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BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is increasing globally, but its molecular features are not well defined. We aimed to identify unique molecular traits characterising NASH-HCC compared to other HCC aetiologies. METHODS: We collected 80 NASH-HCC and 125 NASH samples from 5 institutions. Expression array (n = 53 NASH-HCC; n = 74 NASH) and whole exome sequencing (n = 52 NASH-HCC) data were compared to HCCs of other aetiologies (n = 184). Three NASH-HCC mouse models were analysed by RNA-seq/expression-array (n = 20). Activin A receptor type 2A (ACVR2A) was silenced in HCC cells and proliferation assessed by colorimetric and colony formation assays. RESULTS: Mutational profiling of NASH-HCC tumours revealed TERT promoter (56%), CTNNB1 (28%), TP53 (18%) and ACVR2A (10%) as the most frequently mutated genes. ACVR2A mutation rates were higher in NASH-HCC than in other HCC aetiologies (10% vs. 3%, p <0.05). In vitro, ACVR2A silencing prompted a significant increase in cell proliferation in HCC cells. We identified a novel mutational signature (MutSig-NASH-HCC) significantly associated with NASH-HCC (16% vs. 2% in viral/alcohol-HCC, p = 0.03). Tumour mutational burden was higher in non-cirrhotic than in cirrhotic NASH-HCCs (1.45 vs. 0.94 mutations/megabase; p <0.0017). Compared to other aetiologies of HCC, NASH-HCCs were enriched in bile and fatty acid signalling, oxidative stress and inflammation, and presented a higher fraction of Wnt/TGF-ß proliferation subclass tumours (42% vs. 26%, p = 0.01) and a lower prevalence of the CTNNB1 subclass. Compared to other aetiologies, NASH-HCC showed a significantly higher prevalence of an immunosuppressive cancer field. In 3 murine models of NASH-HCC, key features of human NASH-HCC were preserved. CONCLUSIONS: NASH-HCCs display unique molecular features including higher rates of ACVR2A mutations and the presence of a newly identified mutational signature. LAY SUMMARY: The prevalence of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH) is increasing globally, but its molecular traits are not well characterised. In this study, we uncovered higher rates of ACVR2A mutations (10%) - a potential tumour suppressor - and the presence of a novel mutational signature that characterises NASH-related HCC.
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Carcinoma Hepatocelular/genética , Biología Molecular/estadística & datos numéricos , Enfermedad del Hígado Graso no Alcohólico/genética , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/etiología , Femenino , Humanos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Biología Molecular/métodos , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Factores de RiesgoRESUMEN
Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2'-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum, particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.
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Evolución Molecular , Pyrobaculum/genética , ARN de Archaea/genética , ARN Nucleolar Pequeño/genética , Proteínas Arqueales/genética , Disparidad de Par Base , Genes Duplicados , Genómica , Metilación , Familia de Multigenes , ARN de Archaea/química , ARN de Archaea/clasificación , ARN de Archaea/metabolismo , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/clasificación , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/metabolismo , ARN no Traducido/genética , Alineación de SecuenciaRESUMEN
BACKGROUND: Data from a plethora of high-throughput sequencing studies is readily available to researchers, providing genetic variants detected in a variety of healthy and disease populations. While each individual cohort helps gain insights into polymorphic and disease-associated variants, a joint perspective can be more powerful in identifying polymorphisms, rare variants, disease-associations, genetic burden, somatic variants, and disease mechanisms. DESCRIPTION: We have set up a Reference Variant Store (RVS) containing variants observed in a number of large-scale sequencing efforts, such as 1000 Genomes, ExAC, Scripps Wellderly, UK10K; various genotyping studies; and disease association databases. RVS holds extensive annotations pertaining to affected genes, functional impacts, disease associations, and population frequencies. RVS currently stores 400 million distinct variants observed in more than 80,000 human samples. CONCLUSIONS: RVS facilitates cross-study analysis to discover novel genetic risk factors, gene-disease associations, potential disease mechanisms, and actionable variants. Due to its large reference populations, RVS can also be employed for variant filtration and gene prioritization. AVAILABILITY: A web interface to public datasets and annotations in RVS is available at https://rvs.u.hpc.mssm.edu/.
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Bases de Datos Genéticas , Enfermedad/genética , Variación Genética , Anotación de Secuencia Molecular/métodos , Genoma Humano , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Bases del Conocimiento , Valores de Referencia , Factores de RiesgoRESUMEN
Classical approaches to determine structures of noncoding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, we developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. We probed in two cell types to verify reproducibility. We also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.
