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Hypertension treatment and blood pressure (BP) control reduce cardiovascular disease burden. However, prevalence of controlled BP is overall insufficient and lack of adherence to treatment is a suggested major contributor. This prospective, randomized clinical trial was designed to evaluate whether a specific 3-month (m) action plan to improve therapeutic adherence results in a decrease in BP. Patients with ambulatory 24 h-BP ≥ 130/80 mmHg despite receiving ≥2 antihypertensive drugs and with therapeutic non-compliance confirmed by antihypertensive drugs analyzed in urine were randomized (1:1) to receive a specific 3 m program to improve adherence (INT = intervention) or routine follow-up (C = control). Antihypertensive treatment was not modified and knowledge of non-adherence was only notified to patients randomized to the intervention group. Before randomization and at 3 m all patients underwent urinary screening for antihypertensive drugs and 24 h-ambulatory-BP monitoring. Forty-five patients (36% women, mean age: 58 ± 13 yr) were randomized. At 3 m, mean (95% CI) BP differences (INT vs. C) were 12.2 mmHg (4.3-20.8), adjusted-p = 0.032 and 8.7 mmHg (2.5-14.8), adjusted-p = 0.018 for 24 h-systolic and 24 h-diastolic BP, respectively. Differences (INT vs. C) for office SBP and DBP were 18.4 mmHg (6.8-30.1), adjusted-p = 0.005 and 15.7 mmHg (7.2-24.2), adjusted-p < 0.001. Non-detected antihypertensive drugs were median [IQR]: 40% [25-100] and 0% [0-20] at baseline and 3 m, respectively, in the INT group, and 33.3% [25-63.7] and 33.3% [23.8-57.9], in the C group (p < 0.001 for the 3-month between-group comparison). A combined action plan of notifying knowledge of non-adherence plus a 3-month specific nursing intervention to improve therapeutic adherence results in BP reduction in patients with inadequate therapeutic compliance.
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Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.
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Inversão Cromossômica , Doenças Raras , Humanos , Doenças Raras/genética , Masculino , Feminino , Inversão Cromossômica/genética , Linhagem , Genoma Humano , Sequenciamento Completo do Genoma , Proteína 2 de Ligação a Metil-CpG/genética , Mutação , Proteínas de Homeodomínio/genética , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.
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Variação Genética , Doenças Raras , Humanos , Doenças Raras/diagnóstico , Doenças Raras/genética , Sequenciamento Completo do Genoma , Testes Genéticos , Mutação , Proteínas de Ciclo CelularRESUMO
BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.
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Bronquiectasia , Transtornos da Motilidade Ciliar , Ciliopatias , Síndrome de Kartagener , Humanos , Mutação , Bronquiectasia/diagnóstico , Bronquiectasia/genética , Cílios , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/genética , Ciliopatias/complicações , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genéticaRESUMO
The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor's transcriptional activity. FOXN1's C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.
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PURPOSE: The translation of genome sequencing into routine health care has been slow, partly because of concerns about affordability. The aspirational cost of sequencing a genome is $1000, but there is little evidence to support this estimate. We estimate the cost of using genome sequencing in routine clinical care in patients with cancer or rare diseases. METHODS: We performed a microcosting study of Illumina-based genome sequencing in a UK National Health Service laboratory processing 399 samples/year. Cost data were collected for all steps in the sequencing pathway, including bioinformatics analysis and reporting of results. Sensitivity analysis identified key cost drivers. RESULTS: Genome sequencing costs £6841 per cancer case (comprising matched tumor and germline samples) and £7050 per rare disease case (three samples). The consumables used during sequencing are the most expensive component of testing (68-72% of the total cost). Equipment costs are higher for rare disease cases, whereas consumable and staff costs are slightly higher for cancer cases. CONCLUSION: The cost of genome sequencing is underestimated if only sequencing costs are considered, and likely surpasses $1000/genome in a single laboratory. This aspirational sequencing cost will likely only be achieved if consumable costs are considerably reduced and sequencing is performed at scale.
