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1.
BMC Bioinformatics ; 21(1): 474, 2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33092526

RESUMO

BACKGROUND: Identifying frequently mutated regions is a key approach to discover DNA elements influencing cancer progression. However, it is challenging to identify these burdened regions due to mutation rate heterogeneity across the genome and across different individuals. Moreover, it is known that this heterogeneity partially stems from genomic confounding factors, such as replication timing and chromatin organization. The increasing availability of cancer whole genome sequences and functional genomics data from the Encyclopedia of DNA Elements (ENCODE) may help address these issues. RESULTS: We developed a negative binomial regression-based Integrative Method for mutation Burden analysiS (NIMBus). Our approach addresses the over-dispersion of mutation count statistics by (1) using a Gamma-Poisson mixture model to capture the mutation-rate heterogeneity across different individuals and (2) estimating regional background mutation rates by regressing the varying local mutation counts against genomic features extracted from ENCODE. We applied NIMBus to whole-genome cancer sequences from the PanCancer Analysis of Whole Genomes project (PCAWG) and other cohorts. It successfully identified well-known coding and noncoding drivers, such as TP53 and the TERT promoter. To further characterize the burdening of non-coding regions, we used NIMBus to screen transcription factor binding sites in promoter regions that intersect DNase I hypersensitive sites (DHSs). This analysis identified mutational hotspots that potentially disrupt gene regulatory networks in cancer. We also compare this method to other mutation burden analysis methods. CONCLUSION: NIMBus is a powerful tool to identify mutational hotspots. The NIMBus software and results are available as an online resource at github.gersteinlab.org/nimbus.


Assuntos
Análise Mutacional de DNA/métodos , Mutação/genética , Software , Calibragem , Simulação por Computador , Doença/genética , Genoma Humano , Humanos , Anotação de Sequência Molecular , Taxa de Mutação , Neoplasias/genética , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas , Análise de Regressão , Sequenciamento Completo do Genoma
2.
Cancer Treat Rev ; 89: 102084, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32738738

RESUMO

Accurate identification of patients with solid tumors likely to respond to immunotherapy is crucial. Tumor mutational burden (TMB) measures the number of somatic mutations in a tumor and is an emerging prognostic and predictive biomarker for anti-programmed cell death (PD) 1/anti-PD-ligand 1 therapy and other immunotherapeutic agents. Tumor mutational burden is assessed optimally by whole exome sequencing, but next generation sequencing provides TMB estimates in a more timely and cost-effective manner. Blood-based measurement of TMB in plasma offers an alternative to the need for adequate tumor tissue for molecular testing, and has demonstrated the ability to identify patients who derive benefit from immunotherapy. Tumor mutational burden has diverse prognostic impact in different solid tumor types and also has a demonstrated role in predicting improved survival in patients receiving immunotherapy. There are challenges to TMB adoption into standard clinical practice, including variations in its definition, with the mutational number defining TMB-high appearing to vary across cancer types. The magnitude of TMB also varies across different tumor types, with the highest levels reported in melanoma and other skin cancers (where ultraviolet light is the dominant mutational process), followed by non-small cell lung cancer and other squamous carcinomas. Concerns regarding inter-laboratory and inter-platform variations in analysis methods have been raised, highlighting the need for standardization. Integration of other genomic or pathological biomarkers with TMB may increase its prognostic and predictive capabilities and validation of individual or combination models in prospective trials is warranted.


Assuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA/métodos , Neoplasias/genética , Neoplasias/terapia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase III como Assunto , Testes Genéticos , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Ensaios Clínicos Controlados Aleatórios como Assunto
3.
J Transl Med ; 18(1): 321, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831104

RESUMO

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Assuntos
Betacoronavirus/fisiologia , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/genética , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Betacoronavirus/metabolismo , Sítios de Ligação/genética , Infecções por Coronavirus/etnologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Predisposição Genética para Doença/etnologia , Variação Genética , Geografia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etnologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
4.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831310

RESUMO

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics. The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.


