RESUMO
PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Neoplasias Renais/metabolismo , NF-kappa B/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Humanos , ImunomodulaçãoRESUMO
Aim: The clinical utility of pharmacogenomics (PGx) has been gaining traction alongside growing evidence that adverse drug reactions (ADRs) have significant genetic associations. Nala PGx Core® is a multi-gene qPCR-based panel of 20 allele variants, comprising 18 SNPs and two CYP2D6 copy number markers across four pharmacogenes - CYP2C9, CYP2C19, CYP2D6 and SLCO1B1. Methods: In this study, we validated the performance of Nala PGx Core® against benchmark methods, on the Singaporean and Indonesian populations. Results & conclusion: Nala PGx Core® demonstrated robust and accurate genotyping when compared with other established benchmarks. Furthermore, the panel successfully characterized alleles of clinical relevance, such as CYP2D6*10 and CYP2D6*36, across major ethnic groups present of Singapore and Indonesia, suggesting its potential for adoption in clinical workflows regionally.
Assuntos
Farmacogenética/métodos , Reação em Cadeia da Polimerase/normas , Algoritmos , Povo Asiático , Benchmarking , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Etnicidade , Dosagem de Genes , Genótipo , Humanos , Indonésia , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , SingapuraRESUMO
SARS-CoV-2 is a major threat to global health. Here, we investigate the RNA structure and RNA-RNA interactions of wildtype (WT) and a mutant (Δ382) SARS-CoV-2 in cells using Illumina and Nanopore platforms. We identify twelve potentially functional structural elements within the SARS-CoV-2 genome, observe that subgenomic RNAs can form different structures, and that WT and Δ382 virus genomes fold differently. Proximity ligation sequencing identify hundreds of RNA-RNA interactions within the virus genome and between the virus and host RNAs. SARS-CoV-2 genome binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2'-O-methylated. 2'-O-methylation sites are enriched in viral untranslated regions, associated with increased virus pair-wise interactions, and are decreased in host mRNAs upon virus infection, suggesting that the virus sequesters methylation machinery from host RNAs towards its genome. These studies deepen our understanding of the molecular and cellular basis of SARS-CoV-2 pathogenicity and provide a platform for targeted therapy.
Assuntos
COVID-19/virologia , Interações entre Hospedeiro e Microrganismos , RNA Viral/metabolismo , RNA/metabolismo , SARS-CoV-2/fisiologia , COVID-19/genética , COVID-19/metabolismo , COVID-19/fisiopatologia , Metilação de DNA , Genoma Viral , Humanos , Conformação de Ácido Nucleico , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/genéticaRESUMO
Advanced, late-stage Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) is incurable, and its treatment remains a clinical and therapeutic challenge. Results from a phase II clinical trial in advanced NPC patients employing a combined chemotherapy and EBV-specific T cell (EBVST) immunotherapy regimen showed a response rate of 71.4%. Longitudinal analysis of patient samples showed that an increase in EBV DNA plasma concentrations and the peripheral monocyte-to-lymphocyte ratio negatively correlated with overall survival. These parameters were combined into a multivariate analysis to stratify patients according to risk of death. Immunophenotyping at serial time points showed that low-risk individuals displayed significantly decreased amounts of monocytic myeloid-derived suppressor cells postchemotherapy, which subsequently influenced successful cytotoxic T-lymphocyte (CTL) immunotherapy. Examination of the low-risk group, 2 weeks post-EBVST infusion, showed that individuals with a greater overall survival possessed an increased frequency of CD8 central and effector memory T cells, together with higher levels of plasma interferon (IFN)-γ, and cytotoxic lymphocyte-associated transcripts. These results highlight the importance of the rational selection of chemotherapeutic agents and consideration of their impact on both systemic immune responses and downstream cellular immunotherapy outcomes.
Assuntos
Imunoterapia Adotiva , Células Supressoras Mieloides/imunologia , Carcinoma Nasofaríngeo/imunologia , Carcinoma Nasofaríngeo/terapia , Linfócitos T/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Imunoterapia Adotiva/métodos , Células Supressoras Mieloides/metabolismo , Carcinoma Nasofaríngeo/patologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Falha de Tratamento , Resultado do TratamentoRESUMO
OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.
Assuntos
Anormalidades Múltiplas/genética , Deficiências do Desenvolvimento/genética , Sequenciamento de Nucleotídeos em Larga Escala , Doenças não Diagnosticadas/genética , Anormalidades Múltiplas/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Feminino , Humanos , Lactente , Masculino , Singapura , Doenças não Diagnosticadas/diagnóstico , Adulto JovemRESUMO
Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability.
Assuntos
Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Genoma Humano , Células-Tronco Mesenquimais/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Células Clonais , Genótipo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Homozigoto , Humanos , Células-Tronco Mesenquimais/metabolismo , Transcriptoma/genéticaRESUMO
Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR-Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue.
Assuntos
Antifúngicos/isolamento & purificação , Macrolídeos/isolamento & purificação , Streptomyces/genética , Antifúngicos/metabolismo , Clonagem Molecular , Macrolídeos/metabolismo , Família Multigênica , Streptomyces/metabolismoRESUMO
Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.
Assuntos
Cromossomos Bacterianos , Genes Bacterianos , Metabolismo Secundário/genética , Streptomyces/genética , Sequência de Bases , Mapeamento Cromossômico , Família Multigênica , Plasmídeos/genéticaRESUMO
BACKGROUND: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. RESULTS: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. CONCLUSIONS: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.