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Neoplasias/genética , Doenças Raras/genética , Sequenciamento Completo do Genoma/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Neoplasias/economia , Doenças Raras/economia , Medicina Estatal , Pesquisa Translacional Biomédica , Reino Unido , Sequenciamento Completo do Genoma/instrumentaçãoRESUMO
BACKGROUND: Molecular indicators of colorectal cancer prognosis have been assessed in several studies, but most analyses have been restricted to a handful of markers. We aimed to identify prognostic biomarkers for colorectal cancer by sequencing panels of multiple driver genes. METHODS: In stage II or III colorectal cancers from the QUASAR 2 open-label randomised phase 3 clinical trial and an Australian community-based series, we used targeted next-generation sequencing of 82 and 113 genes, respectively, including the main colorectal cancer drivers. We investigated molecular pathways of tumorigenesis, and analysed individual driver gene mutations, combinations of mutations, or global measures such as microsatellite instability (MSI) and mutation burden (total number of non-synonymous mutations and coding indels) for associations with relapse-free survival in univariable and multivariable models, principally Cox proportional hazards models. FINDINGS: In QUASAR 2 (511 tumours), TP53, KRAS, BRAF, and GNAS mutations were independently associated with shorter relapse-free survival (p<0·035 in all cases), and total somatic mutation burden with longer survival (hazard ratio [HR] 0·81 [95% CI 0·68-0·96]; p=0·014). MSI was not independently associated with survival (HR 1·12 [95% CI 0·57-2·19]; p=0·75). We successfully validated these associations in the Australian sample set (296 tumours). In a combined analysis of both the QUASAR 2 and the Australian sample sets, mutation burden was also associated with longer survival (HR 0·84 [95% CI 0·74-0·94]; p=0·004) after exclusion of MSI-positive and POLE mutant tumours. In an extended analysis of 1732 QUASAR 2 and Australian colorectal cancers for which KRAS, BRAF, and MSI status were available, KRAS and BRAF mutations were specifically associated with poor prognosis in MSI-negative cancers. MSI-positive cancers with KRAS or BRAF mutations had better prognosis than MSI-negative cancers that were wild-type for KRAS or BRAF. Mutations in the genes NF1 and NRAS from the MAPK pathway co-occurred, and mutations in the DNA damage-response genes TP53 and ATM were mutually exclusive. We compared a prognostic model based on the gold standard of clinicopathological variables and MSI with our new model incorporating clinicopathological variables, mutation burden, and driver mutations in KRAS, BRAF, and TP53. In both QUASAR 2 and the Australian cohort, our new model was significantly better (p=0·00004 and p=0·0057, respectively, based on a likelihood ratio test). INTERPRETATION: Multigene panels identified two previously unreported prognostic associations in colorectal cancer involving TP53 mutation and total mutation burden, and confirmed associations with KRAS and BRAF. Even a modest-sized gene panel can provide important information for use in clinical practice and outperform MSI-based prognostic models. FUNDING: UK Technology Strategy Board, National Institute for Health Research Oxford Biomedical Research Centre, Cancer Australia Project, Cancer Council Victoria, Ludwig Institute for Cancer Research, Victorian Government.
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Biomarcadores Tumorais , Neoplasias Colorretais/genética , Mutação , Austrália , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Tecnologia de Impulso Genético , Humanos , Estadiamento de Neoplasias , Prognóstico , Análise de Sequência de DNARESUMO
Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.
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Éxons , Predisposição Genética para Doença , Mutação , Policitemia/genética , Splicing de RNA , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Adolescente , Adulto , Criança , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Policitemia/classificação , Policitemia/patologia , Adulto Jovem , Doença de von Hippel-Lindau/patologiaRESUMO
BACKGROUND: Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing. METHODS AND FINDINGS: We validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care. Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests. Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of "real world" data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds. CONCLUSIONS: This study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.
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Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Colorretais/diagnóstico , Genômica , Melanoma/diagnóstico , Patologia/métodos , Patologia/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/economia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Criança , Tomada de Decisão Clínica , Neoplasias Colorretais/economia , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Melanoma/economia , Melanoma/genética , Melanoma/terapia , Pessoa de Meia-Idade , Programas Nacionais de Saúde , Estudos Prospectivos , Estudos Retrospectivos , Reino Unido , Adulto JovemRESUMO
Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.