Assuntos
Análise Mutacional de DNA/métodos , DNA/genética , Mutação Puntual/genética , Colorimetria , Sondas de DNA/metabolismo , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Espectrofotometria Ultravioleta
5.
Brain Tumor Pathol ; 37(4): 154-158, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32749624

RESUMO

Telomerase reverse transcriptase promoter (TERTp) hotspot mutations are the most frequent mutations in primary glioblastomas (GBM). Previous studies have shown that the combination of TERTp and isocitrate dehydrogenase (IDH) status may serve as a useful diagnostic marker for oligodendroglioma and glioblastoma. In oligodendrogliomas, TERTp and IDH mutations, along with the 1p/19q codeletion, usually coexist and are likely to be founder mutations. However, in contrast to oligodendroglioma, the role of the TERTp status in GBM remains obscure. Here, we used Sanger sequencing, pyrosequencing, and digital PCR (dPCR) to examine the TERTp status in 15 pairs of frozen tissue samples from primary and recurrent IDH wild-type GBM, all of which were operated in a single institute. We showed that the TERTp status was stable between primary and recurrent GBM but this consistency was only detected by dPCR. The results suggest that dPCR is a powerful, highly sensitive tool to detect TERTp mutations, especially in a mixed cell population (e.g., a recurrent GBM tissue) where earlier treatment may have grossly altered the tumor microenvironment.


Assuntos
Neoplasias Encefálicas/genética , Análise Mutacional de DNA/métodos , Glioblastoma/genética , Mutação , Recidiva Local de Neoplasia/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Telomerase/genética , Humanos , Isocitrato Desidrogenase/genética , Sensibilidade e Especificidade
6.
BMC Infect Dis ; 20(1): 518, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677920

RESUMO

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.


Assuntos
Infecções por Campylobacter/diagnóstico , Campylobacter jejuni/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/uso terapêutico , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Substituição de Aminoácidos , Infecções por Campylobacter/tratamento farmacológico , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Criança , Ciprofloxacino/uso terapêutico , Análise Mutacional de DNA/métodos , Diarreia/diagnóstico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Humanos , Isoleucina/genética , Testes de Sensibilidade Microbiana , Peru , Treonina/genética
7.
Eur J Endocrinol ; 183(4): 369-379, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32621582

RESUMO

Background: The '3PAs' syndrome, associating pituitary adenoma (PA) and pheochromocytoma/paraganglioma (PPGL), is sometimes associated with mutations in PPGL-predisposing genes, such as SDHx or MAX. In '3PAs' syndrome, PAs can occur before PPGL, suggesting a new gateway into SDHx/MAX-related diseases. Objective: To determine the SDHx/MAX mutation prevalence in patients with isolated PAs and characterize PAs of patients with SDHx/MAX mutations. Design: Genes involved in PAs (AIP/MEN1/CDKN1B) or PPGLs (SDHx/MAX) were sequenced in patients with isolated PAs. We then conducted a review of cases of PA in the setting of '3PAs' syndrome. Results: A total of 263 patients were recruited. Seven (likely) pathogenic variants were found in AIP, two in MEN1, two in SDHA, and one in SDHC. The prevalence of SDHx mutations reached 1.1% (3/263). Of 31 reported patients with PAs harboring SDHx/MAX mutations (28 published cases and 3 cases reported here), 6/31 (19%) developed PA before PPGL and 8/31 (26%) had isolated PA. The age of onset was later than in patients with AIP/MEN1 mutations. PAs were mainly macroprolactinomas and showed intracytoplasmic vacuoles seen on histopathology. Conclusions: We discovered SDHx mutations in patients bearing PA who had no familial or personal history of PPGL. However, the question of incidental association remains unresolved and data to determine the benefit of SDHx/MAX screening in these patients are lacking. We recommend that patients with isolated PA should be carefully examined for a family history of PPGLs. A family history of PPGL, as well as the presence of intracytoplasmic vacuoles in PA, requires SDHx/MAX genetic testing of patients.