Assuntos
Antifúngicos/química , Depsipeptídeos/química , Proteínas Fúngicas/genética , Saccharomycetales/genética , Sequência de Aminoácidos , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Vias Biossintéticas , Depsipeptídeos/biossíntese , Depsipeptídeos/farmacologia , Genômica , Estrutura Molecular , Família Multigênica , Saccharomycetales/metabolismo , Sequenciamento Completo do GenomaRESUMO
RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms.
Assuntos
Aptâmeros de Nucleotídeos/genética , Bacillus subtilis/genética , Candida albicans/genética , Mononucleotídeo de Flavina/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Pseudomonas aeruginosa/genética , Saccharomyces cerevisiae/genética , Aptâmeros de Nucleotídeos/química , Genoma Bacteriano/genética , Genoma Fúngico/genética , RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in â¼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Cromossomos Humanos Par 1 , Recidiva Local de Neoplasia/genética , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Ácidos Nucleicos Livres/genética , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Reação em Cadeia da Polimerase , Pirimidinas/uso terapêutico , Resultado do TratamentoRESUMO
Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivoVHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1ß heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression.Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305. ©2017 AACR.See related commentary by Ricketts and Linehan, p. 1221This article is highlighted in the In This Issue feature, p. 1201.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Fatores de Transcrição de p300-CBP/genética , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Cromatina , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Oncogenes/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a "phenotype-driven precision-oncology" approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of "screenable" patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive "-omics" interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Medicina de Precisão/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos Endogâmicos NOD , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fenótipo , Fosfoproteínas/genética , Quinazolinas/farmacologia , Fatores de Transcrição , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.
Assuntos
Biomarcadores Tumorais , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.
Assuntos
Cromossomos Bacterianos/genética , Cromossomos Bacterianos/ultraestrutura , DNA Bacteriano/metabolismo , Plasmídeos/genética , Streptomyces/genética , Sequência de Aminoácidos , Replicação do DNA , Dados de Sequência Molecular , Mutação , Recombinação Genética , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Telômero/genéticaRESUMO
Murine double minute 2 (MDM2) is a negative regulator of p53. A single-nucleotide polymorphism (SNP) (rs2279744: c.309T>G) in the promoter region of the MDM2 gene has been shown to result in higher levels of MDM2 RNA and protein. Regarding the contribution of c.309T>G in the MDM2 gene to the lung cancer risk, previous studies are conflicting. In order to evaluate the association between c.309T>G and the lung cancer risk, a case-control study was performed. The MDM2 genotypes were determined in 762 lung cancer patients and in 700 cancer-free control subjects using the Smart Amplification Process. Statistical adjustment was performed for gender, age and pack-years of smoking. The distributions of c.309T>G (T/T, T/G, G/G) were 20.1, 49.7, 30.2% in the case group and 21.7, 47.9, 30.4% in the healthy-control group. There were no overall associations between the MDM2 genotypes and the risk of lung cancer [T/G genotype: Adjusted odds ratio (AOR), 1.30; 95% confidence interval (CI), 0.88-1.93; and G/G genotype: AOR, 1.18; 95% CI, 0.78-1.80]. The subgroup analysis of gender, histology, smoking status and epidermal growth factor receptor mutation status also indicated that there was no association with lung cancer. Additionally, the genotypes did not have an effect on the age at the time of diagnosis of lung cancer (P=0.25). In conclusion, the G allele frequency in the lung cancer cases was 0.551, which was similar to other studies. The results of the present study suggest that the c.309T>G is not significantly associated with lung cancer.
RESUMO
PURPOSE: The transcription factor NRF2 plays a pivotal role in protecting normal cells from external toxic challenges and oxidative stress, whereas it can also endow cancer cells resistance to anticancer drugs. At present little information is available about the genetic polymorphisms of the NRF2 gene and their clinical relevance. We aimed to investigate the single nucleotide polymorphisms in the NRF2 gene as a prognostic biomarker in lung cancer. EXPERIMENTAL DESIGN: We prepared genomic DNA samples from 387 Japanese patients with primary lung cancer and detected SNP (c.-617C>A; rs6721961) in the ARE-like loci of the human NRF2 gene by the rapid genetic testing method we developed in this study. We then analyzed the association between the SNP in the NRF2 gene and patients' overall survival. RESULTS: Patients harboring wild-type (WT) homozygous (c.-617C/C), SNP heterozygous (c.-617C/A), and SNP homozygous (c.-617A/A) alleles numbered 216 (55.8%), 147 (38.0%), and 24 (6.2%), respectively. Multivariate logistic regression models revealed that SNP homozygote (c.-617A/A) was significantly related to gender. Its frequency was four-fold higher in female patients than in males (10.8% female vs 2.7% male) and was associated with female non-smokers with adenocarcinoma. Interestingly, lung cancer patients carrying NRF2 SNP homozygous alleles (c.-617A/A) and the 309T (WT) allele in the MDM2 gene exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021). CONCLUSION: SNP homozygous (c.-617A/A) alleles in the NRF2 gene are associated with female non-smokers with adenocarcinoma and regarded as a prognostic biomarker for assessing overall survival of patients with lung adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/genética , Fator 2 Relacionado a NF-E2/genética , Polimorfismo de Nucleotídeo Único , Adenocarcinoma/diagnóstico , Adenocarcinoma/etnologia , Alelos , Povo Asiático/genética , Feminino , Frequência do Gene , Testes Genéticos/métodos , Genótipo , Humanos , Japão , Estimativa de Kaplan-Meier , Modelos Logísticos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/etnologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Proteínas Proto-Oncogênicas c-mdm2/genética , Fatores Sexuais , FumarRESUMO
BACKGROUND: Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. METHODOLOGY: In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named "Duplex SmartAmp," which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. RESULTS AND CONCLUSIONS: By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.