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Perfilación de la Expresión Génica/métodos , ARN/química , ARN/genética , Animales , Emparejamiento Base , Secuencia de Bases , Mapeo Cromosómico/métodos , Cartilla de ADN , Biblioteca de Genes , Histonas/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neuronas/fisiología , Conformación de Ácido Nucleico , ARN no Traducido/químicaRESUMEN
We describe an open source, portable, JavaScript-based genome browser, JBrowse, that can be used to navigate genome annotations over the web. JBrowse helps preserve the user's sense of location by avoiding discontinuous transitions, instead offering smoothly animated panning, zooming, navigation, and track selection. Unlike most existing genome browsers, where the genome is rendered into images on the webserver and the role of the client is restricted to displaying those images, JBrowse distributes work between the server and client and therefore uses significantly less server overhead than previous genome browsers. We report benchmark results empirically comparing server- and client-side rendering strategies, review the architecture and design considerations of JBrowse, and describe a simple wiki plug-in that allows users to upload and share annotation tracks.
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Genoma/genética , Internet , Programas Informáticos , Animales , Biología Computacional/métodos , Sistemas de Administración de Bases de Datos , Bases de Datos Genéticas , Drosophila melanogaster/genética , Genoma de los Insectos/genética , Diseño de Software , Interfaz Usuario-ComputadorRESUMEN
Ossifying fibromas are very rare tumors that are sometimes seen as part of the hyperparathyroidism-jaw tumor syndrome (HPT-JT), which is caused by inactivating mutations of the HRPT2/CDC73 tumor suppressor gene. CDC73 mutations have been identified in a subset of sporadic cases but aberrant expression of the encoded protein, parafibromin, has not been demonstrated in ossifying fibroma. We sought to determine if loss of parafibromin regularly contributes to the development of sporadic, nonsyndromic ossifying fibroma. We examined a series of 9 ossifying fibromas, including ossifying, cemento-ossifying, and juvenile active variants, for parafibromin protein expression by immunohistochemistry and for CDC73 sequence abnormalities by Sanger sequencing and/or targeted AmpliSeq panel sequencing. Four ossifying fibromas showed a complete absence of nuclear parafibromin expression; loss of parafibromin expression was coupled with aberrant cytoplasmic parafibromin expression in 1 case. CDC73 mutations were detected in 2 cases with aberrant parafibromin expression. These results provide novel evidence, at the level of protein expression, that loss of the parathyroid CDC73/parafibromin tumor suppressor may play a role in the pathogenesis of a subset of ossifying fibromas.
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CONTEXT: Pituitary corticotroph adenomas are rare tumors that can be associated with excess adrenocorticotropin (ACTH) and adrenal cortisol production, resulting in the clinically debilitating endocrine condition Cushing disease. A subset of corticotroph tumors behave aggressively, and genomic drivers behind the development of these tumors are largely unknown. OBJECTIVE: To investigate genomic drivers of corticotroph tumors at risk for aggressive behavior. DESIGN: Whole-exome sequencing of patient-matched corticotroph tumor and normal deoxyribonucleic acid (DNA) from a patient cohort enriched for tumors at risk for aggressive behavior. SETTING: Tertiary care center. PATIENTS: Twenty-seven corticotroph tumors from 22 patients were analyzed. Twelve tumors were macroadenomas, of which 6 were silent ACTH tumors, 2 were Crooke's cell tumors, and 1 was a corticotroph carcinoma. INTERVENTION: Whole-exome sequencing. MAIN OUTCOME MEASURE: Somatic mutation genomic biomarkers. RESULTS: We found recurrent somatic mutations in USP8 and TP53 genes, both with higher allelic fractions than other somatic mutations. These mutations were mutually exclusive, with TP53 mutations occurring only in USP8 wildtype (WT) tumors, indicating they may be independent driver genes. USP8-WT tumors were characterized by extensive somatic copy number variation compared with USP8-mutated tumors. Independent of molecular driver status, we found an association between invasiveness, macroadenomas, and aneuploidy. CONCLUSIONS: Our data suggest that corticotroph tumors may be categorized into a USP8-mutated, genome-stable subtype versus a USP8-WT, genome-disrupted subtype, the latter of which has a TP53-mutated subtype with high level of chromosome instability. These findings could help identify high risk corticotroph tumors, namely those with widespread CNV, that may need closer monitoring and more aggressive treatment.