Assuntos
Adenoma/genética , Mutação em Linhagem Germinativa , Neoplasias Hipofisárias/genética , Succinato Desidrogenase/genética , Adenoma/epidemiologia , Adenoma/patologia , Adolescente , Neoplasias das Glândulas Suprarrenais/epidemiologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idade de Início , Idoso , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Criança , Análise Mutacional de DNA/métodos , Feminino , França/epidemiologia , Predisposição Genética para Doença , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Paraganglioma/epidemiologia , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/epidemiologia , Feocromocitoma/genética , Feocromocitoma/patologia , Neoplasias Hipofisárias/epidemiologia , Neoplasias Hipofisárias/patologia , Prolactinoma/epidemiologia , Prolactinoma/genética , Prolactinoma/patologia , Subunidades Proteicas/genética , Estudos Retrospectivos , Adulto Jovem
8.
Nat Commun ; 11(1): 3166, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576827

RESUMO

Mutational processes acting on cancer genomes can be traced by investigating mutational signatures. Because high sequencing costs limit current studies to small numbers of good-quality samples, we propose a robust, cost- and time-effective method, called mutREAD, to detect mutational signatures from small quantities of DNA, including degraded samples. We show that mutREAD recapitulates mutational signatures identified by whole genome sequencing, and will ultimately allow the study of mutational signatures in larger cohorts and, by compatibility with formalin-fixed paraffin-embedded samples, in clinical settings.


Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/isolamento & purificação , Testes Genéticos/métodos , Mutação , Neoplasias/genética , Biologia Computacional , Primers do DNA , Genes Neoplásicos/genética , Genoma Humano , Humanos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma
9.
PLoS One ; 15(6): e0233900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520976

RESUMO

OBJECTIVES: To identify differences in the mutational profile of endometrial tumours between British White (BW) and South Asian (BSA) women. METHODS: We analysed primary tumours from matched cohorts of British White (BW) and British South Asian (BSA) women resident in Leicestershire diagnosed with EC. Next Generation Sequencing was performed to investigate mutational differences in a panel of 10 genes previously identified as being commonly mutated in EC. The presence of somatic Mismatch Repair (MMR) gene deficiencies was determined by immunohistochemistry. RESULTS: In total, 57 tumours (27 BSA and 30 BW) were sequenced. There was no significant difference in the overall mutation frequency of the 10 genes analysed; however, numerous differences were observed between the groups. There was a positive association between PIK3CA and PTEN mutations in the BSA group, with 78% of PIK3CA-mutant tumours harbouring a PTEN mutation, whereas only 11% of PIK3CA wild-type (wt) tumours were PTEN mutant positive (p = 0.0012). In BW women, 90% of ARID1A mutant tumours had co-existent PI3K pathway mutations versus 50% of wild-type (wt) ARID1A patients (p = 0.0485). This trend was not significant in the BSA group (p = 0.66). The age at diagnosis was significantly higher in the BW group with a somatic MMR gene deficiency compared to those with no deficiency (72.8 years versus 59.6 years, p = 0.007), whereas this difference was not seen in the BSA group (64 years versus 60 years, p = 0.37). CONCLUSION: We have identified differences in the mutational profile of primary EC tumours from BW and BSA women. Further research is needed to confirm these findings and to explore their potential implications for early detection, treatment response and prognosis.


Assuntos
Carcinoma Endometrioide/genética , Enzimas Reparadoras do DNA/genética , DNA de Neoplasias/genética , Neoplasias do Endométrio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Estudos de Coortes , Análise Mutacional de DNA/métodos , Grupo com Ancestrais do Continente Europeu , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pessoa de Meia-Idade , Mutação , Reino Unido
10.
Jpn J Clin Oncol ; 50(9): 1037-1042, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32533146

