycle genes was observed. In global gene expression/ontology analysis of PC3miR-221del cells, cell-cell and cell-substrate adhesion pathways were found to be greatly affected. In addition, reduced levels of adhesion, invasion and motility for PC3miR-221del cells, a change in F-actin localisation and a reduction of EMT markers were observed. RESULTS: The tumour suppressor gene, DIRAS3, was a predicted target of miR-221. In PC3miR-221del cells DIRAS3 was up-regulated at the gene and protein level. Ectopic expression of DIRAS3 in PC3wt cells recapitulated the cellular morphology changes seen in PC3miR-221del cells. DIRAS3 3'UTR was more stable in PC3miR-221del cells, as measured by semi-quantitative PCR and luciferase fusion reporter assays. CONCLUSION: MiR-221 promotes aggressiveness of PC3 cells by down-regulating DIRAS3, and promoting epithelial-to-mesenchymal transition.


Assuntos
Adesão Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Deleção de Sequência/genética , Regiões 3' não Traduzidas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Oncogenes/genética , Células PC-3 , Neoplasias da Próstata/genética , Regulação para Cima/genética , Proteínas rho de Ligação ao GTP/genética
2.
Ideggyogy Sz ; 72(7-8): 273-277, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31517460

RESUMO

Cerebral cavernous malformations (CCMs) represent a relatively rare and heterogeneous clinical entity with mutations identified in three genes. Both sporadic and familial forms have been reported. We present a young female patient with episodic paresthesia and headaches, but without acute neurological deficits. Her mother had a hemorrhaged cavernoma surgically removed 21 years ago. Cranial magnetic resonance imaging revealed multiple cavernous malformations in the size of a few millimeters and the ophthalmologic exam detected retinal blood vessel tortuosity in the proband. Targeted exome sequencing analysis identified a nonsense mutation in exon 16 of the KRIT1 gene, which resulted in a premature stop codon and a truncated protein underlying the abnormal development of cerebral and retinal blood vessels. This mutation with pathogenic significance has been reported before. Our case points to the importance of a thorough clinical and molecular work up despite the uncertain neurological complaints, since life style recommendations, imaging monitoring and genetic counseling may have major significance in the long term health of the patient.


Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico , Proteína KRIT1/genética , Vasos Retinianos/diagnóstico por imagem , Feminino , Cefaleia/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Humanos , Imagem por Ressonância Magnética , Mutação , Parestesia/etiologia , Linhagem , Deleção de Sequência/genética
3.
Medicine (Baltimore) ; 98(27): e16302, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31277167

RESUMO

OBJECTIVE: Researchers have evaluated the associations between mitochondrial DNA (mtDNA) 4977 bp deletion and presbycusis. This study aimed to assess the differences of mtDNA 4977 bp deletion between presbycusis patients and controls by conducting a meta-analysis of published studies. METHODS: Databases, including PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), and Wanfang Data were searched to collect case-control studies on the correlation between mitochondrial DNA 4977 bp deletion and presbycusis. The research findings of related articles were collected according to the inclusion criteria. Pooled odds ratios (ORs) and corresponding confidence intervals (CIs) were calculated. Meanwhile, subgroup analysis was performed to examine the source of heterogeneity. Revman 5.3 and Stata 12.0 software were used for data synthesis. RESULTS: Eight English and Chinese studies were included in the meta-analysis, the results of which showed that mitochondrial DNA 4977 bp deletion could increase the risk of presbycusis (OR = 8.16, 95% CI: 3.51-18.99), and the difference was statistically significant (P <. 01). Analysis of the polled OR showed the incidence of mtDNA 4977 bp deletion was 8.50 times higher in Asians with presbycusis than in the control group. And the OR in the studies of occidentals was 7.24. Sample source analysis was also performed with the sample source divided by temporal bone source and other sources (hair and blood). The OR was 4.18 and 22.36 for the temporal bone and other sources, respectively. CONCLUSION: Mitochondrial DNA 4977 bp deletion could increase the risk of presbycusis.


Assuntos
DNA Mitocondrial/genética , Presbiacusia/genética , Deleção de Sequência/genética , Estudos de Casos e Controles , Humanos
4.
Nature ; 571(7763): 107-111, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31217582

RESUMO

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.