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DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Neoplasias/genética , Biópsia , DNA de Neoplasias/sangue , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Melanoma/diagnóstico , Melanoma/genética , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Erythrocytosis is a rare disorder characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit. Several genetic variants have been identified as causes for erythrocytosis in genes belonging to different pathways including oxygen sensing, erythropoiesis and oxygen transport. However, despite clinical investigation and screening for these mutations, the cause of disease cannot be found in a considerable number of patients, who are classified as having idiopathic erythrocytosis. In this study, we developed a targeted next-generation sequencing panel encompassing the exonic regions of 21 genes from relevant pathways (~79 Kb) and sequenced 125 patients with idiopathic erythrocytosis. The panel effectively screened 97% of coding regions of these genes, with an average coverage of 450×. It identified 51 different rare variants, all leading to alterations of protein sequence, with 57 out of 125 cases (45.6%) having at least one of these variants. Ten of these were known erythrocytosis-causing variants, which had been missed following existing diagnostic algorithms. Twenty-two were novel variants in erythrocytosis-associated genes (EGLN1, EPAS1, VHL, BPGM, JAK2, SH2B3) and in novel genes included in the panel (e.g. EPO, EGLN2, HIF3A, OS9), some with a high likelihood of functionality, for which future segregation, functional and replication studies will be useful to provide further evidence for causality. The rest were classified as polymorphisms. Overall, these results demonstrate the benefits of using a gene panel rather than existing methods in which focused genetic screening is performed depending on biochemical measurements: the gene panel improves diagnostic accuracy and provides the opportunity for discovery of novel variants.
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Mutação , Policitemia/genética , Variação Genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Policitemia/diagnóstico , Policitemia/etiologia , Análise de Sequência de DNARESUMO
BACKGROUND: In mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression. METHODS: MCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses. RESULTS: Hypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer. CONCLUSIONS: Integrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.
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Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Fator 1 Induzível por Hipóxia/genética , MicroRNAs/análise , RNA Mensageiro/análise , Neoplasias da Mama/metabolismo , Hipóxia Celular/genética , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Hypoxia is central to both ischaemic and neoplastic diseases. However, the non-coding transcriptional response to hypoxia is largely uncharacterized. We undertook integrated genomic analyses of both non-coding and coding transcripts using massively parallel sequencing and interfaced this data with pan-genomic analyses of hypoxia-inducible factor (HIF) and RNApol2 binding in hypoxic cells. These analyses revealed that all classes of RNA are profoundly regulated by hypoxia and implicated HIF as a major direct regulator of both the non-coding and coding transcriptome, acting predominantly through release of pre-bound promoter-paused RNApol2. These findings indicate that the transcriptional response to hypoxia is substantially more extensive than previously considered.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , RNA Polimerase II/metabolismo , Transcriptoma , Hipóxia Celular , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. METHODS: MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. RESULTS: Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. CONCLUSIONS: Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.
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Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Homeodomínio/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Família Multigênica , Gradação de Tumores , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The Transforming Growth Factor-ß (TGF-ß) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-ß signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-ß activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341â¼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-ß inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341â¼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-ß/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-ß/Smad2/3 pathway underlie tumor suppression and metastasis, this research also provides a resource for miRNAs involved in cancer.
Assuntos
Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , MicroRNAs/biossíntese , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Família Multigênica , Neoplasias/embriologia , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
OBJECTIVE: To identify novel microRNA (miR) associations in synovial fibroblasts (SF), by performing miR expression profiling on cells isolated from the human tumour necrosis factor (TNF) transgenic mouse model (TghuTNF, Tg197) and patients biopsies. METHODS: miR expression in SF from TghuTNF and wild-type (WT) control mice were determined by miR deep sequencing (miR-seq) and the arthritic profile was established by pairwise comparisons. Quantitative PCR analysis was utilised for profile validation, miR and gene quantitation in patient SF. Dysregulated miR target genes and pathways were predicted via bioinformatic algorithms and validated using gain-of-function coupled with reporter assay experiments. RESULTS: miR-seq demonstrated that TghuTNF-SF exhibit a distinct pathogenic profile with 22 significantly upregulated and 30 significantly downregulated miR. Validation assays confirmed the dysregulation of miR-223, miR-146a and miR-155 previously associated with human rheumatoid arthritis (RA) pathology, as well as that of miR-221/222 and miR-323-3p. Notably, the latter were also found significantly upregulated in patient RA SF, suggesting for the first time their association with RA pathology. Bioinformatic analysis suggested Wnt/cadherin signalling as a putative pathway target. miR-323-3p overexpression was shown to enhance Wnt pathway activation and decrease the levels of its predicted target ß-transducin repeat containing, an inhibitor of ß-catenin. CONCLUSIONS: Using miR-seq-based profiling in SF from the TghuTNF mouse model and validations in RA patient biopsies, the authors identified miR-221/222 and miR-323-3p as novel dysregulated miR in RA SF. Furthermore, the authors show that miR-323-3p is a positive regulator of WNT/cadherin signalling in RA SF suggesting its potential pathogenic involvement and future use as a therapeutic target in RA.