RESUMO

BACKGROUND: An accurate diagnosis is crucial to determine the treatment modality for desmoid-type fibromatosis, although the histopathological diagnosis is occasionally difficult to make. Many desmoid-type fibromatosis have been reported to have hotspot mutation of ß-catenin gene (CTNNB1). In the present study, we performed a systematic review to verify the usefulness of CTNNB1 mutation analysis in the diagnosis of desmoid-type fibromatosis. METHODS: A literature search from January 1990 to August 2017 was conducted. Three reviewers independently assessed and screened the literature for eligibility and determined the final articles to be evaluated. Data regarding the sensitivity, specificity, accuracy and usefulness of CTNNB1 mutation analysis in the diagnosis of desmoid-type fibromatosis were recorded. We rated each report according to the Grading of Recommendations Development and Evaluation approach. RESULTS: The search yielded 90 studies, seven of which were included after the first and second screenings. The positive rate of CTNNB1 mutation in desmoid-type fibromatosis was 86.8%, but the cohort of six of the seven reports was already diagnosed histopathologically as desmoid-type fibromatosis. Therefore, the usefulness of CTNNB1 mutation analysis in a cohort that is difficult to diagnose histopathologically is not clear in this review. Nevertheless, CTNNB1 mutation showed very high specificity in desmoid-type fibromatosis, indicating the usefulness of CTNNB1 mutation analysis in its diagnosis in combination with histological examination. CONCLUSION: Because the lack of data precludes any useful comparison with histological diagnosis, the evidence level is low. However, considering its specificity, CTNNB1 mutation analysis may be useful in cases in which the histopathological diagnosis is difficult.


Assuntos
Análise Mutacional de DNA/métodos , Fibromatose Agressiva/diagnóstico , beta Catenina/genética , Humanos
11.
Mutat Res ; 853: 503195, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522347

RESUMO

Recent years have witnessed an expansion of mutagenesis research focusing on experimentally modeled genome-scale mutational signatures of carcinogens and of endogenous processes. Experimental mutational signatures can explain etiologic links to patterns found in human tumors that may be linked to same exposures, and can serve as biomarkers of exposure history and may even provide insights on causality. A number of innovative exposure models have been employed and reported, based on cells cultured in monolayers or in 3-D, on organoids, induced pluripotent stem cells, non-mammalian organisms, microorganisms and rodent bioassays. Here we discuss some of the latest developments and pros and cons of these experimental systems used in mutational signature analysis. Integrative designs that bring together multiple exposure systems (in vitro, in vivo and in silico pan-cancer data mining) started emerging as powerful tools to identify robust mutational signatures of the tested cancer risk agents. We further propose that devising a new generation of cell-based models is warranted to streamline systematic testing of carcinogen effects on the cell genomes, while seeking to increasingly supplant animal with non-animal systems to address relevant ethical issues and accentuate the 3R principles. We conclude that the knowledge accumulating from the growing body of signature modelling investigations has considerable power to advance cancer etiology studies and to support cancer prevention efforts through streamlined characterization of cancer-causing agents and the recognition of their specific effects.


Assuntos
Carcinógenos/toxicidade , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Neoplasias/induzido quimicamente , Animais , Análise Mutacional de DNA/métodos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Mutagênese/genética , Mutação/genética
12.
Nat Commun ; 11(1): 2493, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427826

RESUMO

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.


Assuntos
Células-Tronco Adultas/metabolismo , Análise Mutacional de DNA/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Adulto , Células-Tronco Adultas/citologia , Algoritmos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Intestinos/citologia , Fígado/citologia , Fígado/metabolismo , Modelos Genéticos , Acúmulo de Mutações , Taxa de Mutação , Medicina Regenerativa/métodos , Sequenciamento Completo do Genoma/métodos
13.
PLoS Genet ; 16(5): e1008743, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463822

RESUMO

Metastasis remains the principle cause of mortality for breast cancer and presents a critical challenge because secondary lesions are often refractory to conventional treatments. While specific genetic alterations are tightly linked to primary tumor development and progression, the role of genetic alteration in the metastatic process is not well-understood. The theory of tumor evolution postulated by Peter Nowell in 1976 has yet to be proven in the context of metastasis. Therefore, in order to investigate how somatic evolution contributes to breast cancer metastasis, we performed exome, whole genome, and RNA sequencing of matched metastatic and primary tumors from pre-clinical mouse models of breast cancer. Here we show that in a treatment-naïve setting, recurrent single nucleotide variants and copy number variation, but not gene fusion events, play key metastasis-driving roles in breast cancer. For instance, we identified recurrent mutations in Kras, a known driver of colorectal and lung tumorigenesis that has not been previously implicated in breast cancer metastasis. However, in a set of in vivo proof-of-concept experiments we show that the Kras G12D mutation is sufficient to significantly promote metastasis using three syngeneic allograft models. The work herein confirms the existence of metastasis-driving mutations and presents a novel framework to identify actionable metastasis-targeted therapies.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genômica/métodos , Mutação , Animais , Células Cultivadas , Evolução Clonal/genética , Análise Mutacional de DNA/métodos , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células HEK293 , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Modelos Biológicos , Metástase Neoplásica , Sequenciamento Completo do Exoma
14.
Yakugaku Zasshi ; 140(5): 657-661, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378667