Assuntos
Diarreia/congênito , Diarreia/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes , Intestinos/fisiologia , Deleção de Sequência/genética , Animais , Cromossomos Humanos Par 16/genética , Modelos Animais de Doenças , Feminino , Genes Reporter , Loci Gênicos/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Linhagem , Fenótipo , Ativação Transcricional , Transcriptoma/genética , Transgenes/genética
5.
PLoS Genet ; 15(6): e1008195, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181062

RESUMO

To characterize the consequences of eliminating essential functions needed for peptidoglycan synthesis, we generated deletion mutations of Acinetobacter baylyi by natural transformation and visualized the resulting microcolonies of dead cells. We found that loss of genes required for peptidoglycan precursor synthesis or polymerization led to the formation of polymorphic giant cells with diameters that could exceed ten times normal. Treatment with antibiotics targeting early or late steps of peptidoglycan synthesis also produced giant cells. The giant cells eventually lysed, although they were partially stabilized by osmotic protection. Genome-scale transposon mutant screening (Tn-seq) identified mutations that blocked or accelerated giant cell formation. Among the mutations that blocked the process were those inactivating a function predicted to cleave murein glycan chains (the MltD murein lytic transglycosylase), suggesting that giant cell formation requires MltD hydrolysis of existing peptidoglycan. Among the mutations that accelerated giant cell formation after ß-lactam treatment were those inactivating an enzyme that produces unusual 3->3 peptide cross-links in peptidoglycan (the LdtG L,D-transpeptidase). The mutations may weaken the sacculus and make it more vulnerable to further disruption. Although the study focused on A. baylyi, we found that a pathogenic relative (A. baumannii) also produced giant cells with genetic dependencies overlapping those of A. baylyi. Overall, the analysis defines a genetic pathway for giant cell formation conserved in Acinetobacter species in which independent initiating branches converge to create the unusual cells.


Assuntos
Acinetobacter/genética , Genes Essenciais/genética , Glicosiltransferases/genética , Peptidoglicano/genética , Acinetobacter/enzimologia , Antibacterianos/biossíntese , Ciclo Celular/genética , Divisão Celular/genética , Parede Celular/enzimologia , Parede Celular/genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Deleção de Genes , Genoma Bacteriano/genética , Peptidoglicano/biossíntese , Peptidil Transferases/genética , Deleção de Sequência/genética
6.
Arch Virol ; 164(8): 2159-2164, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152250

RESUMO

Canine enteric coronaviruses (CCoVs) are important enteric pathogens of dogs. CCoVs with different variations are typically pantropic and pathogenic in dogs. In this study, we isolated a CCoV, designated HLJ-073, from a dead 6-week-old male Pekingese with gross lesions and diarrhea. Interestingly, sequence analysis suggested that HLJ-073 contained a 350-nt deletion in ORF3abc compared with reference CCoV isolates, resulting in the loss of portions of ORF3a and ORF3c and the complete loss of ORF3b. Phylogenetic analysis based on the S gene showed that HLJ-073 was more closely related to members of the FCoV II cluster than to members of the CCoV I or CCoV II cluster. Furthermore, recombination analysis suggested that HLJ-073 originated from the recombination of FCoV 79-1683 and CCoV A76, which were both isolated in the United States. Cell tropism experiments suggested that HLJ-073 could effectively replicate in canine macrophages/monocytes and human THP-1 cells. This is the first report of the isolation of strain HLJ-073 in China, and this virus has biological characteristics that are different from those of other reported CCoVs.


Assuntos
Coronavirus Canino/genética , Deleção de Sequência/genética , Animais , Células Cultivadas , China , Infecções por Coronavirus/virologia , Diarreia/virologia , Doenças do Cão/virologia , Cães , Humanos , Masculino , Filogenia , Análise de Sequência de DNA/métodos , Glicoproteína da Espícula de Coronavírus/genética , Células THP-1
7.
Oncol Rep ; 41(6): 3305-3312, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30942468