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Adenoma Hipofisario Secretor de ACTH/genética , Adenoma/genética , Variaciones en el Número de Copia de ADN , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/genética , Adenoma Hipofisario Secretor de ACTH/epidemiología , Adenoma Hipofisario Secretor de ACTH/patología , Adenoma/epidemiología , Adenoma/patología , Adolescente , Adulto , Estudios de Casos y Controles , Transformación Celular Neoplásica/genética , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/fisiología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Secuenciación del Exoma , Adulto JovenRESUMEN
Adenoid cystic carcinoma (ACC) is a rare cancer type that originates in the salivary glands. Tumors commonly invade along nerve tracks in the head and neck, making surgery challenging. Follow-up treatments for recurrence or metastasis including chemotherapy and targeted therapies have shown limited efficacy, emphasizing the need for new therapies. Here, we report a Drosophila-based therapeutic approach for a patient with advanced ACC disease. A patient-specific Drosophila transgenic line was developed to model the five major variants associated with the patient's disease. Robotics-based screening identified a three-drug cocktail-vorinostat, pindolol, tofacitinib-that rescued transgene-mediated lethality in the Drosophila patient-specific line. Patient treatment led to a sustained stabilization and a partial metabolic response of 12 months. Subsequent resistance was associated with new genomic amplifications and deletions. Given the lack of options for patients with ACC, our data suggest that this approach may prove useful for identifying novel therapeutic candidates.
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The genetic architecture of schizophrenia (SCZ) includes numerous risk loci across a range of frequencies and sizes, including common and rare single-nucleotide variants and insertions/deletions (indels), as well as rare copy number variants (CNVs). Despite the clear heritability of the disease, monozygotic twins are discordant for SCZ at a significant rate. Somatic variants-genetic changes that arise after fertilization rather than through germline inheritance-are widespread in the human brain and known to contribute to risk for both rare and common neuropsychiatric conditions. The contribution of somatic variants in the brain to risk of SCZ remains to be determined. In this study, we surveyed somatic single-nucleotide variants (sSNVs) in the brains of controls and individuals with SCZ (n = 10 and n = 9, respectively). From each individual, whole-exome sequencing (WES) was performed on DNA from neuronal and non-neuronal nuclei isolated by fluorescence activated nuclear sorting (FANS) from frozen postmortem prefrontal cortex (PFC) samples, as well as DNA extracted from temporal muscle as a reference. We identified an increased burden of sSNVs in cases compared to controls (SCZ rate = 2.78, control rate = 0.70; P = 0.0092, linear mixed effects model), that included a higher rate of non-synonymous and loss-of-function variants (SCZ rate = 1.33, control rate = 0.50; P = 0.047, linear mixed effects model). Our findings suggest sSNVs in the brain may constitute an additional component of the complex genetic architecture of SCZ. This perspective argues for the need to further investigate somatic variation in the brain as an explanation of the discordance in monozygotic twins and a potential guide to the identification of novel therapeutic targets.
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Encéfalo/patología , Polimorfismo de Nucleótido Simple , Esquizofrenia/genética , Estudios de Casos y Controles , Variaciones en el Número de Copia de ADN , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Esquizofrenia/patología , Gemelos Monocigóticos/genética , Secuenciación del ExomaRESUMEN
Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor's genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease's genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Genes ras , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Ácido Zoledrónico/administración & dosificación , Animales , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Drosophila/genética , Esquema de Medicación , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Genómica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Medicina de PrecisiónRESUMEN
BACKGROUND: Chemotherapy may exert immunomodulatory effects, thereby combining favorably with the immune checkpoint blockade. The pharmacodynamic effects of such combinations, and potential predictive biomarkers, remain unexplored. OBJECTIVE: To determine the safety, efficacy, and immunomodulatory effects of gemcitabine and cisplatin (GC) plus ipilimumab and explore the impact of somatic DNA damage response gene alterations on antitumor activity. DESIGN, SETTING, AND PARTICIPANTS: Multicenter single arm phase 2 study enrolling 36 chemotherapy-naïve patients with metastatic urothelial cancer. Peripheral blood flow cytometry was performed serially on all patients and whole exome sequencing of archival tumor tissue was performed on 28/36 patients. INTERVENTION: Two cycles of GC followed by four cycles of GC plus ipilimumab. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint was 1-yr overall survival (OS). Secondary endpoints included safety, objective response rate, and progression-free survival. RESULTS AND LIMITATIONS: Grade ≥3 adverse events occurred in 81% of patients, the majority of which were hematologic. The objective response rate was 69% and 1-yr OS was 61% (lower bound 90% confidence interval: 51%). On exploratory analysis, there were no significant changes in the composition and frequency of circulating immune cells after GC alone. However, there was a significant expansion of circulating CD4 cells with the addition of ipilimumab which correlated with improved survival. The response rate was significantly higher in patients with deleterious somatic DNA damage response mutations (sensitivity=47.6%, specificity=100%, positive predictive value=100%, and negative predictive value=38.9%). Limitations are related to the sample size and single-arm design. CONCLUSIONS: GC+ipilimumab did not achieve the primary endpoint of a lower bound of the 90% confidence interval for 1-yr OS of >60%. However, within the context of a small single-arm trial, the results may inform current approaches combining chemotherapy plus immunotherapy from the standpoint of feasibility, appropriate cytotoxic backbones, and potential predictive biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov NCT01524991. PATIENT SUMMARY: Combining chemotherapy and immune checkpoint blockade in patients with metastatic urothelial cancer is feasible. Further studies are needed to refine optimal combinations and evaluate tests that might identify patients most likely to benefit.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/genética , Mutación/genética , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/genética , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/mortalidad , Carcinoma de Células Transicionales/patología , Cisplatino/administración & dosificación , Daño del ADN/efectos de los fármacos , Análisis Mutacional de ADN , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Supervivencia sin Enfermedad , Femenino , Humanos , Ipilimumab/administración & dosificación , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Estudios Prospectivos , Medición de Riesgo , Análisis de Supervivencia , Resultado del Tratamiento , Neoplasias Urológicas/mortalidad , Neoplasias Urológicas/patología , GemcitabinaRESUMEN
Chronic allograft damage, defined by interstitial fibrosis and tubular atrophy (IF/TA), is a leading cause of allograft failure. Few effective therapeutic options are available to prevent the progression of IF/TA. We applied a meta-analysis approach on IF/TA molecular datasets in Gene Expression Omnibus to identify a robust 85-gene signature, which was used for computational drug repurposing analysis. Among the top ranked compounds predicted to be therapeutic for IF/TA were azathioprine, a drug to prevent acute rejection in renal transplantation, and kaempferol and esculetin, two drugs not previously described to have efficacy for IF/TA. We experimentally validated the anti-fibrosis effects of kaempferol and esculetin using renal tubular cells in vitro and in vivo in a mouse Unilateral Ureteric Obstruction (UUO) model. Kaempferol significantly attenuated TGF-ß1-mediated profibrotic pathways in vitro and in vivo, while esculetin significantly inhibited Wnt/ß-catenin pathway in vitro and in vivo. Histology confirmed significantly abrogated fibrosis by kaempferol and esculetin in vivo. We developed an integrative computational framework to identify kaempferol and esculetin as putatively novel therapies for IF/TA and provided experimental evidence for their therapeutic activities in vitro and in vivo using preclinical models. The findings suggest that both drugs might serve as therapeutic options for IF/TA.
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Aloinjertos/patología , Quempferoles/administración & dosificación , Trasplante de Riñón/efectos adversos , Riñón/efectos de los fármacos , Riñón/patología , Umbeliferonas/administración & dosificación , Animales , Línea Celular , Biología Computacional , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Fibrosis , Rechazo de Injerto/tratamiento farmacológico , Humanos , Informática , Enfermedades Renales/genética , Enfermedades Renales/patología , Enfermedades Renales/cirugía , Masculino , Ratones Endogámicos BALB C , Transducción de Señal/efectos de los fármacosRESUMEN
Cushing's disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.
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Adenoma Hipofisario Secretor de ACTH/genética , Proteínas ras/genética , Adenoma/genética , Hormona Adrenocorticotrópica/genética , Adulto , Corticotrofos/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Femenino , Humanos , Mutación , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/genética , Neoplasias Hipofisarias/genética , Receptores de Hormona Liberadora de Corticotropina/genética , Análisis de Secuencia de ADN , Ubiquitina Tiolesterasa/genéticaRESUMEN
Parathyroid carcinoma (PC) is an extremely rare malignancy lacking effective therapeutic intervention. We generated and analyzed whole-exome sequencing data from 17 patients to identify somatic and germline genetic alterations. A panel of selected genes was sequenced in a 7-tumor expansion cohort. We show that 47% (8 of 17) of the tumors harbor somatic mutations in the CDC73 tumor suppressor, with germline inactivating variants in 4 of the 8 patients. The PI3K/AKT/mTOR pathway was altered in 21% of the 24 cases, revealing a major oncogenic pathway in PC. We observed CCND1 amplification in 29% of the 17 patients, and a previously unreported recurrent mutation in putative kinase ADCK1. We identified the first sporadic PCs with somatic mutations in the Wnt canonical pathway, complementing previously described epigenetic mechanisms mediating Wnt activation. This is the largest genomic sequencing study of PC, and represents major progress toward a full molecular characterization of this rare malignancy to inform improved and individualized treatments.