RESUMO

The development of specialized training programs for medical personnel, particularly nurses, clinical laboratory technicians, and pharmacists, is considered critical for the promotion of genomic medicine throughout Japan. Specifically, medical personnel skilled at analyzing and understanding high-throughput genomic data are in high demand. In this symposium, we will introduce the basic knowledge and skills necessary for processing genomic data.


Assuntos
Ciência de Dados/educação , Terapia Genética/métodos , Genoma Humano , Genômica , Corpo Clínico/educação , Neoplasias/genética , Neoplasias/terapia , Equipe de Assistência ao Paciente , Competência Clínica , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Japão , Mutação
15.
J Mycol Med ; 30(2): 100953, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32362445

RESUMO

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.


Assuntos
Aspergilose/microbiologia , Aspergillus flavus/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Voriconazol/uso terapêutico , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/genética , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/isolamento & purificação , Biomarcadores/análise , Sistema Enzimático do Citocromo P-450/genética , Análise Mutacional de DNA/métodos , Regulação Fúngica da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Mutação Puntual , Esterol 14-Desmetilase/genética
17.
AJNR Am J Neuroradiol ; 41(5): 874-881, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32381545

RESUMO

BACKGROUND AND PURPOSE: Recent advances in molecular techniques have characterized distinct subtypes of diffuse intrinsic pontine gliomas. Our aim was the identification of MR imaging correlates of these subtypes. MATERIALS AND METHODS: Initial MRIs from subjects with diffuse intrinsic pontine gliomas recruited for a prospective clinical trial before treatment were analyzed. Retrospective imaging analyses included FLAIR/T2 tumor volume, tumor volume enhancing, the presence of cyst and/or necrosis, median, mean, mode, skewness, kurtosis of ADC tumor volume based on FLAIR, and enhancement at baseline. Molecular subgroups based on EGFR and MGMT mutations were established. Histone mutations were also determined (H3F3A, HIST1H3B, HIST1H3C). Univariate Cox proportional hazards regression was used to test the association of imaging predictors with overall and progression-free survival. Wilcoxon rank sum, Kruskal-Wallis, and Fisher exact tests were used to compare imaging measures among groups. RESULTS: Fifty patients had biopsy and MR imaging. The median age at trial registration was 6 years (range, 3.3-17.5 years); 52% were female. On the basis of immunohistochemical results, 48 patients were assigned to 1 of 4 subgroups: 28 in MGMT-/epidermal growth factor receptor (EGFR)-, 14 in MGMT-/EGFR+, 3 in MGMT+/EGFR-, and 3 in MGMT+/EGFR+. Twenty-three patients had histone mutations in H3F3A, 8 in HIST1H3B, and 3 in HIST1H3C. Enhancing tumor volume was near-significantly different across molecular subgroups (P = .04), after accounting for the false discovery rate. Tumor volume enhancing, median, mode, skewness, and kurtosis ADC T2-FLAIR/T2 were significantly different (P ≤ .048) between patients with H3F3A and HIST1H3B/C mutations. CONCLUSIONS: MR imaging features including enhancement and ADC histogram parameters are correlated with molecular subgroups and mutations in children with diffuse intrinsic pontine gliomas.


Assuntos
Neoplasias do Tronco Encefálico/diagnóstico por imagem , Neoplasias do Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/diagnóstico por imagem , Glioma Pontino Intrínseco Difuso/genética , Neuroimagem/métodos , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Receptores ErbB/genética , Feminino , Histonas/genética , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Mutação , Estudos Prospectivos , Estudos Retrospectivos
18.
Best Pract Res Clin Endocrinol Metab ; 34(2): 101418, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32414619

RESUMO

Cushing syndrome (CS) describes the signs and symptoms caused by exogenous or endogenous hypercortisolemia. Endogenous CS is caused by either ACTH-dependent sources (pituitary or ectopic) or ACTH-independent (adrenal) hypercortisolemia. Several genes are currently known to contribute to the pathogenesis of CS. Germline gene defects, such as MEN1, AIP, PRKAR1A and others, often present in patients with pituitary or adrenal involvement as part of a genetic syndrome. Somatic defects in genes, such as USP8, TP53, and others, are also involved in the development of pituitary or adrenal tumors in a large percentage of patients with CS, and give insight in pathways involved in pituitary or adrenal tumorigenesis.