RESUMO

Histone H2B monoubiquitination has been shown to play critical roles in diverse cellular processes including DNA damage response. Although recent data indicate that H2B monoubiquitination is strongly connected with tumor progression and regulation, the implications of this modification in lung adenocarcinoma are relatively unknown. In the present study, we demonstrated the clinical implication of H2B monoubiquitination and the potential role of tumor necrosis factor receptor­associated factor­interacting protein (TRAIP) in regulating its modification in lung adenocarcinoma. Immunohistochemical analysis showed that H2B monoubiquitination was significantly downregulated in 68 human lung adenocarcinoma patient samples compared to their normal adjacent tissues. Depletion of TRAIP by specific siRNA treatment markedly decreased ionizing radiation (IR)­induced H2B monoubiquitination. In addition, deletion mutants without RING domain or C­terminus of TRAIP diminished the ability to induce H2B monoubiquitination at lysine 120. Notably, the nuclear expression of TRAIP was positively related with H2B monoubiquitination levels in patients with lung adenocarcinoma. Furthermore, statistical analysis indicated that low levels of both TRAIP and H2B monoubiquitination, not each alone, in patients with lung adenocarcinoma were strongly correlated with poor survival. Taken together, these results suggest that TRAIP is a novel regulator of H2B monoubiquitination in DNA damage response and cancer development in lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/genética , Histonas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/radioterapia , Dano ao DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Lisina/genética , Masculino , Domínios Proteicos/efeitos da radiação , RNA Interferente Pequeno/genética , Radiação Ionizante , Deleção de Sequência/genética
8.
Mol Cell ; 74(5): 936-950.e5, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30975459

RESUMO

CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%-60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.


Assuntos
Sistemas CRISPR-Cas/genética , Células-Tronco Embrionárias Humanas , RNA Guia/genética , Deleção de Sequência/genética , Endonucleases/química , Endonucleases/genética , Escherichia coli/genética , Edição de Genes/métodos , Genoma Humano/genética , Genômica , Humanos , Ribonucleoproteínas/genética
9.
Lett Appl Microbiol ; 69(1): 11-15, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004518

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a wide range of hosts, including poultry, and causes acute gastroenteritis in humans that may result in death. Superoxide dismutase (SOD) is an important antioxidant enzyme present in nearly all living cells exposed to oxygen. Recently, we reported the novel roles of SOD in serum resistance and biofilm formation in S. Typhimurium. This study was designed to explore the effect of infection with sodA mutant of S. Typhimurium on the autophagic response of macrophages. Murine macrophage cell line RAW264·7 was infected with wild-type (LSM52), a sodA deletion mutant (LSM52ΔsodA) and complemented strain (LSM52CΔsodA). We found that sodA deletion triggered remarkable autophagic responses in infected cells, shown as higher concentrations of LC3-II or Beclin-1 than those infected with the wild-type or complemented strain during the first hour post-infection in S. Typhimurium. Consistent with these results, the number of viable bacteria in cells infected with the sodA mutant was significantly lower than those infected with wild-type or complemented strains at 1 h, 2 h and 3 h post-infection in S. Typhimurium. All results indicated that infection with sodA mutant of S. Typhimurium leads to up-regulation of autophagy in Raw264·7 macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Autophagy plays an important role in Salmonella infection although the role of autophagy in Salmonella infection remains unclear. This study was designed to explore the effect of sodA on the autophagic response of macrophage. We found that infection with sodA mutant of Salmonella Typhimurium could lead to up-regulation of autophagy in Raw264·7 macrophages.


Assuntos
Autofagia/imunologia , Proteínas de Bactérias/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Salmonella typhimurium/genética , Superóxido Dismutase/genética , Animais , Proteína Beclina-1/metabolismo , Linhagem Celular , Humanos , Camundongos , Células RAW 264.7 , Infecções por Salmonella , Deleção de Sequência/genética , Regulação para Cima , Virulência
10.
Genet Test Mol Biomarkers ; 23(5): 359-362, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30994363

RESUMO

Aim: The amelogenin gene is a widely used gender marker for forensic DNA profiling. Males who have the amelogenin Y (AMELY) allele deletion can be mistakenly identified as females if genotyping is performed only on the amelogenin gene. The aim of this study was to investigate the frequency of the AMELY allele deletion in the Chinese Han population and analyze the possible genetic variation on the Y chromosome. Materials and Methods: The amelogenin gene of 12,735 unrelated males from the Chinese Han population were genotyped using common forensic short tandem repeat (STR) kits. The AMELY allele deletion was verified by redesigned primers and sequencing. Eighteen Y-specific sequence tagged sites (STSs) on the Yp11.2 region were selected to delineate the deletion breakpoints on the Y chromosome. Results: Three males were confirmed to have no AMELY allele. The frequency rate of the AMELY-null allele was 0.236% (3/12,735) in the Chinese Han population of Central China; 2.73 Mb of sequence on the Y chromosome were absent in all the AMELY-negative samples. The deleted region was mapped using SRY, AMELY, 5 Y-STRs, and 18 STSs, which belong to the class I deleted pattern. The three unrelated males shared the same Y-STR haplotype with four males from other Chinese populations, all of whom have the AMELY-null allele. The haplogroup of these males was identified as the O3 haplogroup. Conclusion: The AMELY allele deletion in the Chinese population was accompanied by the deletion of the Y-STR loci on the Yp11.2 region. Therefore, another Y-specific marker should be tested simultaneously when unknown samples are examined as part of a criminal investigation.