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Perfilación de la Expresión Génica , Mutación , Neoplasias de las Paratiroides/genética , Estudios de Cohortes , Ciclina D1/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/genética , Vía de Señalización WntRESUMEN
Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.
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Proliferación Celular/genética , Diabetes Mellitus Tipo 1/terapia , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Neoplasias Pancreáticas/genética , Regeneración/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Diabetes Mellitus Tipo 1/metabolismo , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Insulinoma/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismoRESUMEN
BACKGROUND: Non-coding RNAs (ncRNAs) have a multitude of roles in the cell, many of which remain to be discovered. However, it is difficult to detect novel ncRNAs in biochemical screens. To advance biological knowledge, computational methods that can accurately detect ncRNAs in sequenced genomes are therefore desirable. The increasing number of genomic sequences provides a rich dataset for computational comparative sequence analysis and detection of novel ncRNAs. RESULTS: Here, Dynalign, a program for predicting secondary structures common to two RNA sequences on the basis of minimizing folding free energy change, is utilized as a computational ncRNA detection tool. The Dynalign-computed optimal total free energy change, which scores the structural alignment and the free energy change of folding into a common structure for two RNA sequences, is shown to be an effective measure for distinguishing ncRNA from randomized sequences. To make the classification as a ncRNA, the total free energy change of an input sequence pair can either be compared with the total free energy changes of a set of control sequence pairs, or be used in combination with sequence length and nucleotide frequencies as input to a classification support vector machine. The latter method is much faster, but slightly less sensitive at a given specificity. Additionally, the classification support vector machine method is shown to be sensitive and specific on genomic ncRNA screens of two different Escherichia coli and Salmonella typhi genome alignments, in which many ncRNAs are known. The Dynalign computational experiments are also compared with two other ncRNA detection programs, RNAz and QRNA. CONCLUSION: The Dynalign-based support vector machine method is more sensitive for known ncRNAs in the test genomic screens than RNAz and QRNA. Additionally, both Dynalign-based methods are more sensitive than RNAz and QRNA at low sequence pair identities. Dynalign can be used as a comparable or more accurate tool than RNAz or QRNA in genomic screens, especially for low-identity regions. Dynalign provides a method for discovering ncRNAs in sequenced genomes that other methods may not identify. Significant improvements in Dynalign runtime have also been achieved.
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Mapeo Cromosómico/métodos , Modelos Químicos , Modelos Moleculares , ARN no Traducido/química , Alineación de Secuencia/métodos , Análisis de Secuencia de ARN/métodos , Algoritmos , Inteligencia Artificial , Secuencia de Bases , Simulación por Computador , Transferencia de Energía , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reconocimiento de Normas Patrones Automatizadas/métodosRESUMEN
BACKGROUND: Recent years have seen the emergence of genome annotation methods based on the phylo-grammar, a probabilistic model combining continuous-time Markov chains and stochastic grammars. Previously, phylo-grammars have required considerable effort to implement, limiting their adoption by computational biologists. RESULTS: We have developed an open source software tool, xrate, for working with reversible, irreversible or parametric substitution models combined with stochastic context-free grammars. xrate efficiently estimates maximum-likelihood parameters and phylogenetic trees using a novel "phylo-EM" algorithm that we describe. The grammar is specified in an external configuration file, allowing users to design new grammars, estimate rate parameters from training data and annotate multiple sequence alignments without the need to recompile code from source. We have used xrate to measure codon substitution rates and predict protein and RNA secondary structures. CONCLUSION: Our results demonstrate that xrate estimates biologically meaningful rates and makes predictions whose accuracy is comparable to that of more specialized tools.
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Algoritmos , Inteligencia Artificial , Mapeo Cromosómico/métodos , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Secuencia Conservada , FilogeniaAsunto(s)
Adenoma Hipofisario Secretor de ACTH , Adenoma , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT) , Neoplasias Hipofisarias , Adenoma/genética , Estudios de Cohortes , Endopeptidasas , Complejos de Clasificación Endosomal Requeridos para el Transporte , Humanos , Proteína p53 Supresora de Tumor/genética , Ubiquitina TiolesterasaRESUMEN
High-throughput sequencing of cDNA (RNA-Seq) can be used to generate nuclease accessibility data for many distinct transcripts in the same mixture simultaneously. Such assays accelerate RNA structure analysis and provide researchers with new technologies to tackle biological questions on a transcriptome-wide scale. FragSeq is an experimental assay for transcriptome-wide RNA structure probing using RNA-Seq, coupled with data analysis tools that allow quantitative determination of nuclease accessibility at single-base resolution. We provide a practical guide to designing and carrying out FragSeq experiments and data analysis.