Assuntos
Artrite Reumatoide/genética , MicroRNAs/genética , Transcriptoma , Algoritmos , Animais , Biologia Computacional , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
microRNA expression profiling plays an emerging role in cancer classification and identification of therapeutic strategies. In this study, we have evaluated the benefits of a joint microRNA-mRNA analysis in breast cancer. Matched mRNA and microRNA global expression profiling was conducted in a well-annotated cohort of 207 cases with complete 10-year follow-up. Penalized Cox regression including microRNA expression, mRNA expression, and clinical covariates was used to identify microRNAs associated with distant relapse-free survival (DRFS) that provide independent prognostic information, and are not simply surrogates of previously identified prognostic covariates. Penalized regression was chosen to prevent overfitting. Furthermore, microRNA-mRNA relationships were explored by global expression analysis, and exploited to validate results in several published cohorts (n = 592 with DRFS, n = 1,050 with recurrence-free survival). Four microRNAs were independently associated with DRFS in estrogen receptor (ER)-positive (3 novel and 1 known; miR-128a) and 6 in ER-negative (5 novel and 1 known; miR-210) cases. Of the latter, miR-342, -27b, and -150 were prognostic also in triple receptor-negative tumors. Coordinated expression of predicted target genes and prognostic microRNAs strengthened these results, most significantly for miR-210, -128a, and -27b, whose targets were prognostic in meta-analysis of several cohorts. In addition, miR-210 and -128a showed coordinated expression with their cognate pri-microRNAs, which were themselves prognostic in independent cohorts. Our integrated microRNA-mRNA global profiling approach has identified microRNAs independently associated with prognosis in breast cancer. Furthermore, it has validated known and predicted microRNA-target interactions, and elucidated their association with key pathways that could represent novel therapeutic targets.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , Neoplasias da Mama/genética , Progressão da Doença , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Recidiva Local de Neoplasia/genética , PrognósticoRESUMO
Patients with advanced chronic kidney disease are increasingly elderly with increasing numbers of co-morbidites. Some may not be suitable for dialysis, some will choose to withdraw from treatment after a period of time and some will reach the end of their lives while still on dialysis. Studies have shown nurses' attitudes towards caring for dying patients affect the quality of care. A descriptive study was conducted to explore Spanish nurses' views and attitudes in this context and to assess any relationship between demographic variables and attitudes. Two measurement tools were used: a demographic survey and the Frommelt Attitude Toward Care of the Dying Scale-Form B. Two hundred and two completed questionnaires were returned. Although respondents demonstrated positive attitudes in this domain, 88.9% viewed end-of-life (EOL) care as an emotionally demanding task, 95.3% manifested that addressing death issue require special skills and 92.6% reported that education on EOL care is necessary. This paper suggests strategies which could ease the burden in this area of care.