Assuntos
Amelogenina/genética , Adulto , Alelos , Amelogenina/metabolismo , Amelogenina/fisiologia , Grupo com Ancestrais do Continente Asiático/genética , China , Cromossomos Humanos Y/genética , Impressões Digitais de DNA/métodos , Análise Mutacional de DNA/métodos , Grupos Étnicos/genética , Frequência do Gene/genética , Genótipo , Haplótipos , Humanos , Masculino , Repetições de Microssatélites , Fenótipo , Deleção de Sequência/genética , Análise para Determinação do Sexo/métodos
11.
Mol Pharmacol ; 95(5): 551-562, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30944207

RESUMO

UDP-Glucuronosyltransferase (UGT) plays an important role in the metabolism of endogenous and exogenous compounds. UGT is a type I membrane protein, and has a dilysine motif (KKXX/KXKXX) in its C-terminal cytoplasmic domain. Although a dilysine motif is defined as an endoplasmic reticulum (ER) retrieval signal, it remains a matter of debate whether this motif functions in the ER localization of UGT. To address this issue, we generated systematic deletion mutants of UGT2B7, a major human isoform, and compared their subcellular localizations with that of an ER marker protein calnexin (CNX), using subcellular fractionation and immunofluorescent microscopy. We found that although the dilysine motif functioned as the ER retention signal in a chimera that replaced the cytoplasmic domain of CD4 with that of UGT2B7, UGT2B7 truncated mutants lacking this motif extensively colocalized with CNX, indicating dilysine motif-independent ER retention of UGT2B7. Moreover, deletion of the C-terminal transmembrane and cytoplasmic domains did not affect ER localization of UGT2B7, suggesting that the signal necessary for ER retention of UGT2B7 is present in its luminal domain. Serial deletions of the luminal domain, however, did not affect the ER retention of the mutants. Further, a cytoplasmic and transmembrane domain-deleted mutant of UGT2B7 was localized to the ER without being secreted. These results suggest that UGT2B7 could localize to the ER without any retention signal, and lead to the conclusion that the static localization of UGT results from lack of a signal for export from the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Deleção de Sequência/genética , Animais , Células COS , Calnexina/metabolismo , Linhagem Celular , Cercopithecus aethiops , Citoplasma/metabolismo , Dipeptídeos/metabolismo , Humanos , Proteínas de Membrana , Células Sf9
12.
Mol Biol Rep ; 46(2): 2387-2394, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30848448

RESUMO

Chemokine CC receptor type 5 (CCR5) is a cell surface receptor that has high affinity for chemotropic cytokines called chemokines. The CCR5 gene contains a 32 base pairs (bp) deletion (CCR5Δ32). This deletion may result in a malformed and nonfunctional receptor, reported to be responsible for the development and dissemination of different cancers. CCR5Δ32 exists in two allelic forms i.e. deletion (D) and wild type (WT). This study aims to detect the role of CCR5Δ32 in breast cancer development. Blood samples were collected from breast cancer patients (330) and controls of same gender (306). Along with this histopathologically diagnosed malignant tissue samples were also excised from breast lesions of 100 patients. Genetic variations within the blood and tissue samples were examined by PCR then observed through gel electrophoresis and confirmed by direct DNA sequencing. Obtained DNA sequences were aligned and analyzed by MEGA6 software. Genotypic and association analyses were done by SPSS software version 17.0. Deletion of 32 bp in CCR5 gene has been analyzed. Genotypic variations of CCR5Δ32 are; homozygous wild type (WT/WT), heterozygous deletion (WT/D) and homozygous deletion (D/D). Statistical analyses of CCR5Δ32 data revealed that WT/D was significantly higher in blood samples of breast cancer patients (7.27% (24/330)) as compare to controls (1.30% (4/306)). In tumor tissue samples WT/WT being the most frequent genotype (99.00% (99/100)) with 1.00 (1/100) of D/D which suggested that it may be acquired. Hence, association analysis showed that CCR5Δ32 is positively associated with breast cancer in Pakistan (p < 0.001). The risk ratio of CCR5Δ32 was 5.6610 (95% confidence interval: 2.0377 to 15.7267) and odds ratio was calculated to be 6.0335 (95% confidence interval: 2.1288 to 17.0999) which signifies that deletion also increases the risk of breast cancer development. Moreover, association analyses also revealed that clinicopathological features do not have any impact on the CCR5Δ32 genotype of breast cancer. This suggests that deletion of 32 bp in CCR5 gene may be associated with breast cancer. CCR5 signals the activation and migration of immune cells at the site of tumor formation. Because of deletion; deformed CCR5 receptor might be unable to express and function properly which may subdue the immunity against cancer hence, leading to its progression.