Assuntos
Síndrome de Cushing/genética , Análise Mutacional de DNA , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/epidemiologia , Neoplasias das Glândulas Suprarrenais/genética , Carcinogênese/genética , Carcinogênese/patologia , Síndrome de Cushing/epidemiologia , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/tendências , Estudos de Associação Genética/métodos , Estudos de Associação Genética/tendências , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/fisiologia , Humanos , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/epidemiologia , Neoplasias Hipofisárias/genética , Transdução de Sinais/genética
19.
Genomics Proteomics Bioinformatics ; 18(1): 81-90, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32428603

RESUMO

Accurate detection of low frequency mutations from plasma cell-free DNA in blood using targeted next generation sequencing technology has shown promising benefits in clinical settings. Duplex sequencing technology is the most commonly used approach in liquid biopsies. Unique molecular identifiers are attached to each double-stranded DNA template, followed by production of low-error consensus sequences to detect low frequency variants. However, high sequencing costs have hindered application of this approach in clinical practice. Here, we have developed an improved duplex sequencing approach called SinoDuplex, which utilizes a pool of adapters containing pre-defined barcode sequences to generate far fewer barcode combinations than with random sequences, and implemented a novel computational analysis algorithm to generate duplex consensus sequences more precisely. SinoDuplex increased the output of duplex sequencing technology, making it more cost-effective. We evaluated our approach using reference standard samples and cell-free DNA samples from lung cancer patients. Our results showed that SinoDuplex has high sensitivity and specificity in detecting very low allele frequency mutations. The source code for SinoDuplex is freely available at https://github.com/SinOncology/sinoduplex.


Assuntos
Ácidos Nucleicos Livres/sangue , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA/métodos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Software , Algoritmos , Linhagem Celular , Humanos , Mutação , Sensibilidade e Especificidade
20.
Cancer Sci ; 111(6): 2174-2182, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32314446

RESUMO

Formalin-fixed paraffin-embedded (FFPE) tissues used for routine pathological diagnosis are valuable for cancer genomic analysis; however, the association between mutation status derived from these specimens and prognosis in pancreatic ductal adenocarcinoma (PDAC) remains unclear. We analyzed 50 cancer-related gene mutations including driver genes in PDAC, using next-generation sequencing (NGS) to clarify the association between gene mutations and prognosis. DNA was extracted from FFPE tissues obtained from 74 patients with untreated resectable PDAC who underwent surgery at our institution between 2013 and 2018. Fifty of the 74 patients with DNA extracts from FFPE samples suitable for NGS were analyzed. The prevalence of driver gene mutations was as follows: 84% for KRAS, 62% for TP53, 32% for SMAD4, and 18% for CDKN2A. There were no cases of single SMAD4 mutations; its rate of coincidence with KRAS or TP53 mutations was 30% and 2%, respectively. The combination of KRAS and SMAD4 mutations resulted in significantly shorter relapse-free survival (RFS; median survival time [MST], 12.3 vs. 28.9 months, P = .014) and overall survival (OS; MST, 22.3 months vs. not reached, P = .048). On multivariate analysis, the combination of KRAS and SMAD4 mutations was an independent prognostic factor for RFS (hazard ratio [HR] 4.218; 95% confidence interval [CI], 1.77-10.08; P = .001) and OS (HR 6.730; 95% CI, 1.93-23.43; P = .003). The combination of KRAS and SMAD4 mutations in DNA obtained from FFPE tissues is an independent poor prognostic factor in PDAC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Smad4/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/mortalidade , Análise Mutacional de DNA/métodos , Feminino , Formaldeído , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias Pancreáticas/mortalidade , Inclusão em Parafina , Prognóstico , Fixação de Tecidos
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