Assuntos
Atitude do Pessoal de Saúde , Nefropatias/terapia , Nefrologia , Enfermeiras e Enfermeiros/psicologia , Cuidados de Enfermagem , Especialidades de Enfermagem , Assistência Terminal/psicologia , Adulto , Doença Crônica , Comunicação , Feminino , Humanos , Masculino , Relações Enfermeiro-Paciente , Cuidados Paliativos/psicologia , EspanhaRESUMO
BACKGROUND: Hypoxia is an important mechanism of treatment resistance in head and neck squamous cell carcinoma (HNSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The authors have investigated the role of 3 microRNAs, including the hypoxia-induced hsa-miR-210, as potential markers of hypoxia or prognosis. METHODS: Three hypoxia-related microRNAs, hsa-miR-210, hsa-miR-21, and hsa-miR-10b, were measured in 46 samples from patients with HNSCC. Expression levels were correlated with clinicopathological variables and other markers of hypoxia: a published 99-gene hypoxia metagene, individual hypoxia-related genes such as TWIST1, and immunohistochemical expression of hypoxia-inducible factor 1 and its target gene carbonic anhydrase 9. We then performed survival analyses to investigate the prognostic significance of these microRNAs. RESULTS: Only the level of hsa-miR-210 was significantly correlated with other markers of hypoxia, including the 99-gene hypoxia metagene (rho = 0.67, P < .001). We found no association between hsa-miR-210, hsa-miR-21, or hsa-miR-10b and clinicopathological variables such as tumor size, differentiation, and stage. However, high levels of hsa-miR-210 were associated with locoregional disease recurrence (P = .001) and short overall survival (P = .008). hsa-miR-21 and hsa-miR-10b had no prognostic significance. CONCLUSIONS: Expression of hsa-miR-210 in head and neck cancer correlates with other approaches for assessing hypoxia and is associated with prognosis. This warrants further study as a classification marker of patients for therapies involving modulation of hypoxia.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/análise , Neoplasias de Células Escamosas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Hipóxia/genética , Masculino , Pessoa de Meia-Idade , Neoplasias de Células Escamosas/metabolismo , Prognóstico , Precursores de RNA/análiseRESUMO
BACKGROUND: MicroRNAs (miRNAs) are non-coding RNA molecules involved in post-transcriptional control of gene expression of a wide number of genes, including those involved in glucose homeostasis. Type 2 diabetes (T2D) is characterized by hyperglycaemia and defects in insulin secretion and action at target tissues. We sought to establish differences in global miRNA expression in two insulin-target tissues from inbred rats of spontaneously diabetic and normoglycaemic strains. METHODS: We used a miRNA microarray platform to measure global miRNA expression in two insulin-target tissues: liver and adipose tissue from inbred rats of spontaneously diabetic (Goto-Kakizaki [GK]) and normoglycaemic (Brown-Norway [BN]) strains which are extensively used in genetic studies of T2D. MiRNA data were integrated with gene expression data from the same rats to investigate how differentially expressed miRNAs affect the expression of predicted target gene transcripts. RESULTS: The expression of 170 miRNAs was measured in liver and adipose tissue of GK and BN rats. Based on a p-value for differential expression between GK and BN, the most significant change in expression was observed for miR-125a in liver (FC = 5.61, P = 0.001, Padjusted = 0.10); this overexpression was validated using quantitative RT-PCR (FC = 13.15, P = 0.0005). MiR-125a also showed over-expression in the GK vs. BN analysis within adipose tissue (FC = 1.97, P = 0.078, Padjusted = 0.99), as did the previously reported miR-29a (FC = 1.51, P = 0.05, Padjusted = 0.99). In-silico tools assessing the biological role of predicted miR-125a target genes suggest an over-representation of genes involved in the MAPK signaling pathway. Gene expression analysis identified 1308 genes with significantly different expression between GK and BN rats (Padjusted < 0.05): 233 in liver and 1075 in adipose tissue. Pathways related to glucose and lipid metabolism were significantly over-represented among these genes. Enrichment analysis suggested that differentially expressed genes in GK compared to BN included more predicted miR-125a target genes than would be expected by chance in adipose tissue (FDR = 0.006 for up-regulated genes; FDR = 0.036 for down-regulated genes) but not in liver (FDR = 0.074 for up-regulated genes; FDR = 0.248 for down-regulated genes). CONCLUSION: MiR-125a is over-expressed in liver in hyperglycaemic GK rats relative to normoglycaemic BN rats, and our array data also suggest miR-125a is over-expressed in adipose tissue. We demonstrate the use of in-silico tools to provide the basis for further investigation of the potential role of miR-125a in T2D. In particular, the enrichment of predicted miR-125a target genes among differentially expressed genes has identified likely target genes and indicates that integrating global miRNA and mRNA expression data may give further insights into miRNA-mediated regulation of gene expression.