Assuntos
Neoplasias da Mama/genética , Receptores CCR5/genética , Adulto , Idoso , Alelos , Sequência de Bases/genética , Estudos de Casos e Controles , Estudos Transversais , Feminino , Frequência do Gene/genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Heterozigoto , Humanos , Mutação INDEL/genética , Pessoa de Meia-Idade , Paquistão , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Receptores CCR5/metabolismo , Deleção de Sequência/genética
13.
Invest Ophthalmol Vis Sci ; 60(4): 858-867, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30821811

RESUMO

Purpose: Investigate the effects of the absence of 17 amino acids at the C-terminal end of Aquaporin 0 (AQP0) on lens transparency, focusing property, and homeostasis. Methods: A knockin (KI) mouse model (AQP0ΔC/ΔC) was developed to express AQP0 only as the end-cleaved form in the lens. For this, AQP0 was genetically engineered as C-terminally end-cleaved with amino acids 1 to 246, instead of the full length 1 to 263 of the wild type (WT). After verifying the KI integration into the genome and its expression, the mouse model was bred for several generations. AQP0 KI homozygous (AQP0ΔC/ΔC) and heterozygous (AQP0+/ΔC) lenses were imaged and analyzed at different developmental stages for transparency. Correspondingly, aberrations in the lens were characterized using the standard metal grid focusing method. Data were compared with age-matched WT, AQP0 knockout (AQP0-/-), and AQP0 heterozygous (AQP0+/-) lenses. Results: AQP0ΔC/ΔC lenses were transparent throughout the embryonic development and until postnatal day 15 (P15) in contrast to age-matched AQP0-/- lenses, which developed cataract at embryonic stage itself. However, there was distortion aberration in AQP0ΔC/ΔC lens at P5; after P15, cataract began to develop and progressed faster surpassing that of age-matched AQP0-/- lenses. AQP0+/ΔC lenses were transparent even at the age of 1 year in contrast to AQP0+/- lenses; however, there was distortion aberration starting at P15. Conclusions: A specific distribution profile of intact and end-cleaved AQP0 from the outer cortex to the inner nucleus is required in the lens for establishing refractive index gradient to enable proper focusing without aberrations and for maintaining transparency.


Assuntos
Sequência de Aminoácidos/genética , Aquaporinas/genética , Catarata/genética , Proteínas do Olho/genética , Cristalino/patologia , Erros de Refração/genética , Deleção de Sequência/genética , Animais , Western Blotting , Catarata/embriologia , Catarata/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Erros de Refração/embriologia , Erros de Refração/fisiopatologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção
14.
PLoS Genet ; 15(3): e1007994, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30870431

RESUMO

The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.


Assuntos
Sistemas CRISPR-Cas/genética , Divisão Celular/genética , Genoma/genética , Genômica , Animais , Ciclo Celular/genética , Edição de Genes/tendências , Terapia Genética/tendências , Genótipo , Humanos , Mamíferos , Taxa de Mutação , Deleção de Sequência/genética , Zigoto/crescimento & desenvolvimento
15.
Hum Genet ; 138(5): 509-513, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847549

RESUMO

Startle disease, or hyperekplexia, is a glycinergic disorder characterized by hypertonia and apnea that is triggered by noise and/or touch. Mutations in five genes have been associated with startle disease in humans, dogs, cattle, and mice. We identified a novel recessive startle disease in a family of Spanish greyhounds. Whole genome resequencing of an affected dog revealed a homozygous two base pair deletion in the ninth exon of SLC6A5, encoding the presynaptic glycine transporter. The deletion is predicted to cause a frameshift, p.S460FfsX47, leading to a premature stop codon that truncates over a third of the protein. Family members were genotyped for the deletion, and findings were consistent with an autosomal recessive inheritance pattern. The pathogenic variant was absent from 34 unrelated greyhounds, 659 domestic dogs of pure and mixed breeds, and 54 wild canids, suggesting it occurred recently and may be private to the family. The findings of this study can be used to inform future breeding decisions and prevent dissemination of the deleterious allele in greyhounds.


Assuntos
Doenças do Cão/genética , Mutação da Fase de Leitura/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Rigidez Muscular Espasmódica/genética , Rigidez Muscular Espasmódica/veterinária , Animais , Códon sem Sentido/genética , Modelos Animais de Doenças , Cães , Deleção de Sequência/genética , Sequenciamento Completo do Genoma
16.
Arch Virol ; 164(5): 1287-1295, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30859476

RESUMO

Since 2010, continual outbreaks of highly virulent variants of porcine epidemic diarrhea virus (PEDV) belonging to genotype GII have led to serious economic losses for the Chinese swine industry. To better understand the biological characteristics and pathogenicity of the current prevalent Chinese PEDV field strains, in this study, a highly virulent Chinese genotype GIIa PEDV strain, CH/HBXT/2018, was isolated and serially propagated using Vero cells. Sequencing and phylogenetic analysis showed that strain CH/HBXT/2018 contained novel insertion and deletion mutations in the S gene region relative to the classical strain and belonged to the genotype GIIa, similar to other recently isolated PEDV strains from China and the United States. Pig infection studies indicated that the CH/HBXT/2018 strain was highly virulent in suckling piglets, and the median pig diarrhea dose (PDD50) was 8.63 log10PDD50/3 mL at 7 days postinfection (DPI). The results of the present study are important for future PEDV challenge studies and the development of new PEDV vaccines based on prevalent field strains for the prevention and control of PED in China.


Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Animais , Linhagem Celular , Cercopithecus aethiops , China , Infecções por Coronavirus/virologia , Surtos de Doenças , Genótipo , Mutagênese Insercional/genética , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Deleção de Sequência/genética , Suínos , Doenças dos Suínos/virologia , Células Vero , Vacinas Virais/imunologia , Virulência/genética
17.
PLoS One ; 14(2): e0211864, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753226

RESUMO

Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor ß-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.


Assuntos
Sistemas CRISPR-Cas/genética , Distrofias Hereditárias da Córnea/genética , Proteínas da Matriz Extracelular/genética , Terapia Genética , Fator de Crescimento Transformador beta/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Distrofias Hereditárias da Córnea/patologia , Distrofias Hereditárias da Córnea/terapia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Extremidades/crescimento & desenvolvimento , Extremidades/patologia , Edição de Genes , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Humanos , Cultura Primária de Células , RNA Guia/genética , Deleção de Sequência/genética , Análise de Célula Única , Células-Tronco/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(9): 3853-3862, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755521

RESUMO

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.


Assuntos
Transtorno do Espectro Autista/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Dopamina/genética , Locomoção/genética , Animais , Animais Geneticamente Modificados , Transtorno do Espectro Autista/fisiopatologia , Cristalografia por Raios X , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Espectroscopia de Ressonância de Spin Eletrônica , Medo/fisiologia , Humanos , Relações Interpessoais , Locomoção/fisiologia , Modelos Moleculares , Mutação , Deleção de Sequência/genética
19.
Nat Commun ; 10(1): 759, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770810

RESUMO

Mitochondrial DNA (mtDNA) deletions are associated with mitochondrial disease, and also accumulate during normal human ageing. The mechanisms underlying mtDNA deletions remain unknown although several models have been proposed. Here we use deep sequencing to characterize abundant mtDNA deletions in patients with mutations in mitochondrial DNA replication factors, and show that these have distinct directionality and repeat characteristics. Furthermore, we recreate the deletion formation process in vitro using only purified mitochondrial proteins and defined DNA templates. Based on our in vivo and in vitro findings, we conclude that mtDNA deletion formation involves copy-choice recombination during replication of the mtDNA light strand.


Assuntos
DNA Mitocondrial/genética , Deleção de Sequência/genética , Southern Blotting , Replicação do DNA/genética , Humanos , Proteínas Mitocondriais/genética , Mutação/genética
20.
PLoS Pathog ; 15(2): e1007615, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30779794

RESUMO

Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a "Λ"-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a "Δ"-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies.


Assuntos
Proteínas do Capsídeo/ultraestrutura , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/fisiologia , Animais , Capsídeo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Genoma Viral/genética , Humanos , Camundongos , Muromegalovirus/metabolismo , Muromegalovirus/patogenicidade , Fosfoproteínas/ultraestrutura , Deleção de Sequência/genética , Proteínas da Matriz Viral/ultraestrutura , Vírion , Montagem de Vírus
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